Discovery of substituted (2‐aminooxazol‐4‐yl)isoxazole‐3‐ carboxylic acids as inhibitors of bacterial serine acetyltransferase in the quest for novel potential antibacterial adjuvants

Article abstract of DOI:10.3390/ph14020174

Many bacteria and actinomycetales use L‐cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long‐lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of

Full text of DOI:10.3390/ph14020174

pharmaceuticals
Article
Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-
carboxylic Acids as Inhibitors of Bacterial Serine
Acetyltransferase in the Quest for Novel Potential
Antibacterial Adjuvants
1
2
2,3
1,4
, Päivi Tammela 5
Joana Magalhães , Nina Franko , Samanta Raboni , Giannamaria Annunziato
,
,
1
3,6,7
2
8
8
Agostino Bruno , Stefano Bettati
, Stefano Armao , Costanza Spadini , Clotilde Silvia Cabassi
Andrea Mozzarelli 2
,3,7
, Marco Pieroni 1,4,* , Barbara Campanini 2 and Gabriele Costantino
1,4
1
P4T Group, Department of Food and Drug, University of Parma, 43124 Parma, Italy;
joanarpmagalhaes@gmail.com (J.M.); giannamaria.annunziato@unipr.it (G.A.); agostino84@gmail.com (A.B.);
gabriele.costantino@unipr.it (G.C.)
Laboratory of Biochemistry and Molecular Biology, Department of Food and Drug, University of Parma,
2
43124 Parma, Italy; nina.franko@studenti.unipr.it (N.F.); samanta.raboni@unipr.it (S.R.);
Stefano.armao@unipr.it (S.A.); andrea.mozzarelli@unipr.it (A.M.); barbara.campanini@unipr.it (B.C.)
Institute of Biophysics, CNR, 56124 Pisa, Italy; stefano.bettati@unipr.it
3
4
5
Centro Interdipartimentale Misure (CIM) ‘G. Casnati’, University of Parma, 43124 Parma, Italy
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki,
P.O. Box 56 (Viikinkaari 5 E), FI-00014 Helsinki, Finland; paivi.tammela@helsinki.fi
Department of Medicine and Surgery, University of Parma, Via Volturno, 39, 43125 Parma, Italy
National Institute of Biostructures and Biosystems, 00136 Rome, Italy
Operative Unit of Animals Infectious Diseases, Department of Veterinary Science, University of Parma,
Via del Taglio 10, 43126 Parma, Italy; Costanza.spadini@unipr.it (C.S.); clotildesilvia.cabassi@unipr.it (C.S.C.)
Correspondence: marco.pieroni@unipr.it; Tel.: +39-0521-905054
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Magalhães, J.; Franko, N.;
Raboni, S.; Annunziato, G.; Tammela,
P.; Bruno, A.; Bettati, S.; Armao, S.;
Spadini, C.; Cabassi, C.S.; et al.
Discovery of Substituted
6
7
8
*
(
2-Aminooxazol-4-yl)Isoxazole-3-
carboxylic Acids as Inhibitors of
Bacterial Serine Acetyltransferase in
the Quest for Novel Potential
Antibacterial Adjuvants.
Abstract: Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance
to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset
of antimicrobial resistance that currently represents one of the most menacing threats to public health
worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals;
therefore, its exploitation as a drug target is particularly promising. In this article, we report a series
of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the
rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the
virtual screening of an in-house library of compounds that led to the selection of seven structurally
Pharmaceuticals 2021, 14, 174.
https://doi.org/10.3390/ph14020174
Academic Editor: Mary J. Meegan
Received: 26 January 2021
Accepted: 19 February 2021
Published: 23 February 2021
unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either
from the original library or from medicinal chemistry efforts, were tested to determine a preliminary
structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives,
that was indeed ameliorated by several folds compared to hit compound
5 Despite these progresses,
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
at this stage, the most promising compound failed to interfere with bacterial growth when tested on
a Gram-negative model organism, anticipating the need for further research efforts.
Keywords: adjuvant therapies; antimicrobial resistance; cysteine biosynthesis; non-essential targets;
serine acetyltransferase
Copyright: © 2021 by the authors.
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/
1
. Introduction
Cysteine, which in mammals cannot be obtained via direct synthesis, is the main
building block of many biomolecules and cofactors, such as S-adenosyl methionine, CoA,
biotin, lipoic acid, and thiamin pyrophosphate, and it is the active portion of molecules with
detoxifying properties such as glutathione and mycothiol. Besides being essential to all or-
ganisms, cysteine is particularly important for some facultative intracellular pathogens that
4
.0/).
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https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 174
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must respond to nutrient starvation and/or oxidative stress occurring inside macrophages.
In these challenging conditions, any interference with the adaptation strategies may lead
to a reduction of their infectivity and to an increased susceptibility to antibacterial agents.
Moreover, whereas cysteine biosynthetic enzymes are dispensable during growth in vitro
or acute infection, they become indispensable during persistency [
has been demonstrated that reduction of cysteine biosynthesis or its suppression negatively
affects bacterial fitness and virulence [ ]. For instance, mutants bearing a deletion of cys-
teine biosynthetic enzymes were found to possess reduced virulence as in the case of cysH
of M. tuberculosis [ ] and cysI or cysK of B. melitensis [10], and/or an increased sensitivity
1,2]. In the last years, it
2–8
9
to oxidative stress, like the mutant of cysI of B. melitensis [10]. One of the most intriguing
aspects of cysteine biosynthesis is its role in the development of antibiotic resistance, as
reported in the case of S. typhimurium, where an impaired oxidative stress response due to
the genetic inhibition of cysteine biosynthesis caused a decrease in antibiotic resistance in
both vegetative and swarm cell populations [11].
Altogether, these findings suggest that inhibition of cysteine biosynthesis might have
the two-fold advantage of noticeably improving the action of many marketed antibacterial
agents, counteracting their obsolescence, and preventing the onset of antimicrobial resis-
tance. Since antimicrobial resistance (AMR) is regarded by many experts as the next-to-be
healthcare scourge, the significance of the cysteine biosynthesis inhibition gains further
therapeutic value. Experts estimate a loss of 10 million human lives every year due to an-
timicrobial resistance by 2050, unless innovative research actions are taken [12–14]. Along
with the exploitation of novel druggable targets via proper drug discovery campaigns, a
convenient strategy to counteract AMR would be to pursue unconventional approaches
such as the use of adjuvant agents endowed with weak or absent antimicrobial activity
that engage non-essential targets related to bacterial virulence and/or persistence after
colonization of the host [15–18]. These targets are represented by those proteins which
are dispensable during the normal bacterial life cycle, but become essential during host
infection, as they mitigate the toxic action of antibiotics [18]. Among the many advantages
in pursuing this strategy, adjuvants can suppress intrinsic resistance and therefore expand
the activity spectrum of current antibiotics [19]. Similarly, for those antibiotics showing
toxicity issues, adjuvants can allow for administration at lower doses, therefore diminishing
the side effects [20]. In addition, such targets, being not essential for survival per se, are
less likely to generate mutations and therefore induce resistance [21].
In the final steps of cysteine biosynthesis, the transfer of the acetyl group from acetyl-
CoA to serine, to form O-acetylserine (OAS), is catalyzed by serine acetyltransferase (SAT).
In the following reaction, O-acetylserinesulfhydrylase (OASS) catalyzes the
of OAS with sulfide to give cysteine and acetate. Numerous efforts have been made by
our research group [22 25] and others [26 29] toward the discovery of potent and selective
β-substitution
–
–
OASS inhibitors, to be used either as antibacterials, especially towards M. tuberculosis, or
adjuvants. This has led to the discovery and characterization of the most potent inhibitor
of StOASS known so far; namely, UPAR-415 (Figure 1) [30]. In spite of its potent activity
in the biochemical assay, UPAR-415 was not able to replicate a similar activity in bacterial
cells, even though we have recently gained evidence that UPAR-415 acts as a colistin
adjuvant for pathogen infections (manuscript submitted). According to a preliminary
analysis, the lack of efficacy in bacterial cells was due to permeability issues that prevented
the molecule from reaching the intracellular targets. Indeed, poor permeability is one of the
most common attrition elements in the antibacterial discovery process. Due to these results,
it seemed reasonable to shift our focus also to the previous step of cysteine biosynthesis,
catalyzed by SAT. Inhibition of SAT has been seldom investigated and the molecular tools
reported so far exhibited only limited inhibitory properties towards the enzyme [26,31–33].
SAT catalyzes the transfer of the acetyl group of acetyl-CoA to the hydroxyl group of
L-serine to generate OAS, the activated form of L-Ser. Compared to inhibition of OASS, the
inhibition of SAT is expected not only to exert down-stream cysteine reduction, but also
to inhibit accumulation of OAS that induces the biosynthetic operon upon conversion to

