Novel quinazolinone inhibitors of the Pseudomonas aeruginosa quorum sensing transcriptional regulator PqsR

Article abstract of DOI:10.1016/j.ejmech.2020.112778

Rising numbers of cases of multidrug- and extensively drug-resistant Pseudomonas aeruginosa over recent years have created an urgent need for novel therapeutic approaches to cure potentially fatal infections. One such approach is virulence attenuation where anti-virulence compounds, designed to reduce pathogenicity without affording bactericidal effects, are employed to treat infections. P. aeruginosa uses the pqs quorum sensing (QS) system, to coordinate the expression of a large number of virulence determinants as well as bacterial-host interactions and hence represents an excellent anti-virulence target. We report the synthesis and identification of a new series of thiazole-containing quinazolinones capable of inhibiting PqsR, the transcriptional regulator of the pqs QS system. The compounds demonstrated high potency (IC₅₀ < 300 nM) in a whole-cell assay, using a mCTX:P_pqsA-lux-based bioreporter for the P. aeruginosa PAO1-L and PA14 strains. Structural evaluation defined the binding modes of four analogues in the ligand-binding domain of PqsR through X-ray crystallography. Further work showed the ability of 6-chloro-3((2-pentylthiazol-4-yl)methyl)quinazolin-4(3H)-one (18) and 6-chloro-3((2-hexylthiazol-4-yl)methyl)quinazolin-4(3H)-one (19) to attenuate production of the PqsR-regulated virulence factor pyocyanin. Compounds 18 and 19 showed a low cytotoxic profile in the A549 human epithelial lung cell line making them suitable candidates for further pre-clinical evaluation.

Full text of DOI:10.1016/j.ejmech.2020.112778

European Journal of Medicinal Chemistry 208 (2020) 112778
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel quinazolinone inhibitors of the Pseudomonas aeruginosa
quorum sensing transcriptional regulator PqsR
,
, c,
Scott Grossman a 1, Fadi Soukarieh b 1, William Richardson a, Ruiling Liu a,
Alaa Mashabi a, Jonas Emsley a c, Paul Williams b , Miguel Camara
,
,
,
c
b, c
ꢀ
Michael J. Stocks a
, c, *
a School of Pharmacy, University of Nottingham Biodiscovery Institute, University Park, Nottingham, Nottinghamshire, NG7 2RD, UK
b School of Life Sciences, University of Nottingham Biodiscovery Institute, University Park, Nottingham, Nottinghamshire, NG7 2RD, UK
c National Biofilms Innovation Centre, University of Nottingham Biodiscovery Institute, University Park, Nottingham, Nottinghamshire, NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Rising numbers of cases of multidrug- and extensively drug-resistant Pseudomonas aeruginosa over
recent years have created an urgent need for novel therapeutic approaches to cure potentially fatal in-
fections. One such approach is virulence attenuation where anti-virulence compounds, designed to
reduce pathogenicity without affording bactericidal effects, are employed to treat infections.
P. aeruginosa uses the pqs quorum sensing (QS) system, to coordinate the expression of a large number of
virulence determinants as well as bacterial-host interactions and hence represents an excellent anti-
virulence target.
Received 6 March 2020
Received in revised form
7 August 2020
Accepted 20 August 2020
Available online 28 August 2020
Keywords:
We report the synthesis and identification of a new series of thiazole-containing quinazolinones
capable of inhibiting PqsR, the transcriptional regulator of the pqs QS system. The compounds demon-
strated high potency (IC50 < 300 nM) in a whole-cell assay, using a mCTX:PpqsA-lux-based bioreporter for
the P. aeruginosa PAO1-L and PA14 strains. Structural evaluation defined the binding modes of four an-
alogues in the ligand-binding domain of PqsR through X-ray crystallography. Further work showed the
ability of 6-chloro-3((2-pentylthiazol-4-yl)methyl)quinazolin-4(3H)-one (18) and 6-chloro-3((2-
hexylthiazol-4-yl)methyl)quinazolin-4(3H)-one (19) to attenuate production of the PqsR-regulated
virulence factor pyocyanin. Compounds 18 and 19 showed a low cytotoxic profile in the A549 human
epithelial lung cell line making them suitable candidates for further pre-clinical evaluation.
© 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Pseudomonas aeruginosa
Quorum sensing
Inhibitors
X-ray crystal structure
PqsR
1. Introduction
strategies are required to provide long-lasting solutions, which
drastically reduce the rate of emergence of resistance due to the
Pseudomonas aeruginosa (PA), a Gram-negative pathogenic
bacterium found widely in nature, is a common cause of infection in
immunocompromised patients. Chronic and recurring infections
are widespread, and PA infection is frequently associated with
respiratory failure, reduced pulmonary function and mortality
within cystic fibrosis patients [1e3]. A rise in multidrug resistant
cases of PA has raised this organism to a priority class pathogen of
critical importance by the World Health Organisation [4].
high selective pressure posed by current antibiotic treatments. One
such approach is the use of alternative treatments which can
attenuate virulence within bacterial populations without directly
killing the infectious organisms and/or sensitise these populations
to the action of existing antibiotics [5,6]. The net result is a non-
pathogenic population which can be cleared by the immune sys-
tem or which may become sensitive to existing antimicrobials
[6e9].
In order to combat the threat of antimicrobial resistance, novel
Extensive work associated with this approach has investigated
inhibition of quorum sensing systems within bacterial populations.
Quorum sensing (QS) is a cell-to-cell communication strategy used
by microbes to coordinate the production of numerous traits
including virulence factors in a population-dependent manner. QS
relies on the production and sensing of small diffusible signal
* Corresponding author. School of Pharmacy, University of Nottingham Bio-
discovery Institute, University Park, Nottingham, Nottinghamshire, NG7 2RD, UK.
E-mail address: michael.stocks@nottingham.ac.uk (M.J. Stocks).
1
These authors have contributed equally.
https://doi.org/10.1016/j.ejmech.2020.112778
0223-5234/© 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

2
S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
molecules known as autoinducers (AIs) or QS signal molecules
(QSSMs) [10e12]. Although most commonly observed as an intra-
species event, QS is also seen at the interspecies and inter-kingdom
level through bacteria-bacteria communication systems as well as
in bacteria-fungi mixed populations [13e15].
University of Nottingham Managed Chemical Compound Collection
(MCCC, a collection of 85,000 diverse compounds) gave several hit
compounds bearing this structural motif. One such compound, 5
(Fig. 2), containing a thiazole group proved interesting for further
structure activity relationship (SAR) exploration.
During infection QS in PA controls the production of virulence
traits such as the phenazine, pyocyanin and the siderophore pyo-
verdine, which are capable of cytotoxic effects against mammalian
cells and iron scavenging respectively, as well as those responsible
for bacterial motility and biofilm formation [16e22].
Initial testing of
13.2 2.73 M in a mCTX-PpqsA-lux luminescence based bioreporter
assay. Further optimisation yielded 6, with an improved activity of
1.0 0.42 M. Furthermore, homologous oxadiazole 7 was found to
be inactive at 10 M, providing evidence for the importance of the
5 showed an activity for PAO1-L of
m
m
m
PA utilises three closely interlinked QS systems to fully elicit its
pathogenicity; the las and rhl systems which operate via N-acylho-
moserine lactones as their QSSMs (1, 2), whereas the pqs system uses
alkylquinolones (AQ), namely 2-heptylquinolin-4(1H)-one (HHQ, 3)
and the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4(1H)-
quinolone, PQS, 4) as their cognate signals (Fig. 1) [7,11,23e25].
The pqs QS system of PA requires the pqsABCDE operon for the
biosynthesis of HHQ 3, the precursor of PQS 4, though the pqsE gene
product is also known to play both a biosynthetic and a key regu-
latory role in the regulation of multiple virulence factors [26e31].
HHQ is converted to PQS via the monooxygenase PqsH. The
expression of the pqsABCDE operon is controlled by the LysR-type
transcriptional regulator PqsR upon binding its cognate ligands,
PQS and HHQ [32,33].
