Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand–based drug design

Article abstract of DOI:10.1080/14756366.2018.1512596

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure–Activity Relationship investigation was carried out.

Full text of DOI:10.1080/14756366.2018.1512596

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Discovery of novel fragments inhibiting O-
acetylserine sulphhydrylase by combining scaffold
hopping and ligand–based drug design
Joana Magalhães, Nina Franko, Giannamaria Annunziato, Martin Welch,
Stephen K. Dolan, Agostino Bruno, Andrea Mozzarelli, Stefano Armao, Aigars
Jirgensons, Marco Pieroni, Gabriele Costantino & Barbara Campanini
To cite this article: Joana Magalhães, Nina Franko, Giannamaria Annunziato, Martin Welch,
Stephen K. Dolan, Agostino Bruno, Andrea Mozzarelli, Stefano Armao, Aigars Jirgensons,
Marco Pieroni, Gabriele Costantino & Barbara Campanini (2018) Discovery of novel fragments
inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand–based
drug design, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 1444-1452, DOI:
10.1080/14756366.2018.1512596
To link to this article: https://doi.org/10.1080/14756366.2018.1512596
© 2018 The Author(s). Published by Informa
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Group.
Published online: 17 Sep 2018.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1444–1452
https://doi.org/10.1080/14756366.2018.1512596
RESEARCH PAPER
Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by
combining scaffold hopping and ligand–based drug design
a
b
a
c
c
d
~
Joana Magalhaes , Nina Franko , Giannamaria Annunziato , Martin Welch , Stephen K. Dolan , Agostino Bruno ,
Andrea Mozzarellib,e,f, Stefano Armaog, Aigars Jirgensonsh, Marco Pieronia, Gabriele Costantinoa,i and
Barbara Campaninib
b
aP4T group, Department of Food and Drug, University of Parma, Parma, Italy; Laboratory of Biochemistry and Molecular Biology, Department
c
of Food and Drug, University of Parma, Parma, Italy; Department of Biochemistry, Cambridge University, Cambridge, United Kingdom;
dExperimental Therapeutics Program, IFOM – The FIRC Institute for Molecular Oncology Foundation, Milano, Italy; National Institute of
e
f
g
ꢀ
Biostructures and Biosystems, Rome, Italy; Institute of Biophysics, CNR, Pisa, Italy; Centro Interdipartimentale “Biopharmanet-tec”, Universita
h
i
degli Studi di Parma, Parma, Italy; Latvian Institute of Organic Synthesis, Riga, Latvia; Centro Interdipartimentale Misure (CIM)’G. Casnati’,
University of Parma, Parma, Italy
ABSTRACT
ARTICLE HISTORY
Received 18 July 2018
Revised 10 August 2018
Accepted 10 August 2018
Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise
cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with
mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine bio-
synthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial
infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our
library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for fur-
ther development. This novel chemical tool, endowed with favourable pharmacological characteristics,
was successfully developed, and a preliminary Structure–Activity Relationship investigation was car-
ried out.
KEYWORDS
Scaffold hopping;
fragments; O-acetylserine
sulphhydrylase; medicinal
chemistry; pyrazoles; ligand-
based drug design
Introduction
both bisulphide and thiosulphate6,7. The three-dimensional struc-
tures8–21 of OASS-A and OASS-B isoforms has shown that the
active site is in a cleft between the domains, where a PLP mol-
ecule is covalently bound to a conserved lysine residue. SAT and
OASS-A combine in a tight molecular complex that is called the
cysteine synthase complex, and the SAT C-terminal oligopeptide
shows OASS competitive inhibitory properties. On the other hand,
OASS-B does not interact with SAT, which suggests a different
Since the 1940s, antibacterial agents have greatly reduced illness
and death from infectious diseases. However, the misuse of these
drugs resulted in adaptation of the bacteria to their killing action,
and nowadays antibiotics are less effective with a few of them
being useless (“About Antimicrobial Resistance
j
Antibiotic/
Antimicrobial Resistance j CDC,” 2018; “WHO j Antimicrobial
resistance,” 2018). Therefore, it is necessary to adopt novel strat-
egies to fight antibacterial resistance. Cysteine de novo biosyn-
thesis, present only in bacteria and plants, converts inorganic
sulphur into cysteine, a precursor of several important biomole-
cules such as methionine, Fe-S clusters, thiamin, glutathione,
regulatory role in the reductive sulphur assimilation pathway3,4,22
.
Over the years, inhibitors of both OASS-A and B isoforms have
been developed. Our group was particularly successful in prepar-
ing peptidic23,24 and non-peptidic inhibitors22,25–28 by rational
design inspired by the structure of SAT C-terminal sequence.
Among the many structure–activity relationship (SAR) hints con-
sidered, the carboxylic moiety of Ile267 of SAT was found out to
be essential for the interaction with the target enzyme, and the
inhibitors prepared could not be devoid of this moiety23. Within
the set of synthetic inhibitors with potencies ranging from low
millimolar to low micromolar, ( )-trans-2-[(1E)-prop-1-en-1-yl]cyclo-
propanecarboxylic acid (1, Figure 1), with a Kd of 1.46 lM25, was
selected for further optimization as it suffered from several weak-
nesses, such as poor chemical feasibility and stability.
and biotin1,2
.