Pharmaceuticals 2021, 14, 174
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N-acetylserine (NAS) [34]. From the structural point of view, SAT is a dimer of trimers,
with an
ending with a flexible, disordered C-terminal sequence that binds OASS-A at the active
site and competitively inhibits it [35 36]. Our first attempt to deliver an SAT inhibitor
α-helical N-terminal domain and a carboxyl-terminal left-handed α-helix domain
,
relied on the virtual screening of a focused commercial library of chemical compounds [37].
This strategy was inspired by previous works where similar approaches were carried out
obtaining encouraging results [27
ChemDiv focused libraries containing 91,243 compounds was performed to identify six
compounds displaying an IC50 < 100 M via an indirect assay using Ellman’s reagent [37].
,33]. In our first attempt, the virtual screening of three
µ
In addition, one of the compounds was found to have measurable growth inhibitory effects
against E. coli. We followed a similar approach to virtually assay an in-house library
of chemical compounds that were initially designed to inhibit molecular targets other
than SAT. Over the recent years, drug or chemical libraries repurposing is emerging as
an extremely fruitful strategy to inspire drug discovery, and both big pharmaceutical
companies and academic institutions screen in-house chemical libraries of compounds for
purposes other than those for which they were initially conceived. This approach has the
considerable advantage of potentially turning back to life molecules normally destined
to oblivion, for which a biological/chemical characterization is often already available.
Despite the smaller size of the library compared to the commercial one, the vs. was efficient
in identifying a small set of promising hit compounds (Figure 2), for which the structure–
activity relationships were investigated by means of both analogues already present in the
library and synthesis of novel derivatives.
Figure 1. Schematic representation of the reductive sulfate assimilation pathway in Salmonella. Inorganic sulfate is
transported inside the cell where it is reduced to bisulfide by a multi-step process. L-serine is activated by O-acetylation
by SAT to give the product OAS, which spontaneously tautomerizes to NAS, the inducer of the cysteine regulon. A
-
β
-substitution reaction between OAS and HS , catalyzed by OASS, leads to L-cysteine formation. This amino acid and
the pathways of its biosynthesis influence many processes relevant to infection, like resistance to oxidative stress, biofilm
formation and antibiotic resistance. UPAR415 is a competitive inhibitor of OASS.

