antibiotics

Review
Design and Synthesis of Small Molecules as Potent
Staphylococcus aureus Sortase A Inhibitors
Min Woo Ha 1,2 , Sung Wook Yi 3 and Seung-Mann Paek 4, *
1 College of Pharmacy, Jeju National University, 102, Jejudaehak-ro, Jeju, Jeju-do 63243, Korea;
minuha@jejunu.ac.kr
2 Interdisciplinary Graduate Program in Advanced Convergence Technology & Science,
Jeju National University, 102, Jejudaehak-ro, Jeju, Jeju-do 63243, Korea
3 Department of Chemistry & Biochemistry, University of California Los Angeles, CA 90095, USA;
woogie@chem.ucla.edu
4 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University,
Jinju Daero 501, Jinju, Gyeongsangnam-do 52828, Korea
* Correspondence: million@gnu.ac.kr; Tel.: +82-55-772-2424




Received: 28 August 2020; Accepted: 13 October 2020; Published: 16 October 2020


Abstract: The widespread and uncontrollable emergence of antibiotic-resistant bacteria, especially

methicillin-resistant Staphylococcus aureus, has promoted a wave of efforts to discover a new generation
of antibiotics that prevent or treat bacterial infections neither as bactericides nor bacteriostats. Due to
its crucial role in virulence and its nonessentiality in bacterial survival, sortase A has been considered
as a great target for new antibiotics. Sortase A inhibitors have emerged as promising alternative
antivirulence agents against bacteria. Herein, the structural and preparative aspects of some small
synthetic organic compounds that block the pathogenic action of sortase A have been described.

Keywords: drug resistant; sortase A; Staphylococcus aureus; sortase A inhibitor; small organic molecule;
synthetic chemistry

1. Introduction
Antibiotics are indispensable to the health of humankind [1–3]. Owing to the great contribution
of commendable antibiotics, we might expect to extend our life expectancy over the age of 50 without
substantial difficulties. Antibiotics deactivate bacterial infections by directly killing the bacteria and/or
blocking its growth, ultimately preventing its spread [4]. However, the disconcerted emergence
of antibiotic-resistant bacteria [5], which are resistant to several conventional antibiotics including
β-lactam, cephalosporin, tetracycline, fluoroquinoline, macrolide, vancomycin, aminoglycoside,
and trimethoprim-sulfamethoxazole, has become a serious social problem [6–9]. The greatest concerns
regarding antibiotic-resistant bacteria, especially methicillin resistant Staphylococcus aureus (MRSA)
for several difficult-to-treat infections in humans such as pneumonia, bacteremia, osteomyelitis,
and endocarditis [10,11], resulted in the development of a new generation of antibiotics that target the
alternative enzyme components, fighting bacterial infections not as bactericides or bacteriostats [12,13].
Since the elucidation of sortase, one of the membrane-bound cysteine transpeptidase in
Gram-positive bacteria, in 1999, it has been regarded as a great target in the development of novel types
of antibiotics. Its merits include: (1) its location on the extracellular membrane, (2) its lack of homologs
in humans, and (3) its irrelevancy to bacterial growth [14,15]. Among the 1000+ homologs, sortase A in
Staphylococcus aureus is known as the prototypical subtype [16,17]. To date, several research works have
been published on the structure and mode of action of sortase A in S. aureus [18]. In terms of new drug
design and discovery, a major achievement is associated with the development of sortase A inhibitors

Antibiotics 2020, 9, 706; doi:10.3390/antibiotics9100706 www.mdpi.com/journal/antibiotics

Antibiotics 2020, 9, x FOR PEER REVIEW 2 of 16
Antibiotics 2020, 9, 706 2 of 16
have been published on the structure and mode of action of sortase A in S. aureus [18]. In terms of
new drug design and discovery, a major achievement is associated with the development of sortase
as anti-infective agents [19,20]. Sortase A inhibitors could be free from the consideration of their
A inhibitors as anti-infective agents [19,20]. Sortase A inhibitors could be free from the consideration
penetration inside the bacterial membrane. Sortase A inhibitors reduce the bacterial virulence caused
of their penetration inside the bacterial membrane. Sortase A inhibitors reduce the bacterial virulence
by adhesion of the surface protein that carries disease-causing components to the host. Since sortase
caused by adhesion of the surface protein that carries disease-causing components to the host. Since
A is not required for the growth of S. aureus, drug resistance owing to sortase A inhibitors is less
sortase A is not required for the growth of S. aureus, drug resistance owing to sortase A inhibitors is
expected [21–35].
less expected [21–35].
The enzymatic mode of action of sortase A commenced with the recognition step of the
The enzymatic mode of action of sortase A commenced with the recognition step of the C-
C-terminal amino acid sequence LPXTG (X means any amino acids) motif in the target surface
terminal amino acid sequence LPXTG (X means any amino acids) motif in the target surface protein.
protein. Sulfhydryl functionality of Cys184, one of the conserved residues in the catalytic domain,
Sulfhydryl functionality of Cys184, one of the conserved residues in the catalytic domain, attacks
attacks nucleophilically the carbonyl carbon of Thr in the cell wall sorting signal, breaks down the
nucleophilically the carbonyl carbon of Thr in the cell wall sorting signal, breaks down the Thr and
Thr and Gly linkage, and affords the thioester intermediate. This covalent acyl-enzyme adduct
Gly linkage, and affords the thioester intermediate. This covalent acyl-enzyme adduct undergoes
undergoes another nucleophilic acyl substitution by the amino group of Gly in lipid II, forming the
another nucleophilic acyl substitution by the amino group of Gly in lipid II, forming the rigid amide
rigid amide bond; by virtue of these series of steps of sortase A, the surface protein is incorporated
bond; by virtue of these series of steps of sortase A, the surface protein is incorporated into the
into the peptidoglycan of the cell wall. Therefore, the most reliable strategy for inhibition of the
peptidoglycan of the cell wall. Therefore, the most reliable strategy for inhibition of the enzyme is to
enzyme is to inactivate the thiol functional group with a variety of electrophilic molecules covalently
inactivate the thiol functional group with a variety of electrophilic molecules covalently or non-
or non-covalently. Figure 1 displays the mechanism of sortase A (green) and its inactivation process
covalently. Figure 1 displays the mechanism of sortase A (green) and its inactivation process through
through a sortase A inhibitor (yellow).
a sortase A inhibitor (yellow).