The reductive sulphur assimilation pathway involves five
enzymes that reduce sulphate to bisulfide and is connected with
the serine activation reaction, where L-serine is converted to O-
acetylserine (OAS) by serine acetyltransferase (SAT). O-acetylserine
sulfhydrylase (OASS) catalyzes the b-replacement of the acetoxy
group of OAS by bisulphide, generating cysteine3,4. OASS is a
member of the cysteine synthase superfamily, and it is a pyridoxal
5’-phosphate (PLP)-dependent enzyme that exists in two isoforms:
OASS-A and OASS-B. Both isozymes were firstly identified and
characterised in Salmonella enterica serovar Typhimurium
(Salmonella), although the specific role of OASS-B is still debated5.
OAS is the preferred substrate for both isoforms but while bisul-
Therefore, combining a structure-based and ligand-based drug
design approach, and with the help of spectroscopic methods
such as STD-NMR, we planned and synthesised a series of cyclo-
propanecarboxylic acid derivatives that bind to both OASS
fide is the only sulfur source used by OASS-A, OASS-B can use isoforms at nanomolar concentrations, as in the case of (1S,2S)-
CONTACT Marco Pieroni
marco.pieroni@unipr.it
Department of Food and Drug, University of Parma, Parco area delle Scienze 27, Parma, 43124, Italy
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1445
Figure 1. Chemical structure of the previously identified OASS inhibitors.
1–(4-methylbenzyl)-2-phenylcyclopropanecarboxylic acid (2, Figure
1)28. Despite the remarkably high affinity (Kd-OASS ¼ 0.028 lM), this
compound did not show any activity when tested against
Salmonella in vitro. We were able to demonstrate (unpublished
data) that the lack of efficacy was due to the poor permeability of
this derivative through the cell envelope, that in the case of
Gram-negative bacteria poses particular challenges29. Although
there are no simple rules for predicting Gram-negative bacterial
entry, it is known that molecules such as quinolones, that have
negative ClogP and polar character, are able to accumulate in
Gram-negative bacteria, whereas high-size molecules such as mac-
rolides are not active towards Gram-negative because of the big-
ger size30–32. Keeping in mind these considerations, we have tried
to modify the structure of compound 2, that has high potency
but unfavourable physicochemical characteristics (ClogP ¼ 3.60),
with a focused medicinal chemistry campaign. In a first attempt26
we have reported the design and synthesis of a series of cyclopro-
pane-1,2-dicarboxylic acids variously functionalized (3, Figure 1),
with the aim of enhancing the polar character of the molecules;
the carboxylic moiety was maintained, as well as the cyclopropane
scaffold. Unfortunately, the binding properties of these novel ana-
logues could not be improved, and also in this case, the most
active derivative failed to show activity in bacterial cells (data not
shown). Therefore, we focused our attention toward the design
and synthesis of molecules of smaller size and lower ClogP. We
deemed essential to maintain the carboxylic moiety, as its interac-
tions with the OASS active site have shown to be pivotal for
molecular binding. At the same time, we wanted to bypass the
cyclopropane core, which poses synthetic challenges and restric-
tions in particular with regard to the stereochemistry of the deriv-
atives. To this end, we carried out a scaffold hopping approach
leading to the discovery of a novel fragment, which was further
investigated to optimize its affinity toward the target enzyme. The
computational approach leading to this fragment, and the prelim-
inary structure-activity relationships study around it are
herein reported.
Scheme 1. Synthetic scheme to obtain the title compounds 4a–h and 5d–h.
Reagents and Conditions: (a) Me2NCH(OEt)2, CH2Cl2, rt; (b) R-NH2NH2, MeOH,
20–60 ꢀC; (c) NaOH, EtOH, 80 ꢀC.
Synthetic chemistry
With the exception of compounds 14–16, commercially available,
pyrazole derivatives were synthesized according to a protocol
already reported for the preparation of heterocyclic compounds33.
This procedure was deemed of interest because of its lack of regio-
specificity, that allowed us to obtain simultaneously both the
regioisomers, which could be easily isolated with flash chromatog-
raphy. Ethyl (E)-4-(dimethylamino)-2-oxobut-3-enoate 11 was pre-
pared from commercially available ethyl pyruvate 10, that reacted
with dimethylformamide diethyl acetal in dichloromethane at room
temperature (Scheme 1). Reacting 11 with the properly substituted
hydrazine hydrochloride in methanol afforded both the 1-arylpyra-
zole-3-carboxylic acid ethyl esters 12a–h and the 1-arylpyrazole-5-
carboxylic acid ethyl ester isomers 13d–h, which were therefore
hydrolyzed to obtain the desired title compounds 4a–h and 5d–h.
Enaminone 11 exists in solution in CDCl3 as a single isomer, the
trans-orientated nuclei since the magnitude of the coupling constant
(J ¼ 12.5 Hz). As anticipated, cyclocondensation of compound 11
with the properly substituted hydrazine hydrochloride afforded both
the pyrazole carboxylates 12 and the regioisomers 13.
Methods
Scaffold hopping
Compound 2 (Figure 1) was docked on the chain A of Salmonella
crystal structure (pdb code 1OAS) using the LeadIt software.
Protein preparation was performed using Yazara and LeadIT tools
and the amino acid residues Asn70 and Gln143 were flipped while
Thr73 was rotated through a 90ꢀ angle. In order to preserve the
interactions of the carboxylic acid group, the pharmacophore
Gln143 and Thr73 was applied. Ligand preparation was performed
on Chemaxon software, Marvin. Afterward, using the ReCore tool
of LeadIt software and keeping the tolyl and the carboxylic moi-
eties of the original hit, an alternative scaffold to the cyclopropane
of compound 2 was searched in Zinc database.