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Figure 2. Compounds from the virtual screening.
2
2
. Results
.1. Hit Compounds from Virtual Screening
Since the primary structure of SAT is highly conserved among bacteria, [38] the virtual
screening of our in-house library was performed using the available crystal structures
of EcSAT (pdb code: 1T3D) and HiSAT (pdb code: 1SSM), setting the experimental pa-
rameters as previously reported by us [37]. After this preliminary analysis, a total of
7
compounds were identified and selected to be evaluated on the recombinant enzyme
from S. typhimurium. The enzyme used in these assays has a K = 1.07 0.15 mM, a
37]. The assay conditions for the
m
,
Ser
±
−
1
Km,AcCoA = 0.17
±
0.04 mM, kcat = 3813
±
169 min
[
initial screening were set to improve the sensitivity of the assay towards both competitive
and uncompetitive inhibitors, i.e., the concentration of both substrates was equal to Km.
Interestingly, six out of seven compounds are characterised by the presence of a
carboxylic functional group, a structural requisite that seems to be crucial for properly
interacting with the target. To rank the molecules and select a hit candidate for further
expansion, the inhibitory potency of compounds 1–7 was assayed at a fixed concentration
of 1mM (Table 1). Compounds
by more than 40% at the tested concentration, and they were therefore selected for IC50
determination. Consistent with the preliminary evaluation, compound emerged as the
most potent hit of the set displaying an IC50 of 110 M. On the other hand, compound
showed an IC50 > 2 mM. Further biochemical studies characterised compound as a
competitive inhibitor of the enzyme towards acetyl-CoA with a K of 64 12 M (Figure 3).
5 and 7 were found to inhibit the activity of the enzyme
5
±
0 µ
7
5
±
µ
i
This was further corroborated by the computational investigation that predicted how
the compound is accommodated in both L-serine and acetyl-CoA pockets. The L-shape
allows partial filling of the acetyl-CoA cavity at the tail level, whereas the carboxylic acid
functionality mimics the carboxylic acid group of the natural substrate, L-serine (Figure 4).
This binding mode is compatible with a competitive displacement of acetyl-CoA, although
interactions with other regions of the acetyl-CoA binding pocket are likely to happen.
Compound
5 belongs to a series of antitubercular 2-aminothiazoles and it was initially
designed and synthesised for anti-TB Structure-Activity Relationships (SAR) purposes [39].
Although many of these derivatives have remarkable activities toward M. tuberculosis,
compound
5 was found to be inactive, and this is an advantage when a compound is
repurposed to prevent the occurrence of off-target issues.

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Table 1. Inhibition of SAT by compounds identified in the virtual screening.
Cpd
% Inhibition at 1 mM
IC50 (µM)
Ki (µM)
Mechanism
1
2
3
4
5
6
7
19.4 ± 1
34.9 ± 6
31.4 ± 5
18.7 ± 6
98.6 ± 0
23.6 ± 0
42 ± 5
ND a
ND
ND
ND
ND
ND
ND
ND
110 ± 0
64 ± 12
Competitive with AcCoA
ND
ND
>2 mM
ND
a
ND = Not Determined.
Figure 3. Inhibitory activity of compound
compound . The line through data points is the fitting to equation 1 with IC50 = 110
initial reaction rate on the concentration of acetylCoA in the absence of added inhibitors (grey dots) and in the presence of
different concentrations of compound (green dots 30 M; pink dots 100 M, red dots 300 M). The series of lines through
5. Panel A: dependence of relative initial reaction rate on the concentration of
5
±
0 µM. Panel B: dependence of the
5
µ
µ
µ
data points is the fitting to the linearised form of equation 2 with K = 64 ± 12 µM.
i
Figure 4.
(
A
) docking pose of compound
5
in AcCoA pocket in EcSAT (alignment of structures
) Overlay of the docking pose of compound and
with pdb code 1T3D and pdb code 1SSM). (
B
5
AcCoA structure.

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2
.2. Hit Expansion
The plan to release a valuable hit compound against StSAT consisted of two steps. First,
analogues of compound already available in the in-house library were tested in order to
build a preliminary body of SAR; then, based on the SAR obtained, the rational synthesis
of additional analogues was carried out. A total of 13 in-house analogues of compound
5
5
were selected and tested for their capability to inhibit StSAT (Table 2). In particular, the
carboxylic moiety was maintained either as acid or as its derivatives, namely esters and
amides. In some cases, derivatives bearing an N-methylpyrazole in place of the isoxazole
were considered for testing. Additionally, the effect of small electron withdrawing groups
(
EWGs) in the phenyl ring attached to the 2-aminothiazole was evaluated. The majority of
the derivatives was found to be more potent than the hit compound , with compounds
and 19, where the 2-aminothiazole ring is replaced by a 2-aminooxazole, showing
5
1
8
the most efficient inhibitory activity toward the enzyme. Inspired by these preliminary
findings, in the second step synthetic efforts were devoted toward the synthesis of further
analogues. In particular, a small set of analogues of the 2-aminooxazole derivatives 18 and
1
9
was designed and synthesised, taking advantage from a synthetic protocol that we have
previously reported [40], which proved to be particularly versatile in the case of substituted
-aminooxazoles (Scheme 1). The small set of derivatives consisted of 3-carboxylisoxazoles
characterised by the presence of variously substituted aromatic and heteroaromatic rings
attached at the nitrogen of the 2-aminooxazole (21a 22a ). In this preliminary round
2
–
e
,
–e
of substitution, pyridine and benzene, unadorned or bearing electron-withdrawing or
electron-donor groups, were used as substituents.
Table 2. StSAT Inhibitory activity of compound 5 analogues.
Cpd
8
Structure
IC50 (µM)
Cpd
Structure
IC50 (µM)
18 ± 2
>400
15
9
12 ± 2
9 ± 3
16
17
184 ± 13
21 ± 5
O
S
1
0
N
H
N
N
N
O
H
1
1
2
26 ± 4
18
19
20
1.0 ± 0.2
7 ± 2
O
O
O
1
> 60 *
N
N
O
N
H
1
3
16 ± 3
11 ± 1
1
4
~10 *
*
The IC50 could only be estimated due to solubility issues of the compound.