Figure 1. Mode of action of SrtA and its inactivation through its inhibitor; the surface proteins are
Figure 1. Mode
covalently of action
attached to theofcell
SrtA and
wall its inactivation
peptidoglycan through
with the aiditsofinhibitor; the following
SrtA via the surface proteins are
sequential
covalently attached to the
steps: (1) recognition, cell wall peptidoglycan
(2) thioesterification, and (3)with the aid of SrtAFurther,
transpeptidation. via the they
following sequential
are inhibited for
steps: (1)resulting
display, recognition, (2) thioesterification,
in failure and (3)
of adhesion to specific transpeptidation.
organ tissues, invasion Further,
of hostthey
cells,are inhibited
or the evasionfor
of
display, resulting
host-immune in failure
responses (4).of adhesion to specific organ tissues, invasion of host cells, or the evasion
of host-immune responses (4).
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With the
the attractive
attractive factors
factors of
of sortase
sortase A,
A, several
several strategies
strategies have been explored
explored to isolate
isolate single
single
ingredients with
withinhibitory
inhibitoryactivities
activities against sortase A from natural products and compound
against sortase A from natural products and compound libraries
libraries [36,37].
[36,37]. The The
design design
and and synthesis
synthesis of new molecules
of new molecules inspired
inspired by by their
their own ownskeleton
leading leadinghave
skeleton
also
have
been also been discussed.
discussed. Several small
Several synthetic synthetic smallcompounds
organic organic compounds constructively
constructively prepared prepared by
by chemists
chemists were selected and described in this review. Further, a brief history of the molecular
were selected and described in this review. Further, a brief history of the molecular design and design
and arrangement
arrangement of a of a carbon
carbon skeleton
skeleton is presented.
is presented.

2. Results and
2. Results and Discussion
Discussion
To
To explore
explore the
the practicality
practicality of
of sortase
sortase AA inhibitors,
inhibitors, high-throughput
high-throughput screening
screening (HTS)
(HTS) has
has been
been
preliminarily adopted for constructive syntheses. In addition, further structural
preliminarily adopted for constructive syntheses. In addition, further structural modifications modifications
through structure–activityrelationship
through structure–activity relationship analysis
analysis havehave
been been performed
performed to potential
to identify identify skeletons
potential
skeletons and functionalities.
and functionalities. Several significant
Several significant small organic small organichave
molecules molecules have been
been designed anddesigned and
synthesized,
synthesized, as shown in Figure 2, with promising IC values. Herein, the
as shown in Figure 2, with promising IC50 values. Herein, the concrete synthetic features and the
50 concrete synthetic
features and the analytic
analytic properties properties
for structural for structural
identification identification
are described are described
in the order in the order[38],
of diarylacrylonitriles of
diarylacrylonitriles
pyridazinones [39], [38], pyridazinones [39],
aryl 3-acryloamides [40], aryl 3-acryloamides [41],
dihydro-β-carboline [40], benzisothiazolinones
dihydro-β-carboline [42],[41],
benzisothiazolinones [42], triazolothiadiazoles [43], 2-(2-phenylhydrazinylidene)alkanoates
triazolothiadiazoles [43], 2-(2-phenylhydrazinylidene)alkanoates [44], 2-phenyl-benzo[d]oxazole-7- [44],
2-phenyl-benzo[d]oxazole-7-carboxamide [45], 2-phenyl-benzofuran-7-carboxamide
carboxamide [45], 2-phenyl-benzofuran-7-carboxamide [46], 2-phenylthiazoles[46],[47],
2-phenylthiazoles [47],
2, 5-disubstitued
2, 5-disubstitued thiadiazole [48], and thiadiazolidinedione
thiadiazole [48], and thiadiazolidinedione [49]. [49].

Figure 2. Synthetic small organic

Figure 2. organic molecules
molecules as
as SrtA
SrtA inhibitors
inhibitors (IC 50 values are given in parentheses).
(IC50
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2.1. Preparation
2.1. Preparation of
of Diarylacrylonitriles
Diarylacrylonitriles
Through random
Through randomscreening
screening with thethe
with diverse smallsmall
diverse molecular librarylibrary
molecular of 1000 of
compounds, a molecule
1000 compounds, a
of E-configurated diaryl acrylate was discovered with
molecule of E-configurated diaryl acrylate was discovered 50 an IC of 231 µM by the Kim research
with an IC50 of 231 µM by the Kim group [38].
research
With this
group attractive
[38]. starting
With this lead, a starting
attractive series of lead,
structural modifications
a series of structural as E/Z-geometric
suchmodifications isomerism,
such as E/Z-
the absence of double bond, and substitution of ester for other functionalities was
geometric isomerism, the absence of double bond, and substitution of ester for other functionalities performed,
which
was performed, whichZ-diarylacrylonitriles
finally afforded (5 and 9) with high
finally afforded Z-diarylacrylonitriles inhibitory
(5 and 9) withpotencies S. aureus.
againstpotencies
high inhibitory
To prepare
against the twoTotypes
S. aureus. of double
prepare the twobond structures,
types of double different synthetic strategies
bond structures, different were usedstrategies
synthetic to obtain
the corresponding isomer, as illustrated in Scheme 1 [50].
were used to obtain the corresponding isomer, as illustrated in Scheme 1 [50].