Inhibition of the pqs system can result in a reduction in the
production of pyocyanin and alkylquinolones as well as many other
virulence traits [32,34e37]. As such, PqsR has been validated as a
target for the inhibition of the pqs system and hence virulence using
both in vitro assays and in vivo in mouse infection models [37].
Hereinwe report the discovery of a new series of compounds with
high potency against PqsR in two different PA strains (PAO1-L and
PA14) that represent the two major genomic groups. Binding of
compounds 6, 12, 18 and 19 to this regulatory protein is shown
through co-crystallisation into the ligand-binding domain (LBD) of
PqsR [32,36]. The lead compounds (18, 19) were shown to attenuate
pyocyanin production and demonstrated very low cytotoxicity
against mammalian cells, supporting their suitability for further
studies.
thiazole ring for inhibiting PqsR. As such, a SAR study was con-
ducted around 6, primarily focussing on 2-substitution of the
thiazole.
We envisaged that variation to the isopropyl group of compound
6 was the most promising area to explore with regards to
improving potency. A range of alternate functionalities varying
from alkyl chains to substituted amines and small aromatic groups
provided a diverse set of compounds to explore the space available
within the LBD of the PqsR protein where these ligands were
believed to bind (Fig. 3).
A four-step synthetic procedure (Scheme 1) provided a robust
route to the desired 2,4-disubstituted thiazoles bearing alkyl,
amino and aryl functionalities. Initially, 2-amino-5-chlorobenzoic
acid was condensed in formamide to give the corresponding 6-
chloroquinazolin-4(3H)-one 36. Reaction with chloroacetone gave
the intermediate 37, which was brominated through refluxing in
acetic acid with bromine to afford the a-bromoketone 38. Thiazole
formation in ethanol with a range of thioamides gave the 2,4-
disubstituted thiazoles 6, 8e26.
In addition to this series of 2,4-substituted thiazoles (6, 8e26), a
2,5-substituted thiazole 35 analogous to 6 was synthesised to
observe whether altering the orientation of the thiazole ring could
improve efficacy, as was purported through a ligand docking screen
in silico. Furthermore, in a bid to decrease lipophilicity, a range of 2-
amino-5-substituted thiazoles were synthesised in a two-step
route from 6-chloroquinazolin-4(3H)-one (36).
Synthesis of the 5-substituted thiazole 35 required an alternate
four-step synthetic route (Scheme 2). Initially, bromomalonalde-
hyde was condensed with 2-methylpropanethioamide, to give 2-
isopropylthiazole-5-carbaldehyde, which was immediately
treated with hydroxylamine hydrochloride affording 2-
isopropylthiazole-5-carbaldehyde oxime (39). Reduction of the
oxime using zinc powder and hydrochloric acid gave the primary
amine (40).
2. Results and discussion
2.1. SAR-based design and synthesis of PqsR inhibitors
Previous research has shown that quinazolinone scaffolds can
provide the basis for PqsR antagonists, in part due to the similarity
this core shares with the endogenous ligands PQS and HHQ [32].
Therefore, it was unsurprising that an in silico screen of the
Separately, 2-amino-5-chlorobenzoic acid was treated with
DMFDMA to give methyl 5-chloro-2-(((dimethylamino)methylene)
amino)benzoate (41) [38]. Cyclisation under acidic conditions with
Fig. 1. Known QSSMs in P. aeruginosa include N-acylhomoserine lactones which regulate 1 the rhl and 2 las systems, and 3, 4 HHQ and PQS which regulate the pqs system.

S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
3
Fig. 2. Hit compound 5 found through in silico and subsequent in vitro testing of the MCCC compound library, was optimised to yield 6. Replacing the thiazole moiety with an
oxadiazole, as in 7, yielded an inactive compound when screened at 10 M ligand concentration.
m
Fig. 3. Structures of all synthesised compounds carried through to in vitro testing in PAO1-L.
Scheme 1. Synthetic route to compounds 6, 8e26: (i) formamide, 150 ꢀC, 16 h; (ii) NaH, NMP, 0 ꢀC, 30 min, then chloroacetone, 0 ꢀC, 1 h; (iii) bromine, acetic acid, 65 ꢀC, 16 h; (iv)
thioamide or thiourea, ethanol, 80 ꢀC, 16 h; for complete structure of the compounds, see Fig. 3.

4
S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
Scheme 2. Synthetic route to 35: (i) ethanol, rt, 4 h, then NH2OH$HCl, TEA, rt, 2 h; (ii) HCl, zinc powder, 60 ꢀC, 2 h; (iii) DMFDMA, 100 ꢀC, 2 h; (iv) acetic acid, ethanol, 100 ꢀC, 16 h.
methylamino thiazole (40) gave the desired product 6-chloro-3-
((2-isopropylthiazol-5-yl)methyl)quinazolin-4(3H)-one (35).
The 2-amino-5-substituted thiazoles (27e31) were prepared
from reacting 6-chloroquinazolin-4(3H)-one (36) with 2-chloro-5-
(chloromethyl)thiazole, to give 6-chloro-3-((2-chlorothiazol-5-yl)
methyl)quinazolin-4(3H)-one (34). Displacement of the chlorine at
the 2-position of the thiazole with five different amines under
neutral conditions gave products 27e31 in yields of 21e84%
(Scheme 3).
In addition to variations about the thiazole, changes to the
halogen decorating the quinazolinone core could alter binding to
PqsR significantly. It was believed that the 6-chloro functionality in
6 could bind into a deep pocket within the LBD of PqsR, and
possibly form a hydrogen bond with Thr265, as reported before.
However, compounds reported by Ilangovan et al. contained a
chlorine at the 7-position of the bicyclic core [32]. Therefore, in
order to fully investigate the binding mode of this series, a 7-
chloroquinazolin-4(3H)-one subset was synthesised.
the PpqsA promoter [39]. Consequently, inhibitors of PqsR reduce
bioluminescence levels in these strains. Initially 96-well plates
containing the PAO1-L mCTX:PpqsA-lux bioreporter strain grown in
lysogeny broth (LB) were treated with a single concentration of
10 mM of each compound to determine which compounds were
active. As a threshold level for compound selection, a 50% reduction
of pqs activity compared to the DMSO control was set, where pqs
activity was a ratio of luminescence over OD600. Only compounds
above the 50% threshold progressed for IC50 determination.
Of the 29 compounds tested, 14 compounds showed >50% in-
hibition at a 10
mM concentration and their concentration-dose
response curves were generated. Importantly, the active com-
pounds were found to not inhibit growth compared to the DMSO
control (Fig. S2).
From the SAR study, it was apparent that short, unbranched
alkyl chains led to a loss of activity, (see compounds 8e11).
Furthermore, the addition of heteroatoms into the alkyl chain at the
2-position of the thiazole appeared to reduce activity. Of the seven
Two compounds were synthesised bearing a 7-chloro substi-
tution on the bicyclic ring, with variations on the thiazole: 32
contained a tert-butyl group at the thiazole 2-position, and 33 a
pentyl chain. The synthetic route was similar to that by which the
6-chloro series was synthesised (Scheme 4), though chlorination of
amino alkyl chain containing compounds, only 25 (IC50 ¼ 3.5
m
M in
PAO1-L) had inhibitory activity at a ligand concentration of 10
mM.
The presence of a tertiary amine did not improve activity, and the
inclusion of an additional heteroatom led to no inhibition (com-
pound 24).
quinazolinone 43 yielded the desired
thiazole formation led to the synthesis of 32 and 33.
a
-haloketone 44. Subsequent
Compounds 23 and 26, both containing three heteroatoms in
their side chains, were found to be inactive, further suggesting that
increase in polarity in the side chain leads to inactivity. In the case
of 23 this may be due to the polarity of the nitro group; its charged
state may result in the loss of activity in the whole cell-based assay
as a result of poor cell penetration. Compound 26 had the lowest
ClogP value of all compounds in the series (1.31). Notably, no
compound with a ClogP value under 3.00 showed any activity,
which also suggests that difficulty in penetrating the lipid bilayer
contributes significantly to the inactivity of these compounds.