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Scheme 1. a Reagent and conditions: (a) triethylamine, tetrahydrofuran, rt; (b) N-Bromosuccinimide, p-toluenesulfonic acid,
◦
acetonitrile, reflux; (c) urea, dimethylformamide, MW (120 C, 300 W), 3 min, 49%; (d) suitable bromobenzene derivative or
◦
4
-bromopyridine (21e), Sodium t-butoxide, X-Phos Pd G2, t-butanol, toluene, MW (130 C, 300 W), 15 min, 8–35%; (e) LiOH,
THF/H O/MeOH 3:1:1, rt, 3 h, quantitative.
2
2
.3. Chemistry
a
Scheme 1 Synthesis of compounds 21a–e and 22a–e.
The synthesis of the title compounds 21a–e and 22a–e was carried out via a syn-
thetic protocol optimised in our laboratories relying on the condensation of the proper
-bromoacetophenones with urea followed by the Buchwald–Hartwig cross coupling of
α
the 2-aminooxazole with an aryl halide. Dipolar cycloaddition between 3-butyn-2-one and
ethyl 2-chloro-2-(hydroxyimino)acetate in diethyl ether and trietylamine (TEA) led to the
synthesis of ethyl 5-acetyl-3-isoxazolecarboxylate 25, that was efficiently brominated with
N-bromosuccinimide (NBS) to yield derivative 26. Reaction of 26 with urea in a microwave
◦
reactor at 120 C in dimethylformamide led to the synthesis of building block 27, which
underwent Buchwald–Hartwig cross coupling with variously substituted bromobenzenes
to afford the title compounds 21a-e. Hydrolysis of the ethyl esters with LiOH in a mixture
of THF/MeOH/H O gave the corresponding acids 22a-e in quantitative yields.
2
3. Discussion
3.1. Hints of Structure–Activity Relationships
In this research work, valuable hit compounds able to inhibit StSAT are reported
through a virtual screening and subsequent hit optimization. Since compound
5 emerged
as the most potent hit from the virtual screening, the first body of SAR was obtained
by testing a collection of 13 structurally related compounds already available in the in-
house library. The majority of the derivatives tested was found to be more affine to
StSAT than the hit compound
we first evaluated its abolition. Substitution of the isoxazole-3-carboxylic core with a
pyridine led to loss of the affinity for the enzyme ( , IC50 = >400 M). In general, the
presence of the carboxylic functional group, either as a carboxylic acid (16, IC50 = 184 M,
, IC50 = 21 M, 18 IC50 = 2.6 µM 20, IC50 = 11 M) or its derivatives such as amides
10, IC50 = 9 M, 11, IC50 = 26 M, 12, IC50 = 60 M, 13, IC50 = 16.3 M) and esters
5. To confirm the importance of the carboxylic moiety,
8
µ
µ
17
µ
,
,
µ
(
µ
µ
µ
µ

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(
14,
IC50 = 10 µM
,
15
,
IC50 = 18 µM
,
19, IC50 = 7.3
µM), despite the different substitution
pattern, seems to play a role in maintaining acceptable inhibitory activity, the only exception
being represented by , which shows promising activity despite devoid of the carboxylic
functionality ( IC50 = 12 µM). It might be hypothesised that the electronic pattern of
9
9,
carboxylic acid derivatives (i.e., acid, ester and amides) is favorable for the interaction
with the enzyme. Among the amides, it seems that bulky substituents such as the phenyl
ring and the adamantane attached at the nitrogen confer higher affinity than a methyl or
unsubstituted amide (cfr 10 and 13 vs. 12 and 11). When the isoxazole ring is replaced with
a N-methylpyrazole (16
activity is noticed compared to the isoxazole counterpart
-carboxylic acid derivatives, introduction of electron withdrawing groups (EWGs) in
the phenyl ring attached to the 2-aminothiazole seems to have beneficial effect on the
affinity (20, IC50 = 11 M vs. , IC50 = 110 M), although at this stage this consideration
is quite speculative. An ester moiety can efficiently replace the carboxylic acid group (14
IC50 = 10 M and 15, IC50 = 18 M), regardless the nature of the substituent attached at
the 2-aminothiazole. For compound 14, it must be specified that the IC50 could be only
estimated due to some solubility issue. The difference in the activity for compounds and
cannot be easily explained, unless permeability is brought up. However, this does not
,
IC50 = 184 µM
,
17, IC50 = 21
µM), a reduction of the inhibitory
5
and 20. Among the isoxazole-
3
µ
5
µ
,
µ
µ
5
14
seem to be the case. The synthesis of additional analogues can be of help in explaining
this discrepancy. Replacement of the 2-aminothiazole moiety by a 2-aminooxazole led to
the most potent derivatives of the series (18
a >40-fold improvement in the affinity compared to the 2-aminothiazole counterpart (18
IC50 = 2.6 M vs. , IC50 = 110 M). These findings prompted us to synthesise a small set of
, IC50 = 2.6 µM and 19, IC50 = 7.3 µM), with
,
µ
5
µ
derivatives for further SAR refinement, evaluating the presence of small functional groups
on the phenyl ring attached to the 2-aminooxazole nitrogen and investigating whether
the ethyl ester or the acid had the best affinity for the enzyme. Biochemical evaluation of
compounds 21a–e and 22a–e on StSAT showed that, in general, the SAR obtained with
these small modifications was quite flat (Table 3). Both the carboxylic acid and its ethyl
ester derivatives are equally able to confer adequate inhibitory activity, (compounds 21a–e
vs. compounds 22a–e). Ester derivatives can be considered slightly more efficient than the
acid counterparts, even though compound 22a, an acid, was found to be the most active
derivative obtained from this study. Overall, compound 22a and the other acid derivatives
might be considered valuable hit compounds for further investigation. This might be
due to the fact that acids are generally metabolically more stable than the corresponding
esters, which can undergo hydrolysis. Additionally, acid derivatives have no intrinsic
antitubercular or antibacterial activity [39], an important requirement to avoid off-target
effects during the repurposing process. Introduction of small EWGs (compounds 21b
, 21c,
2
2b 22c) or electron-donor groups (compound 22d) at different positions of the phenyl ring
,
does not interfere significantly with the affinity of the molecules for StSAT, although the
most interesting results are obtained when the phenyl ring is not substituted (compounds
21a and 22a).
Table 3. StSAT Inhibitory activity of synthesised compounds 21a–c, 21e and 22 a–e.
Cmpd
R
StSAT IC50 (µM)
Cmpd
R
StSAT IC50 (µM)
2
2
2
1a
1b
1c
Phenyl
3,5-Dichlorophenyl
4-Fluorophenyl
2.68 ± 0.27
2.52 ± 0.03
3.04 ± 0.37
22a
22b
22c
Phenyl
1.54 ± 0.33
8.03 ± 0.18
2.51 ± 0.34
4.24 ±0.11
12.02 ± 1.25
3,5-Dichlorophenyl
4-Fluorophenyl
3,5-Dimethylphenyl
Pyridine
2
2d
2
1e
Pyridine
3.95 ± 0.65
22e