~
1446
J. MAGALHAES ET AL.
1
Figure 2. Enlargement of the H NMR and HMQC NMR of compounds 12d (a) and 13d (b).
Pyrazole carboxylate 12, which corresponds to the 1,5-isomer, published protocols34 with some modifications. Briefly, protein
is the product obtained in higher yield. The reason of this regiose- expression was induced on bacterial growth in exponential phase
lectivity might be due to the fact that the substituted hydrazine by IPTG for 4 and 5 h for OASS-B and OASS-A, respectively. Cells
attacks the enaminone preferentially through its primary amino were lysed by pulsed sonication in 50 mM NaP, 300 mM NaCl, pH
group instead of the secondary one, as the latter is more hindered 7, with the addition of 1 mg/mL lysozyme. Purification was per-
and, in general, less nucleophilic. Curiously, in only one case formed by immobilized metal affinity column (IMAC) on Co2þ ions
(12a), the cyclocondensation reaction afforded the single 1-aryl- (TalonTM, Clontech Laboratories, Inc., Mountain View, CA). Proteins
pyrazole-5-carboxylate isomer. Although in agreement with the were eluted with 250 mM (His6-OASS-A) or 600 mM (His6-OASS-B)
hypothesis above reported, stereoelectronic reasons might not be imidazol. His-tag was removed from His6-OASS-A by treatment
sufficient to explain this result, since the 4-t-butyl phenylhydrazine with TEV protease during O/N dialysis in 10 mM HEPES pH 8. His-
allows to obtain both the regioisomers. Therefore, to better tag was removed from His6-OASS-B by treatment with TEV prote-
explain the selectivity of the reaction, more information will be ase in 20 mM HEPES, 100 mM NaCl, 1 mM TCEP pH 8 for 24 h at
furnished by the expansion of the series with a higher number of 5 ꢀC. Tag and undigested proteins were removed with batch
substituted hydrazines. The presence and the identification of the IMAC, yielding more than 95% pure protein based on SDS-PAGE
analysis. OASS-A was stored in 10 mM HEPES pH 8 and OASS-B in
two regioisomers (Figure 2) was investigated by NMR analysis
since the proton at Ca in compound 13 is de-shielded compared 20 mM HEPES, 100 mM NaCl, 1 mM TCEP pH 8.
with that in compound 12. Moreover, the 1,3 and 1,5 isomers also
present characteristic13 C spectra where the Ca and Cc present dif-
ferent chemical shields, being Ca more shielded and Cc located
Activity and binding assay
upfield in the case of regioisomer 13 versus regioisomer 12
(Figure 2). In order to refine this conformational analysis, we also
carried out HMQC-NMR experiments (Figure 2).
All of the compounds prepared were then hydrolysed by
means of aqueous sodium hydroxide to afford the carboxylic
group in good yields.
The potency of the compounds was screened at two fixed con-
centrations (1 lM and 1 mM) by a 96-well plate-adapted activity
assay based on the reaction of cysteine with ninhydrin under
acidic conditions28,35. The concentration of bisulfide was saturat-
ing (600 lM), whereas the concentration of OAS was set at Km to
increase the sensitivity. The dissociation constant of selected com-
pounds for StOASS-A and OASS-B was measured by a fluorimetric
method published elsewhere23,24. Briefly, a solution containing
1 mM StOASS-A in 100 mM Hepes pH 7 was titrated with increasing
Protein preparation
The genes cysK and cysM encoding STOASS-A and STOASS-B, concentrations of compound at 20 ꢀC. The fluorescence emission
respectively, were subcloned from pET16b34 to pET19m in order intensity of the PLP cofactor at 505 nm upon excitation at 412 nm
to replace the restriction site for Factor Xa with the one for TEV was collected after each addition, subtracted by the blank and
protease. pET19m-STcysK and pET19m-STcysM were transformed in normalized by the protein dilution. The dependence of the emis-
E. coli RosettaTM (DE3) competent cells for protein expression. sion intensity at 505 nm on the concentration of the compound
Protein expression and purification was performed following was fitted to a binding isotherm23 to calculate the dissociation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1447
constant of the protein-ligand complex. As demonstrated else-
where25,28, the fluorimetric method allows the calculation of the
intrinsic dissociation constant of a competitive inhibitor for the
enzyme and the calculated Kd is in very good agreement with the
IC50 measured by activity assays.
This is due to several considerations. First of all, the analysis of
the key interactions established by compound 4a with OASS back-
bone (Figure 4) anticipates a similar binding pattern as for our
previously reported hits25,27,36
.
After visual inspection, it was possible to envisage also a struc-
tural similarity of 4a when superimposed with some of our previ-
ously reported derivatives bearing a cyclopropane scaffold28, such
as, for instance, compound 9 (Figure 5).
Results and discussion
Moreover, compared to compound 9 and to the most active
derivative compound 2, molecule 4a has a higher total polar sur-
face area (TPSA) and comparable ClogP, anticipating improved
drug-like properties. Finally, we considered the synthetic feasibility
as another advantage, with the possibility to add substituents at
A scaffold hopping using a hit compound previously identified by
us was performed in order to identify a novel inhibitor of OASS to
be further investigated. Our group has already made use of com-
putational tools in order to broaden the body of SAR of
these derivatives36. Among the several scaffolds suggested by the
program (Figure 3), we deemed of particular interest to further the pyrazole core, or to further manipulate the structure with
investigate hit compound 4a, which was characterised by an
N-substituted pyrazole central spacer.
other heterocycles. This compound was thus synthesized and eval-
uated for its capability to bind and inhibit the enzymatic activity
for the two OASS isoforms at two concentrations. At 1 mM com-
pound 4a showed a 50% inhibition of OASS-A activity and a simi-
lar value was found also for OASS-B (Table 1). Although not
remarkable, this result was considered worth of further investiga-
tion, and prompted us to prepare a number of derivatives. As a
preliminary analysis, we wanted to check which was the most
Figure 5. Chemical structure of the previously reported OASS inhibitors 1, 9 and
compound 4a.