For the given series, 6-chloro substitution on the quinazolinone
ring demonstrated improved activity over a 7-chloro substituted
2.2. Inhibition of the pqs system by potential PqsR antagonists
A PqsR-dependent bioreporter assay was used to assess the
degree to which the compounds synthesised in the SAR study could
inhibit the pqs QS system. Introduction of a mCTX:PpqsA-lux tran-
scriptional into the chromosomal CTX sites of PA strains PAO1-L and
PA14 results in the production of bioluminescence at high bacterial
cell densities in cultures where PQS and HHQ have reached their
threshold activation concentrations for PqsR activation and in turn
Scheme 3. Synthetic route to 27e31: (i) KOH, TBAI, 2-chloro-5-(chloromethyl)thiazole, toluene, 70 ꢀC, 1 h; (ii) amine, DMSO, 100e130 ꢀC, 16 h; for complete structure of the
compounds, see Fig. 3.

S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
5
Scheme 4. Synthetic route to 32e34: (i) formamide, 150 ꢀC, 16 h; (i) NaH, NMP, 0 ꢀC, 30 min, then chloroacetone, 0 ꢀC, 1 h; (iii) NCS, sulfuric acid, DCM, 40 ꢀC, 6 h; (iv) thioamide,
acetic acid, ethanol, 80 ꢀC, 16 h; for complete structure of the compounds, see Fig. 3.
scaffold. Compound 32 (IC50 ¼ 2.9
m
M) was only marginally more
evident. It was apparent that an increased chain length improved
potency, with the two most potent inhibitors bearing a hexyl (19,
IC50 ¼ 298 nM) and pentyl chain (18, IC50 ¼ 313 nM) respectively.
Moreover, increased branching at the 1-position of the alkyl chain
greatly improved potency: compound 12 (IC50 ¼ 397 nM) con-
taining a tert-butyl side chain demonstrated potent activity in
strain PAO1-L.
Strain PAO1-L is a moderately virulent laboratory adapted strain
routinely used to screen for PqsR inhibitors [34,42]. However, given
the genomic diversity of PA strains, it is essential to test isolates
belonging to the two major genomic groups. Hence, we also
screened the inhibitors against the highly virulent PA14 strain [43].
It was therefore encouraging to see that activities were comparable
between these strains. Moreover, for a few select compounds
(15e17, 19, 22) potencies were shown to be greater in PA14, sug-
gesting broad activity across the two main PA clades.
Three compounds (18, 19 and 22, IC50 ¼ 244 nM), were selected
for X-ray crystallography based on the high potency across both
strains. Compound 12 was also chosen for further study, due to its
excellent potency in PAO1-L. Furthermore, compound 6 was
selected for X-ray crystallography studies to provide a comparison
between the first optimised compound in the series, and the most
potent compounds.
active than the lead compound 45 (IC50 ¼ 5.0
mM, Fig. 4) of a pre-
viously described 7-chloro substituted scaffold [32]. Furthermore,
compound 33 was inactive when tested at a ligand concentration of
10
mM. Comparable 6-chloro substituted compounds 12
(IC50 ¼ 397 nM) and 18 (IC50 ¼ 313 nM) both showed sub-
micromolar activity, with 12 displaying seven-fold stronger activ-
ity in strain PAO1-L compared with the weakly active 32.
Moreover, inverting the heteroatoms within the thiazole ring
reduced activity, as indicated by the difference in activities of 6
(IC50 ¼ 1.0
at a concentration of 10
ously shown to have an activity of 1.0
m
M) and 35; the 2,5- disubstituted thiazole 35 was inactive
M, whereas the 2,4-analogue 6 was previ-
M. This inversion of the thia-
m
m
zoleringmay have furthercontributed tothe inactivityofcompounds
27e31 in addition to the increased polarity in the side chain.
One argument rationalising the intolerance of heteroatoms may
be poor bacterial uptake, or high rates of transporter-based efflux
[40]. Although a whole cell approach removes the ability to define a
binding event in the context of the assay itself, in combination with
X-ray crystallography this methodology allows for definitive se-
lection of compounds which both bind to the desired molecular
target, and also have a proven efficacy.
Dose response curves were obtained for all 14 compounds
exhibiting >50% inhibition in the 10
mM spot test, with activities
ranging from 244 nM to 3.55 M in the bioreporter strain PAO1-L.
m
2.3. Structure of PqsR ligand-binding domain complexed with
compounds 6, 12, 18 and 19
Active compounds were then assayed against the clinically-
relevant strain PA14 (Table 1). In general, the compounds dis-
played similar activities in both strains PAO1-L and PA14.
The pharmacological evaluation shed further light on the
importance of the 2-substitution of the thiazole. Regarding the
length and branching of the alkyl chain, two clear trends became
Compounds 6, 12, 18 and 19 were successfully soaked into a
truncated form of PqsR (PqsR94-309) containing the ligand-binding
domain of the protein (see S3-6 for additional data including
electron density plots and crystallographic table of data collection
Fig. 4. A previously described PqsR antagonist (45) featured a quinazolinone scaffold with a 7-chloro substituted ring. Removal of the chlorine atom led to a 10-fold drop in activity,
whilst a 6-chlorine substituted ring was inactive. By contrast, the compound series described in this article found 6-chlorine substitution to be optimal. Both 45 and an endogenous
ligand, NHQ (46) were successfully crystallised in the PqsR ligand-binding domain [32].

6
S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
Table 1
In vitro data for all compounds defined as active (inhibits pqs system to below 50% at 10
at 10
M (remaining activity for not active compounds is shown in brackets). Reported values are mean SD of n ¼ 2 replicates. cLogP values were calculated using
MarvinSketch based on the algorithm outlined by Viswanadhan et al. [41].
m
M). NA denotes inactive compounds which showed a remaining activity above 50%
m
Compound
IC50 value in PAO1-L/nM
IC50 value in PA14/nM
Predicted cLogP values
6
8
9
1046 419
NA (83%)
NA (52%)
1578 358.0
NA
NA
NA
3.38
2.14
3.03
2.84
2.09
3.94
3.57
3.73
4.27
4.02
4.02
4.17
4.62
3.10
3.64
4.04
3.98
2.13
3.50
1.31
4.00
4.02
2.17
3.55
2.18
3.94
4.17
2.93
3.43
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
NA (79%)
NA (104%)
397 141.5
1216 44.0
1563 674.0
1112 879.5
1572 1119
1360 501.0
313 156.2
298 182.0
1327 189.5
2048 1241.0
244 49.6
NA (89%)
NA
650 35.1
1333 192.5
1720 882.0
646 90.2
639 73.5
683 156.9
342 39.4
265 3.4
1308 272.0
2916 1005.5
123 20.9
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA (85%)
3545 2934.0
NA (108%)
NA (72%)
NA (83%)
NA (101%)
NA (67%)
NA (85%)
2942 808.0
NA (106%)
NA (95%)
NA
NA
NA
NA
NA (65%)
and refinement). All four ligands occupied a similar space to that
exhibited by a previously described quinazolinone-based inhibitor
45 and two endogenous ligands (HHQ, 3, and NHQ, 46) bearing
quinolone scaffolds [32,44]. In each case, the quinazolinone core
occupies the previously defined B pocket of the LBD [32], and the
alkyl chain extends into the open A pocket (Fig. 5). In addition, no
major conformational changes occurred to the protein backbone in
each of the crystal soaking experiments, as well as the previously
reported PqsR crystal structures.
carbonyl faces towards the B sub-pocket, the electron density maps
suggest that the carbonyl groups of 6, 12, 18 and 19 all face towards
the opposite face of the LBD in the direction of Thr265. The key
result of this is that it enables a hydrogen bond to form between the
carbonyl of each of the four compounds with the hydroxyl group of
Thr265 (Fig. 6). This is further supported by the observation that
the hydroxyl component of Thr265 faces towards the carbonyl in all
four structures, in contrast to 45 where it faces towards the chlorine
atom and elicits a polar interaction with the halogen.