Scheme 1.1.Geometrically
Geometricallyselective synthesis
selective of diarylacrylonitriles
synthesis (IC50 values
of diarylacrylonitriles arevalues
(IC50 given inare
parentheses).
given in
parentheses).
The molecular geometry of the acrylic acid 1 condensed with p-methoxyphenyl acetic acid
and Thep-anisaldehyde in aceticofanhydride
molecular geometry the acrylicand
acidtrimethylamine
1 condensed with was assigned the configuration
p-methoxyphenyl acetic acid and E,
while acrylonitrile
p-anisaldehyde in 5, the adduct
acetic of p-methoxyphenyl
anhydride and trimethylamine acetonitrile and p-anisaldehyde
was assigned under E,
the configuration alkoxide
while
basic condition, was synthesized with the entire Z-isomerism. Further chemical
acrylonitrile 5, the adduct of p-methoxyphenyl acetonitrile and p-anisaldehyde under alkoxide basic replacement via
substitution, reduction, or oxidation was investigated and a few diarylacrylonitrile
condition, was synthesized with the entire Z-isomerism. Further chemical replacement via derivatives (2–8)
were prepared.
substitution, These simple
reduction, and concise
or oxidation synthetic routes
was investigated and awere adopted to obtain the
few diarylacrylonitrile best analog
derivatives (2–8)9
were prepared. These simple and concise synthetic routes were adopted to obtain the best analogin
with 2,5-dimethoxybenzaldehyde. The NMR spectroscopic analysis of olefinic hydrogen (colored 9
with is used to distinguish its E/Z geometry
blue) 2,5-dimethoxybenzaldehyde. The NMR[51,52]; the hydrogen
spectroscopic in the
analysis Z-configurated
of olefinic hydrogenolefin appears
(colored in
to be slightly
blue) is usedmore upfield while
to distinguish E/ZE-geometric
its the geometry hydrogen
[51,52]; theappears
hydrogenmoreindownfield to the left. Even
the Z-configurated in
olefin
this assignment, the comparison of 1 and 7, 2 and 8, and 4 and 5 revealed distinguishable
appears to be slightly more upfield while the E-geometric hydrogen appears more downfield to the information
about
left. the in
Even carbon skeletons [53].
this assignment, the comparison of 1 and 7, 2 and 8, and 4 and 5 revealed distinguishable
information about the carbon skeletons [53].
2.2. Preparation of Pyridazinones
2.2. Preparation
Three typesofof
Pyridazinones
promising heterocycle ring skeletons, rhodanine, pyridazinone, and pyrazolethione,
were introduced by the Jung and Clubb group to the HTS, with over 30,000 organic molecules having
Three types of promising heterocycle ring skeletons, rhodanine, pyridazinone, and
potential IC50 values of up to 3–5 µM [39]. In particular, a further investigation of pyridazinone
pyrazolethione, were introduced by the Jung and Clubb group to the HTS, with over 30,000 organic
derivatives resulted in disulfide 15, which had an IC value of 1.5 µM. Its potency was understood via
molecules having potential IC50 values of up to 3–550µM [39]. In particular, a further investigation of
thiol–disulfide exchange reaction with the SH residue of cysteine of sortase A.
pyridazinone derivatives resulted in disulfide 15, which had an IC50 value of 1.5 µM. Its potency was
With a starting standard 10, the authors intended to investigate the influence of the position of the
understood via thiol–disulfide exchange reaction with the SH residue of cysteine of sortase A.
thiol moiety and proceeded to develop distinct synthetic methods for regioselective substitution with
With a starting standard 10, the authors intended to investigate the influence of the position of
alkoxide to the α or β position of unsaturated ketone. If the relatively mild reaction condition with 1 N
the thiol moiety and proceeded to develop distinct synthetic methods for regioselective substitution
of sodium hydroxide in ethanol proceeded with substrate 11, only β-substituted ethoxide was prepared.
with alkoxide to the α or β position of unsaturated ketone. If the relatively mild reaction condition
However, treatment with ethoxide anion generated in situ 1, 4-dioxane to 11, predominantly providing
with 1 N of sodium hydroxide in ethanol proceeded with substrate 11, only β-substituted ethoxide
the α-substituted counterpart. Moreover, the selective characteristics may depend on the length of
was prepared. However, treatment with ethoxide anion generated in situ 1, 4-dioxane to 11,
the alkoxide carbon chain. Thus, their regioselectivities were determined through the nOe analysis
predominantly providing the α-substituted counterpart. Moreover, the selective characteristics may
between adjacent hydrogens in a molecule; these structural isomers showed their distinguishable
depend on the length of the alkoxide carbon chain. Thus, their regioselectivities were determined
through the nOe analysis between adjacent hydrogens in a molecule; these structural isomers showed
their distinguishable identities on TLC analysis, as shown in Scheme 2. The remaining chlorine atoms
of 12 and 14 were fairly quickly replaced with thiol 13 and 15 and thiol molecule 15 was oxidized to
disulfide 16 in the presence of oxygen gas [54,55].

Antibiotics 2020, 9, 706 5 of 16

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identities on TLC analysis,

their distinguishable as shown
identities on TLCinanalysis,
Scheme as2. shown
The remaining
in Schemechlorine atoms of 12
2. The remaining and 14atoms
chlorine were
fairly
of 12 and 14 were fairly quickly replaced with thiol 13 and 15 and thiol molecule 15 was oxidizedthe
quickly replaced with thiol 13 and 15 and thiol molecule 15 was oxidized to disulfide 16 in to
presence
disulfideof16oxygen gas [54,55].
in the presence of oxygen gas [54,55].

Scheme 2. Synthetic methods for pyridazinone derivatives via regioselective substitution (IC50 values
are given in parentheses).

2.3. Preparation of Aryl 3-Acryloamide

A series of synthetic molecules, presented as aryl 3-acryloamides, were prepared and their
enzyme inhibiting actions were determined by the Velu research group [40]. The authors had
Scheme
performed 2. Synthetic
2.
SchemesystemicSyntheticSARmethods
methods
studiesfor
for pyridazinone
revealedderivatives
pyridazinone
that derivatives via
the promisingviaregioselective
regioselective substitution
substitution
organic materials (IC
(IC5050values
formed values
with the
are
are given
givenin
morpholinobenzoate inparentheses).
parentheses).
group. To examine the effect of the geometry of the double bond on their
activity,
2.3. Z-configurated
Preparation olefin molecules, 18 and 19, and E-configurated molecules, 20 and 21, were
of Aryl 3-Acryloamide
2.3. Preparation
prepared usingof the
Aryloptimized
3-Acryloamide reaction methods presented in Scheme 3. As a result, the E
A series
stereochemistry of synthetic molecules, presented as aryl 3-acryloamides, were prepared and their enzyme
A series of was recognized
synthetic to be
molecules, more important
presented as arylthan the Z configuration
3-acryloamides, were or rigid
prepared triple
andbond.
their
inhibiting
Partial actions
reduction were
with determined
Lindlar by the
catalyst, Velu
which research
is a group [40].
well-established The authors
method had
for performed
the synthesis systemic
of cis-
enzyme inhibiting actions were determined by the Velu research group [40]. The authors had
SAR
olefin,studies
did notthat revealed
provide the the promising
molecule 18, organic materials
presumably due to formed
the with the
poisonous naturemorpholinobenzoate
of the sulfur atom
performed systemic SAR studies that revealed the promising organic materials formed with the
group.
on the To examine for
thiophene the effect of the chemistry.
palladium geometry of the doublethe bond onoftheir activity, Z-configurated olefin
morpholinobenzoate group. To examine theHowever, effect of the use geometry H2 of
gas in double
the the presence
bond of on Pd/C
their
molecules,
afforded 18
the and
desired 19, and E-configurated
cis-alkene 18. For the molecules,
synthesis of20 and 21,
trans-isomer, were prepared
the authors using
utilized the optimized
the already-
activity, Z-configurated olefin molecules, 18 and 19, and E-configurated molecules, 20 and 21, were
reaction
established methods presented
cis-compound as in
theScheme
reactant 3. As a result, the E stereochemistry was recognized to be
prepared using the optimized reaction[56,57].
methods presented in Scheme 3. As a result, the E
moreThe important than
stereochemistry the Z configuration
of E/Z isomers or rigid
can be triple bond.
elucidated Partial
through 1Hreduction
NMR analysis. with Lindlar
On the 1catalyst,
H NMR
stereochemistry was recognized to be more important than the Z configuration or rigid triple bond.
which
spectrum, is a well-established
the olefin method
hydrogens in forand
18 the synthesis
19 have of cis-olefin,
shown didchemical
different not provide theand
shifts molecule
coupling 18,
Partial reduction with Lindlar catalyst, which is a well-established method for the synthesis of cis-
presumably
constants due to the poisonous nature of the sulfur atom on the thiophene for palladium chemistry.
olefin, didcompared
not provide to those of 20 and18,
the molecule 21.presumably
As expected,due thetoapparent coupling
the poisonous constants
nature of thewere sulfursmaller
atom
However,
in the the use of
cis-isomers H2 the
than gas trans-isomers.
in the presenceFurther,
of Pd/C itafforded
has beenthefound
desired cis-alkene
that the For the synthesis
18. hydrogens
olefinic of the
on the thiophene for palladium chemistry. However, the use of H2 gas in the presence of Pd/C
of trans-isomer,
trans-diastereoisomer the authors
are utilized
located the already-established
downfield than those of thecis-compound as
cis-diastereoisomer
afforded the desired cis-alkene 18. For the synthesis of trans-isomer, the authors utilized the already- the reactant
to a certain[56,57].
degree.
established cis-compound as the reactant [56,57].
The stereochemistry of E/Z isomers can be elucidated through 1H NMR analysis. On the 1H NMR
spectrum, the olefin hydrogens in 18 and 19 have shown different chemical shifts and coupling
constants compared to those of 20 and 21. As expected, the apparent coupling constants were smaller
in the cis-isomers than the trans-isomers. Further, it has been found that the olefinic hydrogens of the
trans-diastereoisomer are located downfield than those of the cis-diastereoisomer to a certain degree.