Figure 3. Putative hits obtained after scaffold hopping of the previously identi-
fied compound 2.
Figure 4. Docking results of compound 4a into StOASS-A binding site. (a) StOASS-A (white cartoon and sticks) in complex with compound 9 (orange sticks) and 4a
(cyan sticks); (b) StOASS-A active site (white cartoon and sticks) in complex with compound 9 (orange sticks) and 4a (cyan sticks); (c) alignment of the binding mode
of 9, 4a and 14 highlighting how the compounds accommodate the pharmacophoric features in similar directions; (d) bi-dimensional representation of interactions of
StOASS-A with the cyclopropane scaffold (e) and with compound 4a (c).

~
J. MAGALHAES ET AL.
1448
favourable attachment point for the phenyl ring (N-1 or N-2), introduced at the phenyl ring, as in this case the 1,3-isomer
whether the aromatic ring could be adorned with small electron- resulted twofold more affine to the target enzyme than the corre-
withdrawing groups (EWG) or electron-donating groups sponding 1,5-isomer (4f vs 5f, Table 1). A smaller EDG such as the
(EDG), whether the aromatic ring could be substituted with a methyl does not affect the activity (4f vs 4a, Table 1).
cycloaliphatic structure and, finally, whether the small fragment- Interestingly, substitution of the aromatic ring with a cyclohexane
like pyrazolecarboxylic acid alone was active. First, we investigated is still tolerated, with a percentage of inhibition similar to that of
the 1-arylpyrazole-3-carboxylic acid and the 1-arylpyrazole-5-car- the derivatives bearing an aromatic substituent (see compounds
boxylic acid regioisomers, with the phenyl ring either unsubsti- 4 h and 5h).
tuted or adorned with small functional groups. We took
Despite the range of activity allowed only a rough SAR, these
advantage from a straight synthetic protocol based on a non- results somehow confirmed the presence of a lipophilic pocket in
regiospecific reaction, which allowed the isolation of the two iso- the proximity of the enzyme active site where the pyrazole sub-
mers starting from common starting materials (see Materials and stituent can locate, as previously noticed by us27,36,37. In addition,
Methods section). We then checked the effect of a substituent at the activity shown by compounds 4e and 5f demonstrated that
the phenyl ring, which was arbitrarily introduced at the para pos- the size of the molecule may have some influence on the binding
ition in this preliminary round of modifications. In the cases of to the enzyme pocket. Since these considerations, we were very
small EWGs attached at the phenyl ring, not any difference in the surprised that the 1H-pyrazole-5-carboxylic acid (4c) was found to
potency could be noticed for compounds 4d vs 5d and 4 g vs 5 g be the most potent fragment-like hit of the series, with more than
(Table 1). A bulkier substituent such as the bromine remarkably 90% inhibition at 1 mM and an experimental Kd of 122 lM to
favours the 1,5-isomer compared to the 1,3-isomer (4e vs 5e, OASS-A and a Kd of 272 lM towards OASS-B (Table 2). It must be
Table 1), giving a > 80% inhibition on OASS-A at 1 mM. An oppos- remembered that, as we have already demonstrated,28, in case of
ite pattern is noticed when bulky EDGs such as the tert-Butyl are competitive inhibitors such as the compounds reported, Kd corres-
pond to the Ki. These findings lead to speculate that, although
the presence of the lipophilic pocket has been predicted by com-
putational studies and confirmed by the small set of compounds
reported in this paper; however, its filling with lipophilic moieties
such as aromatic rings seems to be detrimental for the binding.
Further investigation is needed to better select the proper moi-
eties that can interact more efficiently with this side-pocket.
Along with the interesting binding properties, compound 4c
possessed chemical highly desirable characteristics known to be
beneficial for Gram-negative penetration, such as the low ClogP,
high TPSA and the reduced size31,32. Further analysis of the inter-
actions established between this compound and OASS backbone
encouraged us to explore other five-membered heterocycles func-
tionalized with a carboxylic acid functional group, as it was con-
sidered crucial for the interaction at the enzyme active site
(14–16, Table 2).
Table 1. Inhibitory potency of pyrazoles on the activity of recombinant OASS
from Salmonella.
% inhibition OASS-Ac
1 mM 1 mM
nsb
% inhibition OASS-Bc
1 mM 1 mM
ns
Cpda
4a
4b
4c
4d
4e
4f
4g
4h
5d
5e
5f
45
66
94
60
84
47
95
57
63
62
81
73
79
3
1
1
2
1
1
1
1
1
1
1
1
1
65
65
82
56
58
52
62
30
63
35
82
36
43
2
1
1
1
2
5
1
1
1
2
1
3
3
ns
11
ns
3
9
7
ns
ns
ns
9 6
ns
ns
8
1
14
ns
15
ns
11
ns
ns
2
2
6
ns
ns
ns
ns
ns
ns
5g
5h
Surprisingly, all of the compounds tested were found to display
interesting binding properties to StOASS-A, with a noticeable
improvement in the affinity compared to the hit compound
aFor structures see Scheme 1.
bNot Significant.
cAll assays were performed in two replicates.