As with 45, the electron density contours around the halogen
atom in each crystal, suggesting that the chlorine has locked the
ligand into its defined orientation with the quinazolinone core
occupying the B pocket of the PqsR LBD. However, whilst in 45 the
The importance of this hydrogen bond was further emphasised
by the lower activities of compounds 32 and 33, both featuring a 7-
chloro substitution. Repositioning of the chlorine atom removes the
possibility of forming a hydrogen bond between the carbonyl and
Fig. 5. (a) Overlaying the crystal structures of 12 (green), 18 (cyan) and HHQ (3, orange, PDB entry 6Q7U) shows that the core quinazolinone structures occupy almost identical
spaces within the LBD, with the alkyl chains extending out to the LBD entrance. Importantly, the carbonyl groups of 12 and 18 face towards Thr265, enabling a H-bond to form,
whereas the carbonyl of HHQ faces the opposite wall of the LBD in the direction of the B-sub pocket; (b) The same trends are observed between 6 (gold), 19 (magenta) and 45
(yellow, PDB entry 4JVI). The chlorine atoms of 6, 19 and 45 directly overlap, demonstrating its importance in locking the structure’s conformation NHQ (46, PDB entry 4JVD)
followed the above trends, but was omitted for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
7
Fig. 6. The binding of 6 into the PqsR LBD as a surface representation (a), and the key binding interactions observed between the carbonyl of 6 and Thr265, and the sulfur atom of 6
and Tyr258 (b). Below these are analogous representations for 12 (ced), 18 (eef) and 19 (geh). In each surface representation the chlorine atom is buried deep into the B pocket,
anchoring the ligand in place. Binding modes are seen to be similar in each example, with the hydroxyl of Thr265 turned to face the carbonyl of the quinazolinone, thereby eliciting
a H-bond. Furthermore, the sulfur is angled towards the phenol of Tyr258 enabling a polar interaction.
Thr265, hence the significant observed loss of potency.
indicated that increasing the length and branching of the aliphatic
substitution at the 2-position of the thiazole dramatically improved
potency. This is observed in the crystal structures, whereby the
aliphatic chain forms hydrophobic interactions with the side chains
The thiazole moieties were shown to further contribute to the
activity of the series through interactions with Tyr258. In all four
crystal structures, the sulfur atom was shown to orient itself facing
the tyrosine, likely due to the formation of a polar interaction be-
tween the sulfur atom and the phenol group. The inactivity of
oxadiazole 7 suggests that the presence of the sulfur aids in binding
to the LBD, further implicating the thiazole as an integral compo-
nent of the series. The role of the sulfur is further highlighted by the
inactivity of 35, whereby the 2,5-thiazole regioisomer is unable to
form a polar interaction with Tyr258, leading to a lower potency
when compared to the 2,4-thiazole analogue 6.
of residues in the A pocket of the LBD. Compounds 6 (IC50 ¼ 1.0
mM)
and 12 (IC50 397 nM) are weaker inhibitors than 18
¼
(IC50 ¼ 313 nM) and 19 (IC50 ¼ 298 nM), as they have shorter alkyl
chains unable to form as many hydrophobic interactions with the
protein. In particular, 18 and 19 are able to reach Ile186 and Leu189
at the far edge of the A pocket, creating a larger network of hy-
drophobic interactions and improving the activity over shorter
chain analogues such as 6 and 12 (Fig. S4).
The majority of residues in the LBD of PqsR bear aliphatic side
chains. As such, hydrophobic interactions are seen to drive activity
in both endogenous ligands and synthetic inhibitors. The SAR study

8
S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
2.4. Reduction of pyocyanin production by the PqsR inhibitors
mode similar to that seen in previously described quinazolinone-
and quinolone-based inhibitor-bound crystal structures. Further
assays confirmed that the most active compounds 18 and 19 were
capable of significantly reducing production of the toxin pyocyanin,
whilst remaining non-toxic to eukaryotic cells, providing a basis for
further pre-clinical studies.
To demonstrate a link between the inhibitory effects of 18 and
19 on PqsR and hence virulence gene expression, pyocyanin pro-
duction in the PAO1-L was quantified after overnight incubation
with either 18 or 19 at a concentration three times higher than their
IC50, or a vehicle (DMSO) control. Both compounds reduced pyo-
cyanin production to 23% and 36% respectively (Fig. 7), compared
with the control. These results indicate that these compounds can
attenuate the production of this virulence factor through inhibition
of PqsR similar to that shown for other PqsR inhibitors [34,45,46].
4. Experimental
4.1. Chemistry e general methods
Chemicals and solvents were provided by Fisher Scientific UK,
Acros Organics, Sigma-Aldrich, Merck Millipore or Fluorochem. All
reactions were monitored by TLC using Merck Silica Gel 60 Å F254
TLC plates or by LC-MS. Unless otherwise stated, all compounds
were dried under high vacuum either at rt or within an oven at
40 ꢀC. LC-MS data was collected on a Shimadzu UFLCXR HPLC
system coupled to an Applied Biosystems API 2000 LC/MS/MS
electrospray ionization (ESI). The column used was a Phenomenex
2.5. Effect of PqsR inhibitors on cytotoxicity of lung epithelial cells
A cytotoxicity study using A549 lung epithelial cells was con-
ducted with 18 and 19, to establish the suitability of these com-
pounds for further studies. Compounds 18 and 19 tested at
increasing concentrations ranging from 0.1 to 100 mM showed no
significant toxicity (Fig. 8). Testing at higher concentrations was not
possible as neither compound was fully soluble above this con-
centration. However, the data showed that both 18 and 19 are not
cytotoxic up to 100 mM. Therefore, the therapeutic index is > 292-
fold in 18 and > 377-fold in 19 against the A549 cell line relative to
ꢀ
^
Gemini-NX 3
mm-110A C18, 50 ꢁ 2mm at 40 C. The flow rate was
0.5 mL/min, the UV detection was at 220 nm and 254 nm. The LC-
MS ran for 1 min at 5% B; 5e98% B over 2 min, 98% B for 2 min, 98 to
5% B over 0.5 min and then 5% for 1 min where solvent A: 0.1%
formic acid in water; solvent B: acetonitrile. Unless otherwise
stated compounds reported had a purity >95%. NMR spectroscopy
was performed using a Bruker AV(III) HD 400 NMR spectrometer
equipped with a 5 mm BBFOþ probe, recording 1H and 13C NMR at
400.25 MHz and 100.66 MHz respectively; or a Bruker AV(III) 500
NMR spectrometer equipped with a 5 mm dual 1H/13C helium-
cooled cryoprobe, recording 1H and 13C NMR at 500.13 MHz and
125.77 MHz respectively. NMR data was processed using iNMR
(version 5.5.7) referencing spectra to residual solvents. Chemical
the activities calculated for PA14.
3. Conclusion
In summary, a new series of chloro-3-((2-substituted-thiazole)
quinazolin-4(3H)-one compounds was synthesised and tested in a
whole cell bioreporter assay to determine their ability to inhibit
PqsR, and subsequently reduce P. aeruginosa pyocyanin production.
A SAR study showed key contributing functionalities, particularly a
long or branched alkyl chain at the 2-position of the thiazole. These
studies revealed a preference for chlorine substitution on the 6-
position of the quinazolinone ring.
shifts are quoted as d: values in ppm; coupling constants J are given
in Hz and multiplicities are described as follows: s - singlet, d -
doublet, t - triplet, q - quartet, qi - quintet, sep e septet, m e
multiplet, app e apparent, br - broad.
X-ray crystallography into the LBD of PqsR confirmed a binding
4.1.1. General procedure for the preparation of thiazoles 6, 8e23,
32, 33
A solution of the appropriate thioamide or thiourea (0.13 mmol)
and
a-haloketone 38 or 44 (0.06 mmol) in EtOH (4 mL) was
refluxed at 80 ꢀC for 16 h. a After cooling to room temperature, the
reaction mixture was concentrated in vacuo and purified through
flash column chromatography. Chromatography was run with a
gradient of ethyl acetate/petroleum ether (1:2) to pure ethyl ace-
tate. b
a Where compound 44 was used as the a-haloketone, 600 mL of
acetic acid was added to aid in solubilizing the starting material.
b Excepting compounds 11 and 26 which were run in DCM/
methanol (19:1).