Scheme 3.3. Geometrically

Geometrically selective
selective synthesis
synthesis of
of aryl
aryl 3-acryloamides
3-acryloamides (IC50
50 values
values are
are given in
parentheses).
parentheses).

The stereochemistry of E/Z isomers can be elucidated through 1 H NMR analysis. On the 1 H
NMR spectrum, the olefin hydrogens in 18 and 19 have shown different chemical shifts and coupling
constants compared to those of 20 and 21. As expected, the apparent coupling constants were smaller
Scheme 3. Geometrically selective synthesis of aryl 3-acryloamides (IC50 values are given in
parentheses).
Antibiotics 2020, 9, 706 6 of 16

in the cis-isomers than the trans-isomers. Further, it has been found that the olefinic hydrogens of the
trans-diastereoisomer
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66 of
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16

2.4.
2.4. Preparation
Preparation of
of Dihydro-β-Carboline
Dihydro-β-Carboline
A
Anew heterocyclicskeleton,
newheterocyclic skeleton,β-carboline,
β-carboline,
β-carboline,inspired
inspiredby bythethestructures
structuresof ofindole-containing
indole-containingnatural natural
products, was synthesized
products, wassynthesized by the by the
theOhOh
Ohandand
andLeeLee
Leegroupgroup
group[41]. [41].
[41].WithWith 6-hydroxydihydro-β-carboline
With6-hydroxydihydro-β-carboline
6-hydroxydihydro-β-carboline(22) (22)
(22) as
as
as a standard
aastandard
standard andand
and through
through
through a simple
aasimple
simple andand
and fastfast
fast synthetic
synthetic
synthetic routeroute
route [58–61],
[58–61],
[58–61], the the best
thebest
best inhibitor
inhibitor
inhibitor candidate
candidate
candidate 24 24
24with
with
with
an
an IC an
IC 50 IC
50 50 value
value
value of
of 25ofµM
25 25 µM
µM was
was was observed.
observed.
observed. Briefly,
Briefly,
Briefly, condensationbetween
condensation
condensation between5-methoxy
between 5-methoxy tryptamine
5-methoxy tryptamine and and
benzaldehyde
benzaldehydefollowed
benzaldehyde followedbyby
followed ring
by ringclosure
ring waswas
closure
closure veryvery
was smoothly
very performed
smoothly
smoothly according
performed
performed to the Pictet–Spengler
according
according to
to the
the Pictet–
Pictet–
reaction,
Spengleraffording
Spengler reaction, the tetrahydro-β-carboline
reaction,affording
affording the 23. Further,
thetetrahydro-β-carboline
tetrahydro-β-carboline 23.oxidation
23. Further, with the with
Further,oxidation
oxidation aid
withofthe
theaid
the DDQ
aid of reagent
ofthe
the DDQ
DDQ
resulted
reagent in
reagentresulted the generation
resulted in in the of
thegeneration dihydro-β-carboline
generation of of dihydro-β-carboline 24
dihydro-β-carboline 24 with good
24 with yield.
with good As
good yield.
yield. As depicted
As depicted in
depictedin Scheme
in Scheme
Scheme 4,
replacement
4,
4, replacement
replacement of carboxylic
of acidacid
of carboxylic
carboxylic withwith
acid a phenyl
with ring induced
aa phenyl
phenyl ring a marked
ring induced
induced increase
aa marked
marked in the inhibitory
increase
increase in
in the activity
the inhibitory
inhibitory
of sortase
activity
activity ofA.
of sortase
sortase A.A.

Scheme
Scheme4.4.
4. Synthetic methods
Synthetic methods for
methodsfor tetrahydro-β-carboline
fortetrahydro-β-carboline and
tetrahydro-β-carboline and dihydro-β-carboline
anddihydro-β-carboline (IC
dihydro-β-carboline (IC 50 values
(IC50
50 values are
are
givenin
given inparentheses).
parentheses).

2.5.
2.5. Preparation
2.5. Preparation of
Preparation of Benzoisothiazolinones
of Benzoisothiazolinones
Benzoisothiazolinones
From
Fromaaasmall
From smallmolecule
small moleculelibrary
molecule libraryscreening
library screeningthat
screening thatincluded
that includedover
included over 50,000
over 50,000 drug-like
50,000 drug-like compounds,
compounds, aaa new
drug-like compounds, new
new
class
class of
classof sortase
ofsortase
sortaseA A inhibitors,
Ainhibitors,
inhibitors,withwith
with25 25
25asas a representative,
asaarepresentative,
representative,was was identified
wasidentified
identifiedbyby the
bythe Zhulenkovs
theZhulenkovs
Zhulenkovsgroupgroup [16].
group[16].
[16].
The
The skeleton
The skeleton was
skeleton was comprised
was comprised of
comprised of benzo[d]isothiazol-3-(2H)-one
of benzo[d]isothiazol-3-(2H)-one heterocycle,
benzo[d]isothiazol-3-(2H)-one heterocycle, which
heterocycle, which might
which might form
form aaa
might form
covalent
covalent bond
covalent bond with
bond with the
with the sortase
the sortaseA
sortase Aenzyme, the
A enzyme, adamantyl
enzyme, the moiety
the adamantyl
adamantyl moietythat offers
moiety that the hydrophobic
that offers
offers the property,
the hydrophobic
hydrophobic
and an adequate
property,
property, and anlinker.
and an adequate
adequate Owing
linker.to Owing
linker. the
Owingresults
to of results
to the
the the SAR
results ofanalysis,
of the SARthe
the SAR authors
analysis,
analysis, thesynthesized
the authors different
authors synthesized
synthesized
combinations
different of the two
differentcombinations
combinations of ring
ofthe
the twomoieties
two ring with 4–6
ringmoieties
moieties with
withcarboned
4–6
4–6carbonedspacers,
carboned which
spacers,
spacers, ultimately
which
which resulted
ultimately
ultimately resulted
resultedin
an
in IC
in an
an50IC value
IC5050 valueof
value of ~3
of ~3 µM
~3 µM (26).
µM (26). The
(26). The core
The core benzoisothiazolinone
core benzoisothiazolinone structure
benzoisothiazolinone structure was
structure was prepared
was prepared with
prepared with the
with thethe
intermediate,
intermediate, 2-carbamoylbenzenesulfenyl
2-carbamoylbenzenesulfenyl bromide,
bromide, via
via the
the treatment
treatment
intermediate, 2-carbamoylbenzenesulfenyl bromide, via the treatment with bromine to disulfide. with
with bromine
bromine to
to disulfide.
disulfide.
Additionally,
Additionally, the
Additionally, theattachment
the attachment of
attachment ofthe
of theadamantyl
the adamantylfunctionality
adamantyl functionality was
functionality was completed
was completed through
completed through evident
through evident EDC
evident EDC
EDC
coupling
coupling (Scheme
coupling (Scheme
(Scheme 5) 5) [62–64].
5) [62–64].
[62–64].