Table 2. Overall properties of compounds 2 and 4a and dissociation constant plus ligand efficiency of compounds 13–15 determined for StOASS.
Cpd
Structure
MW (g/mol)
266.33
Kd OASS-A (mM)
Kd OASS-B (mM)
LE (OASS-A)b kcal/mol/HA
LE (OASS-B)b kcal/mol/HA
ClogPc
3.60
TPSAc
37.30
2
0.03a
0.049
0.52
0.50
4c
14
112.09
111.1
122
5
272 10
465 50
0.67
0.72
0.61
0.60
0.13
0.66
65.98
53.09
59 11
15
16
113.07
129.14
120 12
548 59
338 14
0.67
0.74
0.60
0.60
0.04
0.17
63.33
50.19
52
6
aWhen SD is not reported, it means that figures are meaningless due to rounding of the decimals.
bLE ¼ (1.37/HA). pIC50 where HA refers to heavy atoms.
cClogP and TPSA were calculated using molinspiration website (http://www.molinspiration.com/).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1449
derived from scaffold hopping (4a), and also better than fragment saturated solution of NaHCO3 and then extracted with chloroform.
4c (Table 2).
The activity shown by the 1H-pyrazole-5-carboxylic acid deriva-
The combined organic layers were washed with brine and dried
over anhydrous magnesium sulphate, filtrated and concentrated
under reduced pressure.
tives is considerably lower than that reported by the most active
derivative 2 used as the reference compound. However, it must
be considered that both the molecules have a similar value of lig-
and efficiency (LE). LE, calculated as described by Chen et al.38, is
a parameter that serves as a valuable metric to analyse the bind-
ing of small molecules and fragment-like compounds and to
assess the viability of a fragment as a starting point for optimisa-
tion. Therefore, together with other favourable drug-like character-
istics39–42, the good LE of compounds 14 and 16 presents these
fragment-like derivatives as suitable candidates to further explore
in the search of novel OASS inhibitors to be used as antibacter-
ial adjuvants.
Ethyl 1-(p-tolyl)-1H-pyrazole-5-carboxylate (Compound 12a)
The product was purified by flash chromatography on silica gel
using 3% ethyl acetate in petroleum ether. The product was
1
obtained as a light brown powder in 48% yield. H NMR (300 MHz,
Chloroform-d) d 7.67 (d, J ¼ 1.9 Hz, 1H), 7.39–7.18 (m, 4H), 7.00 (d,
J ¼ 1.9 Hz, 1H), 4.24 (q, J ¼ 7.1 Hz, 2H), 2.42 (s, 3H), 1.26 (t,
J ¼ 7.1 Hz, 3H).
Ethyl 1-phenyl-1H-pyrazole-5-carboxylate (Compound 12b)
Purification by flash column chromatography on silica gel using
0.2% triethylamine and 2% ethyl acetate in petroleum ether
afforded the isolation of desired product as a yellow oil in 43%
yield. 1H NMR (300 MHz, Chloroform-d) d 7.69 (d, J ¼ 2.0 Hz, 1H),
7.49–7.36 (m, 5H), 7.03 (d, J ¼ 2.0 Hz, 1H), 4.24 (q, J ¼ 7.1 Hz, 2H),
1.24 (t, J ¼ 7.1 Hz, 3H).
Experimental part
General information
All the reagents and some final compounds (14–16) were pur-
chased from Sigma-Aldrich, Alfa-Aesar, and TCI at reagent purity
and, unless stated otherwise, were used without further purifica-
tion. Reactions were monitored by thin-layer chromatography on
silica gel-coated aluminium foils (silica gel on Al foils, SUPELCO Ethyl 1H-pyrazole-5-carboxylate (Compound 12c)
Analytical, Sigma-Aldrich) at 254 and 365 nm. Where indicated, The product was obtained as a dark brown solid in 98% yield. H
1
intermediates and final products were purified by silica gel flash
NMR (300 MHz, Chloroform-d) d 7.75 (d, J ¼ 9.9 Hz, 1H), 6.86 (d,
chromatography (silica gel, 0.040ꢁ0.063 mm), using appropriate
solvent mixtures. 1H NMR and13 C NMR spectra were recorded on
a BRUKER AVANCE spectrometer at 300, 400 and 100 MHz,
respectively, with TMS as internal standard. 1H NMR spectra are
reported in this order: multiplicity and number of protons.
Standard abbreviations indicating the multiplicity were used as
follows: s ¼ singlet, d ¼ doublet, dd ¼ doublet of doublets,
J ¼ 2.1 Hz, 1H), 4.42 (qd, J ¼ 7.1, 1.0 Hz, 2H), 1.41 (t, J ¼ 7.1 Hz, 3H).
Ethyl 1–(4-chlorophenyl)-1H-pyrazole-5-carboxylate
(Compound 12d)
The product was purified by flash chromatography using 2% ethyl
acetate in petroleum ether and it was obtained as a light yellow
t ¼ triplet, q ¼ quadruplet, m ¼ multiplet, and br ¼ broad signal. powder in 23% yield. 1H NMR (400 MHz, Chloroform-d) d 7.69 (d,
HPLC/MS experiments were performed with an Agilent 1100 series
J ¼ 2.0 Hz, 1H), 7.45–7.41 (m, 2H), 7.40–7.36 (m, 2H), 7.03 (d,
HPLC apparatus, equipped with a Waters Symmetry C18, 3.5 lm,
J ¼ 2.0 Hz, 1H), 4.26 (q, J ¼ 7.1 Hz, 2H), 1.28 (t, J ¼ 7.1 Hz, 3H).