4.1.1.1. 6-Chloro-3-((2-isopropylthiazol-4-yl)methyl)quinazolin-
4(3H)-one (6). Obtained 6 (18 mg, 90%) as a white crystal: 1H NMR
(DMSO‑d6, 400 MHz)
d
8.54 (s,1H), 8.05 (d, J ¼ 1.68 Hz,1H), 7.83 (dd,
J ¼ 8.61, 1.72 Hz, 1H), 7.70 (d, J ¼ 8.69 Hz, 1H), 7.39 (s, 1H), 5.25 (s,
2H), 3.24e3.17 (m, 1H), 1.26 (d, J ¼ 6.84 Hz, 6H) ppm; 13C NMR
(DMSO‑d6 101 MHz)
d 177.7, 158.9, 150.0, 148.5, 146.6, 134.5, 131.4,
129.5, 125.1, 122.9, 115.7, 45.4, 32.4, 22.8 ppm; LC-MS (þESI)
calculated for C15H14ClN3OS m/z 320.1 (M þ H), found m/z 320.1
(M þ H).
Fig. 7. Compounds 18 and 19 were shown to significantly reduce pyocyanin produc-
tion to 23% and 36% respectively against a control of 0.1% DMSO at 3 ꢁ the IC50 value in
P. aeruginosa strain PAO1-L.
4.1.1.2. 6-Chloro-3-((2-methylthiazol-4-yl)methyl)quinazolin-4(3H)-
one (8). Obtained 8 (17 mg, 90%) as an orange crystal: 1H NMR

S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
9
Fig. 8. Cytotoxicity data for (left) 18 and (right) 19 indicated that neither shows significant toxicity up to 100 mM in A549 lung epithelial cells. The lethal dose (LD50) value could not
be calculated, as cell viability was not reduced to < 50% (indicated by the red dashed line). It was not possible to test at higher compound concentrations due to insolubility in the
test buffer. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
(CDCl3, 400 MHz)
(m, 2H), 7.20 (s, 1H), 5.22 (s, 2H), 2.66 (s, 3H) ppm; 13C NMR (CDCl3
101 MHz) 159.9,149.4,146.9,146.6,134.7,133.1, 129.2,126.2,123.2,
117.7, 45.4, 29.7, 19.1 ppm; LC-MS (þESI) calculated for
13H10ClN3OS m/z 292.0 (M þ H), found m/z 292.1 (M þ H).
d
8.34 (s, 1H), 8.25 (d, J ¼ 1.24 Hz, 1H), 7.69e7.63
7.86 (dd, J ¼ 8.69, 2.40 Hz, 1H), 7.72 (d, J ¼ 8.69 Hz, 1H), 7.42 (s, 1H),
5.25 (s, 2H), 2.77 (d, J ¼ 7.08 Hz, 2H), 1.95 (qt, J ¼ 6.72, 6.72 Hz, 1H),
d
0.88 (d, J ¼ 6.65 Hz, 6H) ppm; 13C NMR (DMSO‑d6, 101 MHz)
d 170.1,
158.9, 150.1, 148.6, 146.6, 134.5, 131.4, 129.5, 125.1, 122.9, 116.4, 45.3,
41.2, 29.1, 21.9 ppm; LC-MS (þESI) calculated for C16H16ClN3OS m/z
334.1 (M þ H), found m/z 334.1 (M þ H).
C
4.1.1.3. 6-Chloro-3-((2-(trifluoromethyl)thiazol-4-yl)methyl)quina-
zolin-4(3H)-one (9). Obtained 9 (3.5 mg, 16%) as a yellow solid: 1H
4.1.1.8. 3-((2-Butylthiazol-4-yl)methyl)-6-chloroquinazolin-4(3H)-
one (14). Obtained 14 (29 mg, 88%) as a yellow crystal: 1H NMR
NMR (CDCl3, 400 MHz)
d
8.35 (s, 1H), 8.24 (d, J ¼ 1.64 Hz, 1H), 7.71
(s, 1H), 7.69e7.66 (m, 2H), 5.32 (s, 2H) ppm; LC-MS (þESI) calcu-
lated for C13H7ClF3N3OS m/z 346.0 (M þ H), found m/z 346.1
(M þ H).
(400 MHz, CDCl3)
d
8.34 (s, 1H), 8.23 (dd, J ¼ 2.00, 0.87 Hz, 1H),
7.67e7.60 (m, 2H), 7.19 (s, 1H), 5.22 (s, 2H), 2.92 (t, J ¼ 7.74 Hz 2H),
1.71 (tt, J ¼ 7.70, 7.70 Hz, 2H), 1.38 (tq, J ¼ 7.45, 7.45 Hz, 3H), 0.90 (t,
J ¼ 7.38 Hz, 5H) ppm; 13C NMR (101 MHz, DMSO)
d 171.4, 158.9,
150.0, 148.6, 146.7, 134.5, 131.4, 129.6, 125.1, 122.9, 116.2, 45.4, 32.2,
31.4, 21.5, 13.5 ppm; LC-MS (þESI) calculated for C16H16ClN3OS m/z
334.1 (M þ H), found m/z 334.1 (M þ H).
4.1.1.4. 6-Chloro-3-((2-ethylthiazol-4-yl)methyl)quinazolin-4(3H)-
one (10). Obtained 10 (14 mg, 70%) as a white solid: 1H NMR (CDCl3,
400 MHz)
d 8.28 (s, 1H), 8.15 (s, 1H), 7.61e7.55 (m, 2H), 7.14 (s, 1H),
5.16 (s 2H), 2.90 (q, J ¼ 7.52 Hz, 2H),1.27 (t, J ¼ 7.56 Hz, 3H) ppm; 13
C
NMR (CDCl3 101 MHz) d 173.9, 159.7, 149.1, 147.0, 146.5, 134.6, 132.9,
4.1.1.9. 6-Chloro-3-((2-(pentan-2-yl)thiazol-4-yl)methyl)quinazolin-
4(3H)-one (15). Obtained 15 (11 mg, 45%) as a yellow oily solid: 1H
129.1, 126.0, 123.1, 117.1, 45.3, 26.8, 14.0 ppm; LC-MS (þESI) calcu-
lated for C14H12ClN3OS m/z 306.0 (M þ H), found m/z 306.1 (M þ H).
NMR (CDCl3, 400 MHz)
d
8.37 (s, 1H), 8.25 (d, J ¼ 1.52 Hz, 1H),
7.69e7.63 (m, 2H), 7.20 (s, 1H), 5.24 (s, 2H), 3.14 (qt, J ¼ 6.96,
6.96 Hz, 1H), 1.77e1.53 (m, 2H), 1.33e1.25 (m, 5H), 0.88 (t,
4.1.1.5. 2-(4-((6-Chloro-4-oxoquinazolin-3(4H)-yl)methyl)thiazol-2-
J ¼ 7.32 Hz, 3H) ppm; 13C NMR (CDCl3 101 MHz)
d 178.6, 159.8,
yl)acetonitrile (11). Obtained 11 (18 mg, 90%) as a red-orange solid:
1H NMR (CDCl3, 400 MHz)
d
8.32 (s, 1H), 8.23 (d, J ¼ 1.80 Hz, 1H),
7.70e7.64 (m, 2H), 7.43 (s, 1H), 5.24 (s, 2H), 4.06 (s, 2H) ppm; 13C
NMR (CDCl3 101 MHz) 159.8, 158.4, 150.6, 146.6, 146.5, 134.8,
148.8, 147.0, 146.5, 134.7, 133.1, 129.1, 126.2, 123.2, 116.7, 45.3, 39.9,
38.3, 21.4, 20.3, 13.9 ppm; LC-MS (þESI) calculated for
C17H18ClN3OS m/z 348.1 (M þ H), found m/z 348.2 (M þ H).
d
133.3, 129.2, 126.1, 123.1, 119.8, 115.0, 45.3, 22.2 ppm; LC-MS (þESI)
calculated for C14H9ClN4OS m/z 317.0 (M þ H), found m/z 317.2
(M þ H).