Scheme
Scheme 5.5.
5. Synthetic
Synthetic routes
routes for
for benzisothiazolinone-based
for benzisothiazolinone-based inhibitors
benzisothiazolinone-based inhibitors (IC
inhibitors 50 values
(IC50
50 values are
are given
given in
in
parentheses).
parentheses).

2.6.
2.6. Preparation
Preparation of
of Triazolothiadiazoles
Triazolothiadiazoles
After
After the
the acquisition
acquisition of
of the
the hit
hit compound
compound 27
27 with
with aa 3,6-disubstituted
3,6-disubstituted triazole
triazole structure,
structure, aa
synthetic
synthetic optimization
optimization was
was performed
performed byby Schneewind,
Schneewind, Luo,
Luo, and
and Yang
Yang group
group [43],
[43], with
with few
few
Antibiotics 2020, 9, 706 7 of 16

2.6. Preparation of Triazolothiadiazoles

After the acquisition of the hit compound 27 with a 3,6-disubstituted triazole structure, a synthetic
optimization was9, performed
Antibiotics 2020, by Schneewind, Luo, and Yang group [43], with few modifications,
x FOR PEER REVIEW 7 of 16 to
discover the improved activity. The ethyl isonicotinate nucleophilic substitution with hydrazine was
Antibiotics 2020, 9, x FOR PEER REVIEW 7 of 16
modifications,
observed to occur as to planned.
discover the improvedisonicotinohydrazide
Thereafter, activity. The ethyl isonicotinate nucleophilic
was condensed substitution
with carbon disulfide
in thewith hydrazine
presence of was observed
potassium to occur asproducing
hydroxide, planned. Thereafter,
potassium isonicotinohydrazide
dithiocarbazate, was condensed
modifications, to discover the improved activity. The ethyl isonicotinate nucleophilic substitution ring
and a triazole
with
was formed carbon
upon disulfide in the
treatment presence
with of potassium hydroxide, producing potassium dithiocarbazate,
with hydrazine was observed tohydrazine hydrate.
occur as planned. Ultimately,
Thereafter, the thiadiazole
isonicotinohydrazide structure in 28 was
was condensed
and a triazole ring was formed upon treatment with hydrazine hydrate. Ultimately, the thiadiazole
achieved
with with
carbon 4-amino-5-aryl-3-mercapto-1,2,4-triazole and theproducing
disulfide in the presence of potassium hydroxide, corresponding benzoyl
potassium chloride in the
dithiocarbazate,
structure in 28 was achieved with 4-amino-5-aryl-3-mercapto-1,2,4-triazole and the corresponding
and aoftriazole
presence phosphorusring was formed upon
oxychloride treatment
(Scheme with hydrazine hydrate. Ultimately, the thiadiazole
6) [65].
benzoyl chloride in the presence of phosphorus oxychloride (Scheme 6) [65].
structure in 28 was achieved with 4-amino-5-aryl-3-mercapto-1,2,4-triazole and the corresponding
benzoyl chloride in the presence of phosphorus oxychloride (Scheme 6) [65].

Scheme 6. Optimized
Scheme 6. Optimizedsynthetic
syntheticscheme
schemeof
ofthe
the 3,
3, 6-disubstituted triazolothiadiazole
6-disubstituted triazolothiadiazole scaffold
scaffold (IC50(IC50
values are given
values in parentheses).
are given in parentheses).
Scheme 6. Optimized synthetic scheme of the 3, 6-disubstituted triazolothiadiazole scaffold (IC50
values are given in parentheses).
2.7. 2.7. Preparation of 2-(2-Phenylhydrazinylidene)Alkanoates
Preparation of 2-(2-Phenylhydrazinylidene)Alkanoates
The The Maggio
Maggio
2.7. Preparation of and
and Raffaresearch
Raffa research group
grouphas hasstudied
2-(2-Phenylhydrazinylidene)Alkanoates studied the the
1,3-dicarbonyl core skeleton
1,3-dicarbonyl with an aryl
core skeleton with an
hydrazinylidene
aryl hydrazinylidene at the α-position
at Raffa
the α-positionof β-keto ester
of β-keto as a novel
esterthe class of sortase
as 1,3-dicarbonyl A
a novel class coreinhibitors
of sortase [44]. As shown
The Maggio and research group has studied skeletonAwithinhibitors
an aryl [44].
in Scheme
As shown 7, the diazonium salt prepared from the substituted aniline was reacted with β-keto ester
hydrazinylidene at the α-position of β-keto ester as a novel class of sortase A inhibitors [44]. As shown with
in Scheme 7, the diazonium salt prepared from the substituted aniline was reacted
29 or 30 [66,67]. Thereafter, basic hydrolysis was carried out, affording the acids 33 and 34. During
in ester
β-keto Scheme 297,orthe [66,67]. Thereafter,
30diazonium salt prepared basic
fromhydrolysis was carried
the substituted out,reacted
aniline was affording
withthe acids
β-keto 33 and
ester
the synthesis of a series of analogs of the screened molecule, 35, with an IC50 value of 192 µM, an issue
34. During
29 or 30the synthesis
[66,67]. of a basic
Thereafter, serieshydrolysis
of analogs wasofcarried
the screened molecule,
out, affording 35, with
the acids 33 andan34.
ICDuring
50 value of
ensued regarding the assignment of the geometry of the C=N double bond for 31 and 32 [68–71].
the synthesis
192 µM, an issue of a seriesregarding
ensued of analogs of
thethe screened molecule,
assignment 35, with an of
of information
the geometry IC50 value of 192 µM, an issuefor 31
Through a comparison of the 1H NMR spectroscopic for thethe C=N
reference double bond
molecules, the
ensued
and 32 regarding
[68–71]. Through the assignment of the geometry
1 of the C=N double bond for 31 and 32 [68–71].
obtained materials, 31aand
comparison
32,1 were of the H NMR
confirmed to be E spectroscopic information
and Z, respectively [72–74].forFor
thethe
reference
E-
Through
molecules, thea comparison
obtained of the H 31
materials, NMR andspectroscopic
32,ofwere information tofor
be the reference molecules, the
configurated molecule, 31, the hydrogen NH confirmed
and the oxygen E and
of Z, respectively
the acetyl moiety form [72–74].
obtained materials, 31 and 32, were confirmed to be E and Z, respectively [72–74]. For the E-
For the E-configurated
intramolecular molecule,
hydrogen bonding, the hydrogen
31, depicting of NHdownfield
an apparent and the oxygen
shift of NHof the
andacetyl
methylmoiety
signalsform
configurated molecule, 31, the hydrogen of NH and the oxygen of the acetyl moiety form
compared tohydrogen
intramolecular the Z-geometry
bonding,[75,76].
depicting an apparent downfield shift of NH and methyl signals
intramolecular hydrogen bonding, depicting an apparent downfield shift of NH and methyl signals
compared to the Z-geometry [75,76].
compared to the Z-geometry [75,76].