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Having in mind that compound
5
is a competitive inhibitor of acetyl CoA predicted by
the computational studies to be accommodated in both pockets of acetyl CoA and L-serine,
it is possible that the structure we are currently exploring is only occupying a part of the
pocket and the substitutions we are performing are all tolerable due to the empty space in
the cavity.
3.2. Stability of the Isoxazole-Oxazole Nucleus
Upon noticing changes in the appearance of the DMSO solution over time at room
temperature, we deemed it of interest to investigate the chemical stability of the compounds.
1
Therefore, in order to evaluate it, H NMR analysis at different time points (1 h, 2.5 h, and
2
0 h) of the DMSO solution (5 mg in 500 µL of DMSO-d6) was performed (Supporting
Information). Since the time required to observe changes in the colour of the solution
was dependent of the substituent at position R2 of series 22, to establish the effect of
the substituent on the overall stability of the compound, analogue 22a (R2 = phenyl)
which contains the non-substituted phenyl group, analogue 22b (R2 = 3,5-dichlorophenyl),
substituted with an EWG and analogue 22d (R2 = 3,5-dimethylphenyl) substituted with
an electron donating group (EDG) were selected to perform the chemical stability studies.
The stability of the compounds increases in the following order: 22b (stability in DMSO
<
2.5 h) < 22a (stability in DMSO < 20 h) < 22d (stability in DMSO < 7 days). Thus, it is
possible to conclude that EWGs like the simple phenyl or the phenyl replaced with two
chlorines produce fewer stable molecules. On the other hand, the phenyl replaced with
EDGs contributes to the overall stability of the molecule. It is hypothesised that the mild
oxidative properties of DMSO are able to interfere with the oxazole scaffold, which leads
to the opening of the ring and then full degradation of the compound in study [41].
3.3. Antibacterial Activity
Considering the stability issues above described, compound 22a did not undergo
biological evaluation. Rather, compound 22d, which is only two-fold less active that 22a
in the biochemical assay, was tested for its MIC against E. coli both in standard Mueller
Hinton broth (MHB) and in Middlebrook 9 (M9), which is deprived of cysteine [42].
Unfortunately, in both these conditions, no antibacterial activity could be measured at con-
centration
≤
128 µM on two bacterial strains, namely E. coli ATCC25922 and S. Typhimurium
ATCC14028. A plausible cause for the lack of activity might be the poor permeability
of this compound through the bacterial outer membrane and cell wall, which is one of
the most common issues related to antibacterial drug discovery, especially toward Gram-
negative species.
4. Materials and Methods
4.1. Enzyme Preparation and Activity Assay
SAT from Salmonella enterica serovar Typhimurium was expressed as trxA fusion
protein as previously described [37] and was more than 95% pure based on SDS-PAGE
analysis. The enzyme activity was measured by a spectrophotometric indirect assay that
exploits the signal at 412 nm generated by 5,5’-Dithiobis(2-nitrobenzoic acid (Ellmn’s
reagent) reduction by the reaction product coenzyme A [37]. A total of 5% DMSO was
present in all the assays and was proven not to perturb enzyme activity (data not shown).
m
The concentration of substrates was set equal to K in order to improve the sensitivity
of the assay for both competitive and uncompetitive mechanism of inhibition [43]. The
dependence of the relative reaction rate on the concentration of compounds was fitted to
Equation (1) to calculate the IC :
50
vi
v0
1
=
[
I]
1 +
IC50