4.1.1.10. 6-Chloro-3-((2-(2-methylbutyl)thiazol-4-yl)methyl)quina-
zolin-4(3H)-one (16). Obtained 16 (20 mg, 91%) as a colourless oil:
1H NMR (CDCl3, 400 MHz)
7.69e7.63 (m, 2H), 7.21 (s, 1H), 5.24 (s, 2H), 2.94 (dd, J ¼ 14.61,
6.08 Hz, 1H), 2.75 (dd, J ¼ 14.61, 8.09, 1H), 1.89e1.80 (m, 1H),
1.46e1.14 (m, 2H), 0.92e0.87 (m, 6H) ppm; 13C NMR (CDCl3
d
8.35 (s, 1H), 8.25 (d, J ¼ 1.64 Hz, 1H),
4.1.1.6. 3-((2-(Tert-butyl)thiazol-4-yl)methyl)-6-chloroquinazolin-
4(3H)-one (12). Obtained 12 (9.0 mg, 43%) as a white crystal: 1H
NMR (CDCl3, 400 MHz)
7.70e7.65 (m, 2H), 7.19 (s, 1H), 5.26 (s, 2H), 1.40 (s, 9H) ppm; 13C
NMR (CDCl3 101 MHz) 182.4, 159.9, 148.9, 147.1, 146.6, 134.7, 133.1,
d
8.43 (s, 1H), 8.27 (d, J ¼ 1.80 Hz, 1H),
101 MHz) d 171.6, 159.7, 149.2, 146.9,146.6,134.7, 129.2, 126.2, 123.0,
117.3, 45.4, 40.3, 36.0, 29.7, 29.1, 19.0, 11.3 ppm; LC-MS (þESI)
calculated for C17H18ClN3OS m/z 348.1 (M þ H), found m/z 348.2
(M þ H).
d
129.2, 126.2, 123.2, 116.7, 45.3, 37.8, 30.9 ppm; LC-MS (þESI)
calculated for C16H16ClN3OS m/z 334.1 (M þ H), found m/z 334.2
(M þ H).
4.1.1.11. 6-Chloro-3-((2-isopentylthiazol-4-yl)methyl)quinazolin-
4.1.1.7. 6-Chloro-3-((2-isobutylthiazol-4-yl)methyl)quinazolin-
4(3H)-one (17). Obtained 17 (13 mg, 71%) as a white crystal: 1H
4(3H)-one (13). Obtained 13 (11 mg, 53%) as a silver crystal: 1H
NMR (CDCl3, 400 MHz)
7.69e7.63 (m, 2H), 7.20 (s, 1H), 5.23 (s, 2H), 2.95 (t, J ¼ 7.61 Hz, 2H),
d
8.36 (s, 1H), 8.25 (d, J ¼ 1.48 Hz, 1H),
NMR (DMSO‑d6, 400 MHz)
d
8.54 (s, 1H), 8.07 (d, J ¼ 2.36 Hz, 1H),

10
S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
1.65e1.62 (m, 3H), 0.93 (d, J ¼ 6.04 Hz, 6H); 13C NMR (CDCl3
4.1.1.18. 6-Chloro-3-((2-((2-methoxyethyl)amino)thiazol-4-yl)
101 MHz)
d
172.9, 159.8, 149.2, 146.9, 134.7, 133.0, 129.2, 126.2,
methyl)quinazolin-4(3H)-one (24). Obtained 24 (34 mg, 51%) as a
123.3, 117.2, 45.4, 38.9, 31.5, 29.7, 27.7, 23.3 ppm; LC-MS (þESI)
calculated for C17H18ClN3OS m/z 348.1 (M þ H), found m/z 348.2
(M þ H).
cream solid: 1H NMR (DMSO‑d6, 400 MHz)
d 8.47 (s, 1H), 8.08 (d,
J ¼ 2.67 Hz, 1H), 7.85 (dd, J ¼ 9.06, 2.70 Hz, 1H), 7.72e7.67 (m, 2H),
6.42 (s, 1H), 5.00 (s, 2H), 3.41 (t, J ¼ 5.39 Hz, 2H), 3.32 (t, J ¼ 4.91 Hz,
2H), 3.21 (s, 3H) ppm; 13C NMR (DMSO‑d6, 101 MHz)
d 169.1, 158.9,
148.6, 146.6, 146.3, 134.5, 131.3, 129.5, 125.1, 122.9, 102.8, 70.1, 57.9,
45.5, 43.9 ppm; LC-MS (þESI) calculated for C15H15ClN4O2S m/z
351.1 (M þ H), found m/z 350.6 (M þ H).
4.1.1.12. 6-Chloro-3-((2-pentylthiazol-4-yl)methyl)quinazolin-4(3H)-
one (18). Obtained 18 (20 mg, 90%) as a white crystal: 1H NMR
(CDCl3, 400 MHz)
d
8.35 (s, 1H), 8.26 (d, J ¼ 1.92 Hz, 1H), 7.69e7.63
(m, 2H), 7.20 (s, 1H), 5.23 (s, 2H), 2.93 (t, J ¼ 7.68 Hz, 2H), 1.77e1.69
(m, 2H), 1.37e1.33 (m, 4H), 0.90 (t, J ¼ 6.93 Hz, 3H) ppm; 13C NMR
4.1.1.19. 3-((2-(Butylamino)thiazol-4-yl)methyl)-6-chloroquinazolin-
4(3H)-one (25). Obtained 25 (16 mg, 24%) as a white crystal: 1H
(CDCl3 101 MHz) d 172.8,159.9,149.1, 147.0, 146.6,134.7,133.1, 129.2,
126.2, 123.2, 117.2, 45.4, 33.4, 31.3, 29.6, 22.3, 13.9 ppm; LC-MS
(þESI) calculated for C17H18ClN3OS m/z 348.1 (M þ H), found m/z
348.2 (M þ H).
NMR (DMSO‑d6, 400 MHz)
d
8.46 (s, 1H), 8.08 (d, J ¼ 2.65 Hz, 1H),
7.86 (s, dd, J ¼ 8.68, 2.65 Hz, 1H), 7.72 (d, J ¼ 8.68 Hz, 1H), 7.61 (t,
J ¼ 5.67 Hz, 1H), 6.42 (s, 1H), 5.00 (s, 2H), 3.11 (dt, J ¼ 6.87, 5.20 Hz,
2H), 1.46 (tt, J1 ¼ J2 ¼ 7.07 Hz), 1.28 (tq, J ¼ 7.33, 7.33 Hz, 2H), 0.83 (t,
J ¼ 7.32 Hz, 3H) ppm; 13C NMR (DMSO‑d6, 101 MHz)
d 169.4, 158.9,
4.1.1.13. 6-Chloro-3-((2-hexylthiazol-4-yl)methyl)quinazolin-4(3H)-
one (19). Obtained 19 (15 mg, 66%) as a white crystal: 1H NMR
148.6, 146.7, 146.5, 134.5, 131.3, 129.5, 125.1, 122.9, 102.4, 45.5, 44.2,
30.7, 19.5, 13.6 ppm; LC-MS (þESI) calculated for C16H17ClN4OS m/z
349.1 (M þ H), found m/z 348.9 (M þ H).
(CDCl3, 400 MHz)
d
8.36 (s, 1H), 8.25 (d, J ¼ 1.72 Hz, 1H), 7.69e7.64
(m, 2H), 7.21 (s, 1H), 5.23 (s, 2H), 2.94 (t, J ¼ 7.89 Hz, 2H), 1.74 (tt,
J ¼ 7.64, 7.64 Hz, 2H), 1.40e1.33 (m, 2H), 1.30e1.27 (m, 4H), 0.86 (t,
J ¼ 6.93 Hz, 3H) ppm; 13C NMR (CDCl3 101 MHz)
d 172.8, 159.8,
4.1.1.20. 2-(4-((6-Chloro-4-oxoquinazolin-3(4H)-yl)methyl)thiazol-
2-yl)guanidine (26). Obtained 26 (13 mg, 62%) as a cream solid: 1H
149.1, 146.9, 146.5, 134.7, 133.1, 129.1, 126.1, 123.2, 117.2, 45.4, 33.4,
31.4, 29.9, 28.7, 22.4, 14.0 ppm; LC-MS (þESI) calculated for
NMR (DMSO‑d6, 400 MHz)
d
8.55 (s, 1H), 8.08 (d, J ¼ 2.40 Hz, 1H),
C
18H20ClN3OS m/z 362.1 (M þ H), found m/z 362.3 (M þ H).