Scheme 7. Synthetic methods for 2-(2-phenylhydrazinylidene)alkanoates (IC50 values of 33 and 34 are

given in parentheses).
Scheme
Scheme 7. Synthetic
7. Synthetic methods
methods forfor 2-(2-phenylhydrazinylidene)alkanoates (IC
2-(2-phenylhydrazinylidene)alkanoates 50 values
(IC of 33ofand
50 values 33 34
andare34 are
givengiven in parentheses).
in parentheses).
Antibiotics 2020,9,9,706
Antibiotics2020, x FOR PEER REVIEW 88 of
of 16
16

2.8. Preparation of Benzo[d]Oxazoles and Benzofuran

2.8. Preparation of Benzo[d]Oxazoles and Benzofuran
During the search for a new frame of sortase A inhibitors, the Jiang and Fu research group
During the search for a new frame of sortase A inhibitors, the Jiang and Fu research group
developed L-shaped kinked molecules that mimic the sortase A substrate, LPXTG, sequentially
developed L-shaped kinked molecules that mimic the sortase A substrate, LPXTG, sequentially
designing 2-phenyl-benzo[d]oxazole-7-carboxamide and 2-phenyl-benzofuran-3-carboxamide
designing 2-phenyl-benzo[d]oxazole-7-carboxamide and 2-phenyl-benzofuran-3-carboxamide [45,46].
[45,46]. Sortase A is known to recognize the LPXTG sorting signal (X means any amino acids) which
Sortase A is known to recognize the LPXTG sorting signal (X means any amino acids) which is arranged
is arranged in an L-shape mode by the L (Leu) and P (Pro) residues. The authors introduced an
in an L-shape mode by the L (Leu) and P (Pro) residues. The authors introduced an isobutyl amide
isobutyl amide functionality at the C-7 position of benzo[d]oxazoles and the C-3 position of
functionality at the C-7 position of benzo[d]oxazoles and the C-3 position of benzofuran. Further,
benzofuran. Further, the core bicyclic rings were regarded as the displacement of pyrrolidine, and a
the core bicyclic rings were regarded as the displacement of pyrrolidine, and a right-side assembly of
right-side assembly of carbons and oxygens was expected to produce some spatial effect, mimicking
carbons and oxygens
the X amino acids in was expected to
the substrate. produce
The some spatial
constructive effect,
methods mimicking
of the the described
objects are X amino acids in the
in Scheme
substrate.
8 [77]. The constructive methods of the objects are described in Scheme 8 [77].

O
H2N O R O
H
N N
N N CO2H
H H
O
OH
LPXTG

OH

HN O OH HN O
OH
7 3
O O OH O
O O OH
2 2
N O

36 (19.8 M) 37 (30.8 M)

NaOH, DMF, 45 oC OH NaOH, DMF

6 h, 22 - 76 % rt, 3 h, 23 %
HN O OH HN O

Cl OH
O Br + HO OH + HO
O O OH
N O

i) SOCl2, reflux, 2 h 1. HATU, Et3N, rt, 90 %

ii) Amine, Et3N, rt, 2 h, 69 - 76 % 2. SOCl2, CH2Cl2, reflux, 78 %

HO O NH2 +
NH2 + CO2H
O Br OH

N O
1. i) Acyl chloride, Et3N, CH2Cl2, rt, 1 h OH
ii) TsOH, xylene, reflux, 20 h, 84 % 1. NaOAc, DMF, 90 oC
2. LiOH, THF, H2O, rt, 4 h, 87 % 2. NaOH, MeOH, reflux, 86 % I2, K2CO3, THF, rt, 94 %
MeO O CO2CH3
OH Br
Cl Br
+ O O
NH2 O
NBS, AIBN, CCl4 DMF, POCl3,ClCH2CH2Cl
1. H2SO4, MeOH, reflux, 18 h, 98 % reflux, 75 % reflux, 78 %
2. HNO3, H2SO4, CH2Cl2, 0 oC, 20 min, 35 %
CO2CH3 CHO
3. Pd/C, H2, MeOH, rt, 20 h, 90 %

CO2H
OH O O
1. NH2SO3H, NaClO2, MeOH/H 2O, rt, 83 %
2. H2SO4, MeOH, reflux, 90 %

Scheme8.8. Construction
Scheme Construction of
ofthe
thebenzo[d]oxazole
benzo[d]oxazoleand
andbenzofuran
benzofuranskeleton
skeletonmolecules
molecules(IC
(IC5050 values
values are
are
given in parentheses).
given in parentheses).

In
Inorder
ordertoto
prepare the the
prepare molecule 36, the
molecule 36,authors used salicylic
the authors acid as a acid
used salicylic starting
as material.
a startingFollowing
material.
the protection
Following the of carboxylic
protection ofacid to methyl
carboxylic acidester of salicylic
to methyl esteracid, a nitration
of salicylic acid,reaction wasreaction
a nitration carried was
out
to introduce a nitryl group to an aryl ring [78]. Transformation of the nitryl functionality to
carried out to introduce a nitryl group to an aryl ring [78]. Transformation of the nitryl functionality amine
under
to aminea hydrogen atmosphere
under a hydrogen in the presence
atmosphere in theofpresence
Pd/C enabled
of Pd/Ctheenabled
2-hydroxyl aniline to react
the 2-hydroxyl with
aniline to
react with 4-methyl benzoyl bromide via intramolecular cyclization, resulting in benzoxazole. The
Antibiotics 2020,
Antibiotics 2020, 9,
9, 706
x FOR PEER REVIEW 99 of 16
of 16