Pharmaceuticals 2021, 14, 174
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The inhibition mechanism was determined by fitting the dependence of the initial
reaction rate on the concentration of acetyl CoA (at fixed, 1 mM L-Ser concentration)
1
in the presence of different concentrations of inhibitor ( IC50 ≥ [inhibitor] ≤
3IC50) to
3
the equation for competitive, uncompetitive and non-competitive mechanisms [43]. The
equation that fits a competitive mechanism (Equation (2)) was used to calculate K for
i
compound 5:
Vmax·[acetyl − CoA]
v0 =
[
acetyl − CoA] + (Km·α)
ꢀ
ꢁ
[
comp5]
Ki
where α = 1 +
.
4.2. Chemistry
All the reagents were purchased from Sigma-Aldrich, Alfa-Aesar, and Enamine at
reagent purity and, unless otherwise noted, were used without any further purification.
Dry solvents used in the reactions were obtained by distillation of technical grade mate-
rials over appropriate dehydrating agents. Microwave reactions were performed using
CEM microwave synthesizer Discover model. Reactions were monitored by thin layer
chromatography on silica gel coated aluminium foils (silica gel on Al foils, SUPELCO
Analytical, Sigma-Aldrich) at both 254 and 365 nm wavelengths. When indicated, inter-
mediates and final products were purified through silica gel flash chromatography (silica
1
13
gel, 0.040–0.063 mm), using appropriate solvent mixtures. H NMR and C NMR spectra
were recorded on a BRUKER AVANCE spectrometer at 300, 400, and 100 MHz, respectively,
1
with TMS as internal standard. H NMR spectra are reported in this order: multiplicity
and number of protons. Standard abbreviation indicating the multiplicity was used as
follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quadruplet,
m = multiplet and br = broad signal. HPLC/MS experiments were performed with HPLC
instrumentation (Agilent 1100 series, equipped with a Waters Symmetry C18, 3.5 µm,
4
.6 mm × 75 mm column) and MS instrumentation (Applied Biosystem/MDS SCIEX, with
API 150EX ion source). HRMS experiments were performed with LTQ ORBITRAP XL
THERMO. All compounds were tested as 95% purity samples or higher (by HPLC/MS).
Analytical data for compounds 8-20, 23-27 have been already published [39,40,44].
Ethyl 5-acetylisoxazole-3-carboxylate (25): To a solution of butynone 23 (2.3 mL,
29 mmol) and ethyl-2-chloro-2-(hydroxyimino)acetate 24 (4.39 g, 29 mmol) in THF (37.5 mL)
a solution of triethylamine (4 mL, 29 mmol) in THF (10 mL) was added dropwise. Re-
action mixture was allowed to stir at room temperature for 3h and then volatiles were
evaporated. Extractions with ethyl acetate were performed (3
organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under
reduced pressure to be purified by flash column chromatography on silica gel using ethyl
×
50 mL) and the combined
acetate/petroleum ether (7:93). The product was obtained as a white powder in 50% yield.
1
H NMR (400 MHz, CDCl3)
.43 (t, J = 7.1 Hz, 3H).
δ 7.26 (s, J = 2.0 Hz, 1H), 4.47 (q, J = 7.1 Hz, 2H), 2.65 (s, 1H),
1
Ethyl 5-(2-bromoacetyl)isoxazole-3-carboxylate (26). To a solution of compound 25
2.65 g, 14.47 mmol) in acetonitrile (26.8 mL) stirring on an ice bath, p-toluenesulfonic acid
(
monohydrate (2.49 g, 14.47 mmol) and N-bromosuccinimide (2.58 g, 14.47 mmol) were
added portion-wise. Reaction mixture was stirred at reflux for 5 h. After volatiles were
evaporated and reaction mixture was purified by flash column chromatography on silica
gel using a gradient of ethyl acetate in petroleum ether. The product was obtained as a
1
yellow powder in 58% yield. H NMR (300 MHz, DMSO)
δ
7.89 (s, 1H), 4.87 (s, 2H), 4.41
(
dt, J = 12.4, 4.5 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).
Ethyl 5-(2-aminooxazol-4-yl)isoxazole-3-carboxylate (27). A mixture of compound 26
(
315 mg, 1.2 mmol) and urea (721 mg, 12 mmol) were solubilised in anhydrous dimethyl-
formamide (3.14 mL) and stirred at reflux for 2 h, and then cooled until room temperature.
After, a solution of LiCl 5% in water (32 mL) was added to the reaction mixture and
extractions with ethyl acetate were performed (4
×
10 mL). The reaction mixture was