4.6 mm ꢂ 75 mm column and an MS: Applied Biosystem/MDS
SCIEX instrument, with API 150EX ion source. All compounds were
Ethyl 1–(4-chlorophenyl)-1H-pyrazole-3-carboxylate
(Compound 13d)
tested as 95% purity or higher (by HPLC/MS).
The product was obtained as a light yellow powder in 10% yield.
1H NMR (400 MHz, cdcl3) d 7.90 (d, J ¼ 2.5 Hz, 1H), 7.70 (d,
J ¼ 9.0 Hz, 2H), 7.55–7.40 (m, 2H), 6.99 (d, J ¼ 2.5 Hz, 1H), 4.44 (q,
J ¼ 7.1 Hz, 2H), 1.42 (t, J ¼ 7.1 Hz, 3H).
Ethyl (E)-4-(dimethylamino)-2-oxobut-3-enoate (11)
Ethyl pyruvate (17.22 mmol, 2g) was solubilised in dichlorome-
thane (34 ml) and to the previous solution was added dropwise
dimethylformamide dimethyl acetal (17.22 mmol, 2g). Reaction
mixture was stirred at room temperature for 4 h and after the vol-
atiles were evaporated under reduced pressure to obtain a dark Ethyl 1–(4-bromophenyl)-1H-pyrazole-5-carboxylate
brown oil that was purified by flash column chromatography
using 1% MeOH in dichloromethane to obtain the product as a
brown yellowish oil in 54% yield. H NMR (300 MHz, Chloroform-d)
d 7.83 (d, J ¼ 12.5 Hz, 1H), 5.81 (d, J ¼ 12.5 Hz, 1H), 4.30 (qd,
J ¼ 7.1, 0.9 Hz, 2H), 3.18 (s, 3H), 2.95 (s, 3H), 1.36 (td, J ¼ 7.1,
0.9 Hz, 3H).
(Compound 12e)
The product was purified by flash column chromatography on sil-
ica gel using 1.7% triethylamine and 3.4% of ethyl acetate in pet-
roleum ether. The product was obtained as light yellow powder in
35% yield. 1H NMR (300 MHz, Chloroform-d) d 7.69 (d, J ¼ 2.0 Hz,
1H), 7.64–7.53 (m, 2H), 7.38–7.26 (m, 2H), 7.03 (d, J ¼ 2.0 Hz, 1H),
4.26 (q, J ¼ 7.2 Hz, 2H), 1.28 (t, J ¼ 7.1 Hz, 3H).
1
General procedure for the synthesis of ethyl 1R-pyrazole-5-
carboxylate
Ethyl 1–(4-bromophenyl)-1H-pyrazole-3-carboxylate
(Compound 13e)
To a solution of ethyl (E)-4-(dimethylamino)-2-oxobut-3-enoate
(130 mg, 0.76 mmol) in methanol (580 ll) was added the properly
substituted hydrazine (0.76 mmol). Reaction mixture was stirred at
60 ꢀC for 6 h. The volatiles were evaporated under reduced pres-
1
The product was obtained as light yellow powder in 23% yield. H
NMR (300 MHz, Chloroform-d) d 7.91 (dd, J ¼ 2.5, 0.6 Hz, 1H),
7.70–7.51 (m, 4H), 7.00 (dd, J ¼ 2.6, 0.6 Hz, 1H), 4.59–4.34 (m, 2H),
sure and the residue was solubilized in chloroform, treated with a 1.42 (td, J ¼ 7.1, 0.6 Hz, 3H).

~
J. MAGALHAES ET AL.
1450
Ethyl 1–(4-(tert-butyl)phenyl)-1H-pyrazole-5-carboxylate
(Compound 12f)
1-(p-tolyl)-1H-pyrazole-5-carboxylic acid (Compound 4a)
The product was obtained as a white solid in 66% yield. 1H NMR
(300 MHz, Methanol-d4) d 7.70 (d, J ¼ 2.0 Hz, 1H), 7.29 (s, 4H), 7.04
(d, J ¼ 2.0 Hz, 1H), 2.43 (s, 3H).13 C NMR (101 MHz, Methanol-d4) d
161.70, 140.44, 140.01, 139.29, 135.67, 130.15, 126.95, 113.42,
21.17. HRMS (ESI): calculated for C11H9O2N2 [M-H] 201.0670
found 201.06691.
The product was purified by flash column chromatography on sil-
ica gel using 0.2% triethylamine and 2% ethyl acetate in petrol-
eum ether as eluent. The product was obtained as a yellow oil in
42% yield. 1H NMR (300 MHz, Chloroform-d) d 7.67 (d, J ¼ 2.0 Hz,
1H), 7.51–7.41 (m, 2H), 7.41–7.29 (m, 2H), 7.01 (d, J ¼ 2.0 Hz, 1H),
4.24 (q, J ¼ 7.2 Hz, 2H), 1.36 (s, 9H), 1.24 (t, J ¼ 7.1 Hz, 3H).
1-phenyl-1H-pyrazole-5-carboxylic acid (Compound 4b)
The product was obtained as a yellow powder in 78% yield. 1H
NMR (300 MHz, Methanol-d4) d 7.67 (d, J ¼ 2.0 Hz, 1H), 7.50–7.33
(m, 5H), 7.02 (d, J ¼ 2.0 Hz, 1H).13 C NMR (101 MHz, Methanol-d4) d
161.62, 141.73, 140.64, 135.65, 129.74, 129.65, 127.16, 113.60.