deprotected free carboxylic acid derived via hydrolysis was then converted to acyl chloride.
4-methyl
Thereafter,benzoyl bromidesubstitution
nucleophilic via intramolecular cyclization,
with isobutyl resulting
amine in benzoxazole.
was performed, The deprotected
followed by a final
free carboxylic acid derived via hydrolysis was then converted
connection of the ester component via an undoubtful SN2 reaction [79]. to acyl chloride. Thereafter, nucleophilic
substitution with isobutyl
To synthesize amine was performed,37,
2,3-substituted-benzofuran followed by a final connection of prepared
(E)-2-(4-methylstyryl)phenol the ester component
via Wittig
via an undoubtful
olefination SN 2 reaction
was treated [79]. under basic conditions. Ring closure by iodination was then
with iodine
To synthesize
followed 2,3-substituted-benzofuran
by a Vilsmeier 37, (E)-2-(4-methylstyryl)phenol
reaction, resulting in a formyl moiety at the C-3 position.preparedFollowingvia Wittig
stepwise
olefination was treatedesterification,
reactions, oxidation, with iodine under basic conditions.
bromination, Ringamide
hydrolysis, closurecoupling,
by iodination
and was then followed
substitution were
by a Vilsmeier reaction, resulting in a formyl moiety at the C-3 position. Following stepwise reactions,
performed.
oxidation, esterification, bromination, hydrolysis, amide coupling, and substitution were performed.
2.9. Preparation of Phenylthiazoles
2.9. Preparation of Phenylthiazoles
A study on the structural investigation of sortase A inhibitors through Bemis–Murcko scaffold
A study
analysis on the
revealed structural
that investigation
the scaffolds preferred of
forsortase
sortaseAAinhibitors
inhibitorsthrough
contain at Bemis–Murcko scaffold
least two ring systems
analysis revealedorthat
bonded directly the scaffolds
merged preferred
in a condensed for (Scheme
cycle sortase A9).
inhibitors contain
The Araniciu and atPalage
least two ringdesigned
group systems
bonded directly or merged in a condensed cycle (Scheme 9). The Araniciu and
a new framework of five aromatic rings linked directly and inspired by Bemis–Murcko clustering, Palage group designed
aultimately
new framework of fivea aromatic
synthesizing rings linked directly
series of 2-phenyl-thiazole and inspired
derivatives byBriefly,
38 [47]. Bemis–Murcko clustering,
thiobenzamide and
ultimately synthesizing a series of 2-phenyl-thiazole derivatives 38 [47].
4-(2-bromoacetyl)benzonitrile were intramolecularly cyclized in one pot, affording the key Briefly, thiobenzamide and
4-(2-bromoacetyl)benzonitrile were intramolecularly cyclized
intermediate, 4-(2-phenylthiazol-4-yl)benzonitrile. Treatmentin one pot,hydrogen
with affording the key intermediate,
sulfide gas under
4-(2-phenylthiazol-4-yl)benzonitrile.
triethylamine condition transformed nitrile Treatment
into with hydrogen
thioamide via asulfide
furthergas underring
thiazole triethylamine
formation
condition transformed nitrile into thioamide via a further thiazole ring formation
reaction. Structures of the molecules were confirmed based on the distinct hydrogen signals on the reaction. Structures
of
1Hthe
NMRmolecules
spectra were confirmed
and sharp signals based on the
between distinct
3111 hydrogen
and 3103 cm−1 onsignals
the IR the 1 H[80].
onspectra NMR spectra and
Although the
sharp signals −1
authors have between 3111 and the
not demonstrated 3103ICcm on the IR spectra [80]. Although the authors have not
50 values of these sorts of new inhibitors, the antimicrobial
demonstrated the ICshowed
activity evaluation 50 values of these
that sorts of new
the potential inhibitors,
scaffold the antimicrobial
has reduced activity
activity against evaluation
bacterial cell
showed
viability.that the potential scaffold has reduced activity against bacterial cell viability.

Scheme
Scheme 9. Synthesis of
9. Synthesis of 2-phenylthiazole
2-phenylthiazole derivatives
derivatives within
within a
a pentacyclic
pentacyclic system.
system.

2.10. Preparation of Thiadiazole

2.10. Preparation of Thiadiazole
The Olsson group identified a new class of inhibitors against S. aureus sortase A [48]. Through
The Olsson group identified a new class of inhibitors against S. aureus sortase A [48]. Through
small-molecule libraries screening and PAINS (pan-assay interference compounds) study, top hit
small-molecule libraries screening and PAINS (pan-assay interference compounds) study, top hit
structures emerged. The hit compound 40 was selected as the starting point for structural hit-to-lead
structures emerged. The hit compound 40 was selected as the starting point for structural hit-to-lead
optimization. A series of modifications of 40 commenced with the removal of the oxadiazole unit
optimization. A series of modifications of 40 commenced with the removal of the oxadiazole unit
which has been shown to cause cytotoxicity. Synthetic modifications included elongation of the sulfide
which has been shown to cause cytotoxicity. Synthetic modifications included elongation of the
substituent and addition of various substituted benzyl derivatives to validate some electronic effects.
sulfide substituent and addition of various substituted benzyl derivatives to validate some electronic
After all, N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide 41 (IC50 = 3.8 µM) was identified
effects. After all, N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide 41 (IC50 = 3.8 µM) was
as a potent inhibitor of sortase A. As illustrated in Scheme 10, 5-amino-1,3,4-thiadiazole-2-thiol was
identified as a potent inhibitor of sortase A. As illustrated in Scheme 10, 5-amino-1,3,4-thiadiazole-2-
reacted with the appropriate bromide under basic conditions, affording the desired products in good
thiol was reacted with the appropriate bromide under basic conditions, affording the desired
yields. The primary amine was acylated, which was achieved upon treatment with nicotinoyl chloride
products in good yields. The primary amine was acylated, which was achieved upon treatment with
39 that was induced from nicotinic acid in situ [81–83].
nicotinoyl chloride 39 that was induced from nicotinic acid in situ [81–83].
Antibiotics 2020, 9, 706 10 of 16
Antibiotics 2020, 9, x FOR PEER REVIEW 10 of 16

Scheme 10.
Scheme 10. Synthetic
Synthetic process
process for
for 2,
2, 5-substituted
5-substituted thiadiazole
thiadiazole (IC
(IC50
50 values are given in parentheses).
50