7.85e7.83 (m, 1H), 7.70 (dd, J ¼ 8.77, 2.36 Hz, 1H), 7.04 (br s, 4H),
5.06 (s, 2H) ppm; LC-MS (þESI) calculated for C13H11ClN6OS m/z
335.0 (M þ H), found m/z 335.2 (M þ H).
4.1.1.14. 6-Chloro-3-((2-(furan-2-yl)thiazol-4-yl)methyl)quinazolin-
4(3H)-one (20). Obtained 20 (17 mg, 80%) as a white crystal: 1H
NMR (CDCl3, 400 MHz)
d
8.40 (s, 1H), 8.26 (d, J ¼ 1.88 Hz, 1H), 7.67
4.1.2. General procedure for the preparation of 3-((2-amino)
thiazol-5-yl)methyl)quinazolin-4(3H)-one compounds 27e31 from
34
(d, J ¼ 2.20 Hz, 1H), 7.66 (s, 1H), 7.50 (d, J ¼ 1.09 Hz, 1H), 7.32 (s, 1H),
6.97 (d, J ¼ 3.64 Hz, 1H), 6.52 (dd, J ¼ 3.36, 1.80 Hz, 1H), 5.29 (s, 2H)
ppm; 13C NMR (CDCl3 101 MHz)
d 159.9, 158.9, 150.6, 148.5, 146.9,
A
solution of 34 (0.32 mmol) and appropriate amine
(0.96 mmol) in DMSO (0.5 mL) was refluxed at 100e130 ꢀC
(dependent on the boiling point of the amine) for 16 h. The reaction
mixture was cooled to room temperature, diluted with water and
extracted with ethyl acetate (3 ꢁ 20 mL). The combined organic
layers were dried over brine and concentrated in vacuo. Purification
through flash column chromatography in ethyl acetate/petroleum
ether (4:1) to ethyl acetate to methanol/ethyl acetate (19:1) yielded
products 27e31 in yields of 21e84%.
146.6, 143.9, 134.8, 133.2, 129.2, 126.2, 123.2, 117.3, 112.3, 109.5,
45.4 ppm; LC-MS (þESI) calculated for C16H10ClN3O2S m/z 344.0
(M þ H), found m/z 344.1 (M þ H).
4.1.1.15. 6-Chloro-3-((2-(2-(furan-2-yl)vinyl)thiazol-4-yl)methyl)
quinazolin-4(3H)-one (21). Obtained 21 (12 mg, 51%) as a cream
solid. A minor impurity remained, giving a final purity of 91.7% as
determined by 1H NMR: 1H NMR (DMSO‑d6, 400 MHz)
d 8.60 (s,
1H), 8.09 (d, J ¼ 2.32 Hz, 1H), 7.88 (dd, J ¼ 8.73, 2.36 Hz, 1H),
7.77e7.73 (m, 2H), 7.54 (s, 1H), 7.29 (d, J ¼ 16.09 Hz, 1H), 7.08 (d,
J ¼ 16.09 Hz,1H), 6.79 (d, J ¼ 3.24 Hz,1H), 6.59 (dd, J ¼ 2.88,1.64 Hz,
4.1.2.1. 6-Chloro-3-((2-(4-methylpiperidin-1-yl)thiazol-5-yl)methyl)
quinazolin-4-(3H)-one (27). Obtained 27 (86 mg, 72%) as a yellow
1H), 5.30 (s, 2H) ppm; 13C NMR (101 MHz, DMSO)
d 166.0, 159.0,
solid: 1H NMR (DMSO‑d6, 400 MHz)
d 8.57 (s, 1H), 8.12 (d,
151.7, 151.1, 148.6, 146.7, 144.5, 134.6, 131.5, 129.6, 125.1, 122.9, 121.5,
J ¼ 2.28 Hz, 1H), 7.88 (dd, J ¼ 8.82, 2.67 Hz, 1H), 7.72 (d, J ¼ 8.70 Hz,
1H), 7.26 (s, 1H), 5.20 (s, 2H), 3.80e3.77 (m, 2H), 2.92 (ddd, J ¼ 3.02,
3.02,12.44 Hz, 2H), 1.65e1.61 (m, 2H),1.58e1.51 (m, 1H),1.10 (dddd,
118.3, 117.0, 112.7, 112.5, 45.5 ppm; LC-MS (þESI) calculated for
C
18H12ClN3O2S m/z 370.0 (M þ H), found m/z 370.2 (M þ H).
J ¼ 4.14, 4.14, 4.14, 11.93 Hz, 2H), 0.89 (d, J ¼ 6.16 Hz, 3H) ppm; 13
C
NMR (DMSO‑d6, 101 MHz)
d 171.7, 159.0, 147.8, 146.6, 140.2, 134.6,
4.1.1.16. 6-Chloro-3-((2-phenylthiazol-4-yl)methyl)quinazolin-
4(3H)-one (22). Obtained 22 (20 mg, 92%) as a white solid: 1H NMR
131.6, 129.6, 125.0, 122.6, 119.8, 48.2, 42.2, 32.7, 30.0, 21.5 ppm; LC-
MS (þESI) calculated for C18H19ClN4OS m/z 375.1 (M þ H), found m/
z 374.6 (M þ H).
(CDCl3, 400 MHz)
(m, 2H), 7.70e7.64 (m, 2H), 7.43e7.41 (m, 3H), 7.36 (s, 1H), 5.32 (s,
2H) ppm; 13C NMR (CDCl3 101 MHz)
169.1, 159.9, 150.7, 147.1,
d
8.47 (s, 1H), 8.27 (d, J ¼ 1.72 Hz, 1H), 7.92e7.89
d
146.6,134.7,133.1,133.1,130.4,129.2,129.0,126.5,126.2,123.2,117.9,
45.5 ppm; LC-MS (þESI) calculated for C18H12ClN3OS m/z 354.0
(M þ H), found m/z 354.2 (M þ H).
4.1.2.2. 6-Chloro-3-((2-(cyclohexylamino)thiazol-5-yl)methyl)quina-
zolin-4-(3H)-one (28). Obtained 28 (25 mg, 21%) as a brown solid:
1H NMR (DMSO‑d6, 400 MHz)
d
8.55 (s, 1H), 8.11 (d, J ¼ 2.52 Hz, 1H),
8.67 (dd, J ¼ 8.55, 2.21 Hz, 1H), 7.72 (d, J ¼ 8.78 Hz, 1H), 7.50 (d,
J ¼ 7.48 Hz, 1H), 7.11 (s, 1H), 5.15 (s, 2H), 3.39e3.34 (m, 1H),
1.89e1.84 (m, 2H), 1.69e1.64 (m, 3H), 1.56e1.51 (m, 1H), 1.31e1.11
4.1.1.17. 6-Chloro-3-((2-(4-nitrophenyl)thiazol-4-yl)methyl)quinazo-
lin-4(3H)-one (23). Obtained 23 (15 mg, 48%) as a white crystal: 1H
NMR (400 MHz, DMSO‑d6)
d
8.66 (s, 1H), 8.36e8.28 (m, 2H), 8.15 (d,
(m, 4H) ppm; 13C NMR (DMSO‑d6, 101 MHz)
d 169.0, 159.0, 147.9,
J ¼ 2.08 Hz, 1H), 8.14e8.06 (m, 2H), 7.91e7.83 (m, 2H), 7.75 (d,
146.6, 139.5, 134.6, 131.6, 129.6, 125.0, 122.7, 117.9, 53.0, 42.3, 32.3,
25.3, 24.4 ppm; LC-MS (þESI) calculated for C18H19ClN4OS m/z
375.1 (M þ H), found m/z 374.6 (M þ H).