HRMS (ESI): calculated for C10H7O2N2 [M-H] 187.0513
found 187.05122.
Ethyl 1–(4-(tert-butyl)phenyl)-1H-pyrazole-3-carboxylate
(Compound 13f)
The product was obtained as a yellow oil in 46% yield. 1H NMR
(300 MHz, Chloroform-d)
d
7.90 (d, J ¼ 2.5 Hz, 1H), 7.66 (d,
J ¼ 8.9 Hz, 2H), 7.47 (d, J ¼ 8.8 Hz, 2H), 6.98 (d, J ¼ 2.5 Hz, 1H), 4.44
(q, J ¼ 7.1 Hz, 2H), 1.42 (t, J ¼ 7.1 Hz, 3H), 1.35 (s, 9H).
1H-pyrazole-5-carboxylic acid (Compound 4c)
The product was obtained as a pearl solid in 40% yield. 1H NMR
(300 MHz, Methanol-d4) d 7.69 (d, J ¼ 2.6 Hz, 1H), 6.81 (d, J ¼ 2.7 Hz,
1H).13 C NMR (101 MHz, Methanol-d4) d 164.62, 142.92, 133.87,
109.03. HRMS (ESI): calculated for C4H3O2N2 [M-H] 111.0200
found 111.0200.
Ethyl 1–(4-fluorophenyl)-1H-pyrazole-5-carboxylate
(Compound 12g)
The product was purified by flash column chromatography on sil-
ica gel using 0.2% triethylamine and 2% ethyl acetate in petrol-
eum ether. The product was obtained as a yellow powder in 47%
yield. 1H NMR (300 MHz, Chloroform-d) d 7.68 (d, J ¼ 2.0 Hz, 1H),
7.50–7.34 (m, 2H), 7.21–7.07 (m, 2H), 7.02 (d, J ¼ 2.0 Hz, 1H), 4.25
(q, J ¼ 7.1 Hz, 2H), 1.27 (t, J ¼ 7.1 Hz, 3H).
1–(4-chlorophenyl)-1H-pyrazole-5-carboxylic acid (Compound 4d)
The product was obtained as a white powder in 77% yield. 1H
NMR (300 MHz, Methanol-d4) d 7.72 (d, J ¼ 2.0 Hz, 1H), 7.54–7.37
(m, 4H), 7.05 (d, J ¼ 2.0 Hz, 1H).13 C NMR (101 MHz, Methanol-d4) d
161.74, 142.78, 140.98, 140.42, 135.40, 129.72, 128.69, 113.74.
HRMS (ESI): calculated for C10H6ClO2N2 [M-H] 221.0123
found 221.01221.
Ethyl 1–(4-fluorophenyl)-1H-pyrazole-3-carboxylate
(Compound 13g)
The product was obtained as a yellow powder in 20% yield. 1H
NMR (300 MHz, Chloroform-d) d 7.87 (d, J ¼ 2.5 Hz, 1H), 7.82–7.62
(m, 2H), 7.23–7.10 (m, 2H), 6.99 (d, J ¼ 2.5 Hz, 1H), 4.44 (q,
J ¼ 7.1 Hz, 2H), 1.42 (t, J ¼ 7.1 Hz, 3H).
1–(4-chlorophenyl)-1H-pyrazole-3-carboxylic acid (Compound 5d)
The product was obtained as a white powder in 87% yield. 1H
NMR (300 MHz, Methanol-d4) d 8.32 (d, J ¼ 2.5 Hz, 1H), 7.94–7.77
(m, 2H), 7.62–7.43 (m, 2H), 6.99 (d, J ¼ 2.5 Hz, 1H).13 C NMR
(101 MHz, Methanol-d4) d 165.07, 146.75, 139.71, 134.20, 130.72,
130.63, 122.22, 111.38. HRMS (ESI): calculated for C10H6ClO2N2 [M-
H] 221.0123 found 221.01237.
Ethyl 1-cyclohexyl-1H-pyrazole-5-carboxylate (Compound 12h)
Purification by flash column chromatography on silica gel using
0.2% triethylamine and 2% ethyl acetate in petroleum ether
allowed the isolation of the desired product in 39% yield as a col-
orless oil. 1H NMR (300 MHz, Methanol-d4) d 7.52 (d, J ¼ 2.0 Hz,
1H), 6.86 (d, J ¼ 2.0 Hz, 1H), 5.16 (dq, J ¼ 10.4, 5.4, 4.1 Hz, 1H), 4.36
(q, J ¼ 7.1 Hz, 2H), 2.09–1.69 (m, 7H), 1.58–1.15 (m, 6H).
1–(4-bromophenyl)-1H-pyrazole-5-carboxylic acid (Compound 4e)
The product was obtained as a light yellow powder in 91% yield.
1H NMR (300 MHz, Methanol-d4)
d
7.73 (d, J ¼ 2.0 Hz, 1H),
7.69–7.58 (m, 2H), 7.42–7.30 (m, 2H), 7.07 (d, J ¼ 2.0 Hz, 1H).13
C
NMR (101 MHz, Methanol-d4) d 161.48, 141.03, 140.85, 135.66,
132.77, 128.99, 123.32, 113.93. HRMS (ESI): calculated for
C10H6BrO2N2 [M-H] 264.9618 found 264.96170.