2.11.
2.11. Preparation
Preparation of of Thiadiazolidinedione
Thiadiazolidinedione
In
In an
an attempt
attempt to to discover
discover innovative
innovative scaffolds
scaffolds for
for sortase
sortase AA inhibitors,
inhibitors, the
the S. Yang and
S. Yang and C.-G
C.-G Yang
Yang
research
research groups employed FRET-based HTS on a library consisting of over 2400 clinical drugs and
groups employed FRET-based HTS on a library consisting of over 2400 clinical drugs and
candidates
candidates [49].[49]. Tideglusib
Tideglusib (TD),(TD), aa drug
drug candidate
candidate for for myotonic
myotonic dystrophy
dystrophy [84],
[84], was
was identified
identified as
as aa
hit
hit structure
structure withwith anan ICIC50 value of 5.9 µM. The authors prepared 40 sorts of TD analogs which were
50 value of 5.9 µM. The authors prepared 40 sorts of TD analogs which were
50
constructed
constructed with a thiadiazolidinedione skeleton
with a thiadiazolidinedione skeleton and
and analyzed
analyzed their
their structure–activity
structure–activity relationships
relationships
for
for sortase A inhibition. Several molecules were explored, changing two
sortase A inhibition. Several molecules were explored, changing two N-substitutions
N-substitutions withwith aa
variety
variety of of functionalities
functionalities [85], [85],and
andititwas
wasobserved
observed that
that thethe most
most promising
promising compound,
compound, 42, bears
42, bears 3,5-
3,5-dimethylisoxazole
dimethylisoxazole substituent, retains inhibitory activities compared to that of TD, shows better
substituent, retains inhibitory activities compared to that of TD, shows better
solubility,
solubility, andand minimally
minimally inhibits
inhibits the
the growth
growth of of the
the S.S. aureus.
aureus. When
When it it comes
comes to to the
the preparation
preparation ofof
TD analogs, a concise synthetic route illustrated in Scheme 11 was adopted.
TD analogs, a concise synthetic route illustrated in Scheme 11 was adopted. The reaction system with The reaction system
with an isothiocyanate,
an isothiocyanate, an isocyanate,
an isocyanate, and same andequivalent
same equivalent molar amount
molar amount of sulfuryl
of sulfuryl chloride chloride
(SO22Cl22)
(SO
even2 Cl 2 ) even generated
generated the unstable
the unstable S-chloroisothiocarbamoyl
S-chloroisothiocarbamoyl chloride,chloride,
and via and via the assembly
the assembly with
with chloro-
chloro-intermediates, the desired scaffold was established with
intermediates, the desired scaffold was established with good conversion efficiency.good conversion efficiency.

Scheme 11. Construction

Scheme 11. Construction of
of the
the thiadiazolidinedione
thiadiazolidinedione scaffold
scaffold through
through chloro-intermediates
chloro-intermediates (IC
(IC5050
50

values
values are
are given
given in
in parentheses).
parentheses).

3. Conclusions
3. Conclusions
Novel approaches for the development of antibiotics includes targeting virulence via toxin
Novel approaches for the development of antibiotics includes targeting virulence via toxin
production and virulence factor secretion, impeding bacterial adhesion to host cells and biofilm
production and virulence factor secretion, impeding bacterial adhesion to host cells and biofilm
formation, interrupting or inhibiting bacterial communication, and downregulating virulence. As part
formation, interrupting or inhibiting bacterial communication, and downregulating virulence. As
of the struggle against drug resistance, the development of sortase A inhibitor has emerged as an
part of the struggle against drug resistance, the development of sortase A inhibitor has emerged as
alternative to conventional antibiotics, with a mechanism that involves blocking the pathogenic
an alternative to conventional antibiotics, with a mechanism that involves blocking the pathogenic
processes of bacterial infection. The ideal inhibitors for sortase A should have minimal impacts
processes of bacterial infection. The ideal inhibitors for sortase A should have minimal impacts on
on bacterial viability, which can be validated by their minimal inhibitory concentration (MIC) test.
bacterial viability, which can be validated by their minimal inhibitory concentration (MIC) test. A
A number of influential advances in the development of sortase A inhibitors were comprehensively
number of influential advances in the development of sortase A inhibitors were comprehensively
studied and reviewed by a variety research groups, but the interest and focus on their structural and
Antibiotics 2020, 9, 706 11 of 16

studied and reviewed by a variety research groups, but the interest and focus on their structural and
preparative aspects are insufficient so far. It is of great interest to the medicinal chemists and associates
how the core scaffolds are established and the tangent molecular groups are chemically installed.
We the authors focus on the synthetic area and highlight significant small organic molecules with high
inhibitory potencies against S. aureus, eliciting the greatest IC50 values up to 1.5 µM.
The organic molecular group, aryl(β-amino)ethyl ketones, mainly abbreviated as AAEK and one
of the promising S. aureus sortase A inhibitors, is known to their inhibitory mechanism [86]. In virtue
of the kinetic analysis and mass spectrometry, it is suggested that the initial deprotonation of α-position
of AAEK by an appropriate base of sortase A facilitates the generation of an enolate which is stabilized
by the guanidinium residue in the enzyme and then β-elimination, which means that the cleavage of
the C-N bond is undergone, releasing the separate moiety dimethylamine. Therefore, AAEK is now
considered as an α,β-unsaturated ketone at which 1,4-Michael type addition can be easily performed
by the nucleophilic attack of the thiol group of cysteine in sortase A [87–90]. Through the irreversible
covalent bond formation between sortase A and its inhibitor, the enzymatic action was attenuated. In a
similar context, the α,β-unsaturated carbonyl system of sortase A inhibitors is regarded as a Michael
donor. Moreover, there are some sulfur containing sortase A inhibitors which inhibit sortase A by
forming a disulfide bond with the cysteine residue, and it seems that most of the potent chemicals for
sortase A bear sulfur (S) atoms in the molecules.
In summary, high-throughput screening and in silico virtual screening techniques from the
small-molecule libraries have been widely used to discover novel types of scaffold sortase A inhibitors.
As discussed above, we can determine the tendency that many compounds have α,β-unsaturated
carbonyl core or sulfur-associated functionalities. In the text, 12 sorts of skeletons, diarylacrylonitriles,
pyridazinones, aryl 3-acryloamide, dihydro-β-carboline, benzisothiazolinones, triazolothiadiazoles,
2-(2-phenylhydrazinylidene)alkanoates, 2-phenyl-benzo[d]oxazole-7-carboxamide, 2-phenyl-
benzofuran-7-carboxamide, 2-phenylthiazoles, 2, 5-disubstitued thiadiazole, and thiadiazolidinedione,
are demonstrated from a synthetic point of view. These cumulative achievements regarding sortase
A inhibitors have provided the molecular basis for designing and synthesizing sortase A inhibitors.
Their systematic development is thus expected to result in a novel class of antibiotics that would serve
as antivirulence agents in the near future.

Author Contributions: M.W.H.: conceptualization, investigation and writing original draft. S.W.Y.: investigation
and writing original draft. S.-M.P.: conceptualization, writing review and editing, and supervision. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported by the Korea Basic Science Institute (National Research Facilities and
Equipment Center) through a grant funded by the Ministry of Education (grant No.: 2020R1A6C101A188).
This research was funded by Basic Science Research Program to Research Institute for Basic Sciences (RIBS) of
Jeju National University through the National Research Foundation of Korea (NRF) funded by the Ministry of
Education. (2019R1A6A1A10072987).
Conflicts of Interest: The authors declare no conflict of interest.

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