Pharmaceuticals 2021, 14, 174
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then quenched with water and the mixture extracted with ethyl acetate (3
×
10 mL). The
organic layers were washed with water, brine and dried over Na SO . After filtration,
2
4
the solvent was removed in vacuo and the crude material was purified by flash column
1
chromatography. The product was obtained as a yellow powder in 77% yield. H NMR
(300 MHz, DMSO)
δ
8.15 (s, 1H), 7.07 (s, 2H), 6.89 (s, 1H), 4.37 (dd, J = 13.8, 6.8 Hz, 2H),
1.32 (t, J = 6.9 Hz, 3H).
General procedure for the Buchwald–Hartwig reaction. Synthesis of compounds
21a-e. In a 10 mL test tube for microwave, a solution of the proper 2-aminooxazole
(
1 equiv), the suitable aryl halide (0.5 equiv), the base (1 equiv) in a mixture of anhydrous
toluene (2.5 mL/mmol) and t-butanol (0.5 mL/mmol) was stirred under argon flux for
5 min. After this time, the suitable catalyst (0.1 equiv) was added and the reaction
1
mixture was irradiated in a microwave reactor after setting the parameters as follows:
◦
temperature = 130 C
,
time = 15 min, pressure = 250 psi, power = 300 W, power max = off
.
The reaction was monitored by TLC. After the complete consumption of the starting
material, as revealed by TLC (95:5 dichloromethane/methanol), water was added, and the
mixture extracted with ethyl acetate (3
×
10 mL). The organic layers were washed with
water, brine and dried over Na SO . After filtration, the solvent was removed in vacuo
2
4
and the crude material was purified by flash column chromatography.
Ethyl 5-(2-(phenylamino)oxazol-4-yl)isoxazole-3-carboxylate (21a). Purification by
flash column chromatography using ethyl acetate/petroleum ether (1:9) afforded the
1
product as a yellow solid in 24% yield. H NMR (400 MHz, Acetone)
δ 9.41 (s, 1H), 8.17 (s,
1
1
1
H), 7.76 (d, J = 7.9 Hz, 2H), 7.36 (t, J = 7.9 Hz, 2H), 7.12–6.75 (m, 2H), 4.43 (q, J = 7.1 Hz, 2H),
.39 (t, J = 7.1 Hz, 3H). 13C-NMR (100.6 MHz; Acetone):
= 20.8; 62.4; 101.5; 117.4; 122.4;
29.8; 132.7; 136.9; 156.5; 156.6; 158.1; 159.3; 165.1. HRMS (ESI) calculated for C H N O
δ
1
5
13
3
4
+
[
M + H] , 300.0906 found 300,1112.
Ethyl 5-(2-((3,5-dichlorophenyl)amino)oxazol-4-yl)isoxazole-3-carboxylate (21b). Pu-
rification by flash column chromatography using ethyl acetate/petroleum ether (1:9) af-
1
forded the product as an orange powder in 30% yield. H NMR (300 MHz, CDCl )
δ
7.82
3
(
s, 1H), 7.51 (d, J = 1.6 Hz, 2H), 7.07 (s, 1H), 7.02 (s, 2H), 4.48 (q, J = 7.1 Hz, 2H), 1.45 (t,
J = 7.1 Hz, 3H). 13C-NMR (100.6 MHz; CDCl3):
δ = 18.5; 62.1; 101.3; 108.8; 119.8; 120.2;
1
28.0; 139.9; 150.1; 154.8; 156.8; 158.2; 160.5. HRMS (ESI) calculated for C H Cl N O
15 11 2 3 4
+
[
M + H] , 368.0126 found 368.0520.
Ethyl 5-(2-((4-fluorophenyl)amino)oxazol-4-yl)isoxazole-3-carboxylate (21c). Purifica-
tion by flash column chromatography using ethyl acetate/petroleum ether (1:9) afforded
1
the product as a slightly orange powder in 8% yield. H NMR (300 MHz, CDCl )
δ 7.77 (s,
3
1
H), 7.51 (dd, J = 8.4, 4.2 Hz, 2H), 7.07 (t, J = 8.5 Hz, 2H), 6.98 (s, 1H), 6.92 (s, 1H), 4.47 (q,
J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H). 13C-NMR (100.6 MHz; CDCl3):
δ = 18.9; 62; 100.8;
1
08.2; 121.0; 121.2; 127.8; 139.9; 150.1, 154.8; 157.3; 159.2; 160.8. HRMS (ESI) calculated for
+
C H FN O [M + H] , 318.0811 found 318.0822.
15
12
3
4
Ethyl 5-(2-((3,5-dimethylphenyl)amino)oxazol-4-yl)isoxazole-3-carboxylate (21d). Pu-
rification by flash column chromatography using ethyl acetate/petroleum ether (1:9) af-
1
forded the desired product as a yellow powder in 35% yield. H NMR (300 MHz, DMSO-d )
6
δ
10.28 (s, 1H), 8.42 (s, 1H), 7.25 (s, 2H), 7.10 (s, 1H), 6.63 (s, 1H), 4.39 (q, J = 7.1 Hz, 2H), 2.26
(
s, 6H), 1.34 (t, J = 7.1 Hz, 3H). 13C-NMR (100.6 MHz; DMSO-d6):
δ
= 18.8; 21.8, 21.9, 61.8;
1
00.8; 112.8; 121.4; 126.2; 138.8; 140.1; 150.8, 155; 156.8; 159.2; 160.8. HRMS (ESI) calculated
+
for C H N O [M + H] , 328.1219 found 328.1401.
17
17
3
4
Ethyl 5-(2-(pyridin-4-ylamino)oxazol-4-yl)isoxazole-3-carboxylate (21e). Purification
by flash column chromatography using ethyl acetate/petroleum ether (1:1
the product as a yellow powder in 15% yield. H NMR (400 MHz, Acetone)
→
100%) afforded
8.47 (d,
1
δ
J = 6.2 Hz, 2H), 8.27 (s, 1H), 7.73 (d, J = 6.3 Hz, 2H), 7.07 (s, 1H), 4.44 (q, J = 7.1 Hz, 2H), 1.40
1
3
(
1
t, J = 7.1 Hz, 3H). C-NMR (100.6 MHz; Acetone):
δ
= 21; 62.4; 101.3; 109,0; 125.4; 139.2;
+
50.1, 155.6; 156.6; 158.1; 159.3; 160.5. HRMS (ESI) calculated for C H N O [M + H] ,
14
12
4
4
301.0858 found 301,0999.