Ethyl 1-cyclohexyl-1H-pyrazole-3-carboxylate (Compound 13h)
The product was obtained as a yellow oil in 39% yield. 1H NMR
(300 MHz, Methanol-d4) d 7.75 (d, J ¼ 2.5 Hz, 1H), 6.78 (d, J ¼ 2.4 Hz,
1H), 4.37 (q, J ¼ 7.2 Hz, 2H), 4.32–4.09 (m, 1H), 2.12 (d, J ¼ 14.1 Hz,
2H), 1.99–1.63 (m, 8H), 1.39 (t, J ¼ 7.2 Hz, 3H).
1–(4-bromophenyl)-1H-pyrazole-3-carboxylic acid (Compound 5e)
The product was obtained as a light yellow powder in 73%
yield.1H NMR (300 MHz, Methanol-d4) d 8.32 (d, J ¼ 2.6 Hz, 1H),
7.84–7.75 (m, 2H), 7.71–7.63 (m, 2H), 6.99 (d, J ¼ 2.6 Hz, 1H).13
C
General procedure for the synthesis of
1-R-pyrazole-5-carboxylic acid
NMR (101 MHz, Methanol-d4) d 165.07, 146.79, 140.17, 133.75,
130.59, 122.47, 121.91, 111.41. HRMS (ESI): calculated for
C10H6BrO2N2 [M-H] 264.9618 found 264.96161.
To
a solution of the proper ethyl 1R-pyrazole-5-carboxylate
(0.203 mmol) solubilized in ethanol (0.6 ml) was added sodium
hydroxide 6 M (36 ll, 1.2 mmol). Reaction mixture was stirred at
80 C for 2 h. The solvent was evaporated under reduced pressure, 1–(4-(tert-butyl)phenyl)-1H-pyrazole-5-carboxylic acid
water was added and the residue acidified with HCl 1 M until pH
1–2. The precipitate was filtrated and washed with petrol-
eum ether.
(Compound 4f)
The product was obtained as a cream powder in 47% yield. 1H
NMR (300 MHz, Methanol-d4) d 7.70 (d, J ¼ 2.1 Hz, 1H), 7.58–7.47

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1451
(m, 2H), 7.40–7.27 (m, 2H), 7.02 (d, J ¼ 2.1 Hz, 1H), 1.38 (s, 9H).13
C
methodologies with a ligand-based medicinal chemistry approach.
NMR (101 MHz, Methanol-d4) d 162.45, 153.17, 140.64, 139.45, Although the small set of compounds tested against recombinant
136.48, 126.78, 113.34, 111.64, 35.82, 31.95. HRMS (ESI): calculated
for C14H15O2N2 [M-H] 243.1139 found 243.11357.
enzyme from Salmonella, we successfully identified two small frag-
ments (1H-pyrrole-2-carboxylic acid and thiazole-2-carboxylic acid)
with promising StOASS inhibitory characteristics, which will serve
as chemical templates for improved cysteine biosynthesis blockers.
In addition to these promising biological characteristics, it must
be considered that these heterocyclic carboxylic acids have physi-
cochemical properties that indicate potential for penetration
through the Gram-negative bacterial cell wall, and are endowed
with considerable drug-like characteristics, as they do not show
any violation of the four physicochemical characteristics defined
by the Lipinski rule of five. The chemical manipulation and bio-
logical evaluation in cell of these inhibitors are currently ongoing
in our laboratories.
1–(4-(tert-butyl)phenyl)-1H-pyrazole-3-carboxylic acid
(Compound 5f)
The product was obtained as a cream powder in 64% yield. 1H
NMR (300 MHz, Methanol-d4) d 8.19 (d, J ¼ 2.5 Hz, 1H), 7.82–7.64
(m, 2H), 7.62–7.44 (m, 2H), 6.91 (d, J ¼ 2.4 Hz, 1H), 1.36 (d,
J ¼ 2.3 Hz, 9H).13 C NMR (101 MHz, Methanol-d4) d 167.56, 151.63,
149.16, 138.90, 129.94, 127.44, 120.42, 110.52, 35.45, 31.70. HRMS
(ESI): calculated for C14H15O2N2 [M-H] 243.1139 found 243.11377.
1–(4-fluorophenyl)-1H-pyrazole-5-carboxylic acid (Compound 4g)
The product was obtained as a yellow powder in 93% yield. 1H
NMR (300 MHz, Methanol-d4) d 7.67 (d, J ¼ 2.0 Hz, 1H), 7.47–7.34
(m, 2H), 7.24–7.10 (m, 2H), 7.02 (d, J ¼ 2.0 Hz, 1H).13 C NMR
(101 MHz, Methanol-d4) d 165.16, 161.49, 140.73, 137.94, 135.77,
129.37, 129.28, 116.47, 116.24, 113.65. HRMS (ESI): calculated for
C10H6FO2N2 [M-H] 205.0419 found 205.04185.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was funded under the MSCA-ITN-2014-ETN project
INTEGRATE [grant number 642620].
1–(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid (Compound 5g)
The product was obtained as a yellow powder in 36% yield. 1H
NMR (400 MHz, Methanol-d4) d 8.25 (d, J ¼ 2.6 Hz, 1H), 7.90–7.76
(m, 2H), 7.31–7.18 (m, 2H), 6.96 (d, J ¼ 2.5 Hz, 1H).13C NMR
(101 MHz, Methanol-d4) d 164.44, 161.99, 146.52, 137.49, 137.46,
130.78, 123.02, 122.93, 117.44, 117.21, 111.23. HRMS (ESI): calcu-
lated for C10H6FO2N2 [M-H] 205.0419 found 205.04185.
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