Pharmaceuticals 2021, 14, 174
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General procedure for the hydrolysis. Synthesis of compounds 22a-e. The suitable ester
(
1
1 equiv) and LiOH
·
H O (4 equiv) were dissolved in a mixture of THF/MeOH/H O (3:1:1,
2
2
mL/mmol) and stirred at room temperature until consumption of the starting material as
indicated by TLC (7:3 petroleum ether/ethyl acetate, then 9:1 Dichloromethane/methanol). The
reaction mixture is then evaporated under reduced pressure, and the crude obtained is
taken up with H O, acidified with 2N HCl and extracted with ethyl acetate (3
ter evaporation of the solvent, the desired acid derivatives are obtained in sufficient purity.
×
10 mL). Af-
2
5
-(2-(phenylamino)oxazol-4-yl)isoxazole-3-carboxylic acid (22a). Yellow powder (37%
1
yield); H NMR (400 MHz, DMSO-d )
J = 7.8 Hz, 2H), 7.34 (t, J = 7.9 Hz, 2H), 7.00 (dd, J = 15.0, 7.6 Hz, 2H). C NMR (101 MHz,
δ 10.45 (s, 1H), 9.47 (s, 1H) 8.42 (s, 1H), 7.66 (d,
6
13
DMSO-d6)
δ
164.5; 160.6; 157.4; 138.8; 132.1; 129.0; 128.9; 121.6; 116.8; 101.2. HRMS (ESI)
+
calculated for C H N O [M + H] , 272.0593 found 272.0606.
13
9
3
4
5
-(2-((3,5-dichlorophenyl)amino)oxazol-4-yl)isoxazole-3-carboxylic acid (22b). Light
1
yellow powder (60% yield); H NMR (400 MHz, DMSO-d )
δ 11.01 (s, 1H), 8.49 (s, 1H),
6
7
.72 (d, J = 1.8 Hz, 2H), 7.19 (t, J = 1.8 Hz, 1H), 7.03 (s, 1H). 13C NMR (101 MHz, DMSO-d6)
δ
160.4; 156.2; 141.0; 134.2; 132.5; 127.9; 124.0; 120.5; 114.7; 101.2. HRMS (ESI) calculated for
+
C H Cl N O [M + H] , 339.9814 found 339.9986.
13
7
2
3
4
5
-(2-((4-fluorophenyl)amino)oxazol-4-yl)isoxazole-3-carboxylic acid (22c). Yellow
1
powder (81% yield); H NMR (400 MHz, Acetone)
δ
9.48 (s, 1H), 8.18 (s, 1H), 7.85–7.77
166.0; 161.0; 160.1;
(
m, 2H), 7.19–7.11 (m, 2H), 7.01 (s, 1H). 13C NMR (101 MHz, Acetone)
δ
1
58.6; 158.0; 136.3; 132.3; 129.8; 119.6; 119.5; 116.4; 116.2; 102.2. HRMS (ESI) calculated for
+
C H FN O [M + H] , 290.0499 found 290.0509.
13
8
3
4
5
-(2-((3,5-dimethylphenyl)amino)oxazol-4-yl)isoxazole-3-carboxylic acid (22d). Yellow
1
powder (68% yield); H NMR (400 MHz, DMSO-d )
δ
10.28 (s, 1H), 8.41 (s, 1H), 7.26 (s,
165.0; 161.0;
6
2
H), 7.04 (s, 1H), 6.64 (s, 1H), 2.27 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
δ
1
57.9; 157.7; 139.1; 138.4; 132.4; 128.5; 123.8; 115.0; 101.6; 21.6. HRMS (ESI) calculated for
+
C H N O [M + H] , 300.0906 found 300.1002.
15
13
3
4
5
-(2-(pyridin-4-ylamino)oxazol-4-yl)isoxazole-3-carboxylic acid (22e). Yellow powder
1
(
2
1
2
63% yield); H NMR (300 MHz, DMSO-d )
δ
12.58 (s, 1H), 8.71 (s, 1H), 8.66 (d, J = 7.2 Hz
,
6
H), 8.12 (d, J = 5.6 Hz, 2H), 7.20 (s, 1H). 13C NMR (101 MHz, DMSO-d6)
55.0; 143.0; 134.2; 128.2; 112.2; 102.0. HRMS (ESI) calculated for C H N O [M + H] ,
δ
163.6; 160.5; 157.5;
+
1
2
8
4
4
73.0545 found 273.0628.
4.3. Biology
All reference bacterial strains (4–5 morphologically similar colonies) were inoculated
◦
in MHB e M9 and incubated at 37 C, in agitation at 240 rpm until log phase was reached.
◦
The bacterial suspension was then centrifuged 20 min at 2000 rpm and 4 C, then the pellet
was resuspended in MHB and M9 media. The turbidity of the bacterial suspension was
immediately measured and adjusted by spectrophotometry. At 600 nm, the OD range
8
0
.08–0.13 was considered to correspond to a bacterial concentration of 10 CFU/mL [45].
1
00
µ
L of the obtained suspension were further diluted in 9.9 mL of MHB and M9 media to
6
obtain a final bacterial concentration of 10 CFU/mL. An amount of 50
µl of the bacterial
suspension were inoculated within 30 min into the wells of a microtiter plate to obtain
5
5
× 10 CFU/mL, according with CLSI guidelines [45]. Antimicrobial activity of 22a
,
22b
,
22d was assessed by Minimal Inhibitory Concentration (MIC) assay. Briefly, in a
L of M9 or MHB media. 1 L of
each compounds originally diluted in DMSO was then added to each well, thus testing
concentrations ranging from 128 g/mL to 0.25 g/mL with a final concentration of
L. Growth and sterility
microtiter plate 10 wells were inoculated with 49
µ
µ
µ
µ
5
× 105 CFU/mL of bacterial suspension in a volume of 100
µ
controls were performed. For each bacterial strain three independent experiments, with
three replicates each, were performed.

Pharmaceuticals 2021, 14, 174
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5. Conclusions
Through an in-house library virtual screening combined with a medicinal chemistry
campaign, we were able to identify the most potent inhibitor of StSAT known so far. This
compound was however found to show instability issues when solubilised in DMSO;
therefore, other derivatives, endowed with higher stability, were used for the following
cellular assay. The most promising compound of the series (22d) was tested against E. coli
in a minimum medium characterised by the absence of cysteine. Despite the remarkable
activity in the biochemical assay, the compound was not able to interfere with the bacterial
growth, likely for its inability to cross the bacterial membrane. This investigation allowed
us to delineate a preliminary SAR, although the synthesis of further analogues is currently
ongoing to further expand the set of structural information and, more in particular, to
afford molecules with ameliorated permeability.
Supplementary Materials: The following are available online at https://www.mdpi.com/1424-824
7
/14/2/174/s1.
Author Contributions: Conceptualization, M.P. and B.C.; Data curation, J.M., P.T., S.B., C.S. and
C.S.C.; Formal analysis, G.A.; Funding acquisition, G.C.; Investigation, M.P. and B.C.; Methodology,
J.M., N.F., S.R., G.A., A.B., S.B., S.A. and C.S.; Validation, P.T.; Writing—original draft, M.P. and B.C.;
Writing—review & editing, A.M., M.P. and G.C. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was funded under the MSCA-ITN-2014-ETN project INTEGRATE [grant number
6
42620]. PT thanks the Academy of Finland (Grant. no. 277001) for financial support.
Data Availability Statement: The data presented in this study are available in this article and related
Supplementary Material.
Acknowledgments: The Centro Interdipartimentale Misure “G. Casnati” is kindly acknowledged
for the contribution in the analytical determination of the molecules synthesised.
Conflicts of Interest: The authors declare no conflict of interest.
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