J ¼ 8.74 Hz, 1H), 5.39 (s, 2H) ppm; LC-MS (þESI) calculated for
C
18H11ClN4O3S m/z 399.0 (M þ H), found m/z 399.1 (M þ H).

S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
11
4.1.2.3. 6-Chloro-3-((2-(2-hydroxyethyl(methyl)amino)thiazol-5-yl)
141.3, 135.4, 134.2, 134.0, 129.6, 126.3, 123.0, 43.1 ppm; LC-MS
(þESI) calculated for C12H7Cl2N3OS m/z 312.0 (M þ H), found m/z
311.8 (M þ H).
methyl)quinazolin-4-(3H)-one (29). Obtained 29 (94 mg, 84%) as a
yellow solid: 1H NMR (DMSO‑d6, 400 MHz)
d 8.57 (s, 1H), 8.12 (d,
J ¼ 2.52 Hz, 1H), 7.88 (dd, J ¼ 8.3, 2.51 Hz, 1H), 7.72 (d, J ¼ 8.8 Hz,
1H), 7.24 (s, 1H), 5.20 (s, 2H), 4.75 (t, J ¼ 5.74, 1H), 3.55 (dt, J ¼ 6.31,
6.31 Hz, 2H), 3.43 (t, J ¼ 5.75, 2H), 2.99 (s, 3H) ppm; 13C NMR
4.1.2.9. 6-Chloro-3-((2-isopropylthiazol-5-yl)methyl)quinazolin-
4(3H)-one (35). To
0.58 mmol) in acetonitrile (5 mL) was added acetic acid (500 m
a
solution of intermediate 41 (140 mg,
L)
(DMSO‑d6, 101 MHz)
d
171.1, 159.0, 147.8, 146.6, 140.3, 134.6, 131.6,
129.5, 125.0, 122.6, 119.0, 58.1, 54.7, 42.3, 40.4 ppm; LC-MS (þESI)
calculated for C15H15ClN4O2S m/z 351.1 (M þ H), found m/z 350.6
(M þ H).
and 40 (55 mg, 0.29 mmol) and stirred at 100 ꢀC for 2 h. The re-
action mixture was cooled to room temperature, neutralised with
saturated NaHCO3, and extracted with ethyl acetate (3 ꢁ 10 mL).
The combined organic layers were dried over brine, concentrated in
vacuo and purified through HPLC, yielding 35 (2 mg, 2%) as a white
4.1.2.4. 3-((2-(Butylamino)thiazol-5-yl)methyl)-6-chloroquinazolin-
4(3H)-one (30). Obtained 30 (46 mg, 41%) as a yellow solid: 1H
solid: 1H NMR (400 MHz, DMSO‑d6)
d 8.63 (s, 1H), 8.12 (d,
NMR (DMSO‑d6, 400 MHz)
d
8.54 (s, 1H), 8.11 (d, J ¼ 2.22 Hz, 1H),
J ¼ 2.51 Hz, 1H), 7.88 (dd, J ¼ 8.71, 2.53 Hz, 1H), 7.77 (s, 1H), 7.72 (d,
7.85 (dd, J ¼ 8.03, 2.22 Hz, 1H), 7.70 (d, J ¼ 7.75 Hz, 1H), 7.56 (t,
J ¼ 5.54 Hz, 1H), 7.13 (s, 1H), 5.15 (s, 2H), 3.13 (dt, J ¼ 6.96, 6.96 Hz,
2H), 1.46 (tt, J ¼ 7.01, 7.01 Hz, 2H), 1.29 (tq, J ¼ 7.36, 7.36 Hz, 2H),
J ¼ 8.69 Hz, 1H), 5.37 (s, 2H), 3.20 (sept, J ¼ 6.87 Hz, 1H), 1.27 (d,
J ¼ 6.85 Hz, 6H) ppm; 13C NMR (126 MHz, DMSO)
d 178.2, 165.8,
147.9, 146.6, 142.2, 134.8, 131.7, 131.6, 129.7, 125.1, 122.7, 41.8, 32.6,
22.7 ppm; LC-MS (þESI) calculated for C15H14ClN3OS m/z 320.1
(M þ H), found m/z 320.2 (M þ H).
0.85 (t, J ¼ 7.41 Hz, 3H) ppm; 13C NMR (CDCl3, 101 MHz)
d 172.0,
160.1, 146.7, 145.9, 139.8, 135.0, 133.5, 129.4, 126.3, 123.2, 118.8, 46.0,
43.2, 31.4, 20.1, 13.8 ppm; LC-MS (þESI) calculated for C16H17ClN4OS
m/z 349.1 (M þ H), found m/z 348.7 (M þ H).
4.1.2.10. 6-Chloroquinazolin-4(3H)-one (36). A solution of 2-amino-
5-chlorobenzoic acid (4.99 g, 29.00 mmol) and formamide (20 mL)
was refluxed at 150 ꢀC for 16 h, forming a brown precipitate. The
reaction mixture was cooled to room temperature and 20 g ice was
added. The mixture was left to stand for 1 h, then the precipitate
filtered and concentrated in vacuo, yielding 36 (5.13 g, 97%) as
4.1.2.5. 6-Chloro-3-((2-((2-methoxyethyl)amino)thiazol-5-yl)
methyl)quinazolin-4-(3H)-one (31). Obtained 31 (24 mg, 22%) as a
yellow solid: 1H NMR (DMSO‑d6, 400 MHz)
d 8.55 (s, 1H), 8.11 (d,
J ¼ 2.04 Hz, 1H), 8.08 (s, br, 1H), 7.86 (dd, J ¼ 8.85, 2.85 Hz, 1H), 7.71
(d, J ¼ 8.85 Hz, 1H), 7.21 (s, 1H), 5.16 (s, 2H), 3.43 (t, J ¼ 5.37 Hz, 2H),
3.38e3.34 (m, 2H), 3.23 (s, 3H) ppm; 13C NMR (CDCl3, 101 MHz)
brown microcrystals: 1H NMR (DMSO‑d6, 400 MHz)
d 12.44 (br s,
1H), 8.12 (s, 1H), 8.06 (d, J ¼ 2.40 Hz, 1H), 7.85 (dd, J ¼ 8.65, 2.52 Hz,
d
171.7, 160.2, 146.9, 145.7, 136.6, 135.1, 133.6, 129.4, 126.3, 123.0,
118.0, 70.5, 59.0, 45.9, 43.2 ppm; LC-MS (þESI) calculated for
15H15ClN4O2S m/z 351.1 (M þ H), found m/z 350.7 (M þ H).
1H), 7.70 (d, J ¼ 8.77 Hz, 1H) ppm; 13C NMR (DMSO‑d6, 101 MHz)
d
160.3, 152.6, 146.5, 134.9, 131.5, 130.0, 125.3, 124.4 ppm; LC-MS
C
(þESI) calculated for C8H5ClN2O m/z 181.0 (M þ H), found m/z
181.2 (M þ H).
4.1.2.6. 3-((2-Tert-butyl)thiazol-4-yl)methyl)-7-chloroquinazolin-
4(3H)-one (32). Obtained 32 (2.0 mg, 11%) as a white solid: 1H NMR
4.1.2.11. 6-Chloro-3-(2-oxopropyl)quinazolin-4(3H)-one
(37).
(400 MHz, CDCl3)
J ¼ 1.99 Hz, 1H), 7.47 (dd, J ¼ 8.55, 2.01 Hz, 1H), 7.22 (s, 1H), 5.28 (s,
2H), 1.43 (s, 9H) ppm; 13C NMR (126 MHz, CDCl3)
182.9, 160.3,
148.7, 148.7, 148.3, 140.8, 128.4, 128.1, 127.0, 120.6, 117.1, 45.2, 38.0,
31.0 ppm; LC-MS (þESI) calculated for C16H16ClN3OS m/z 334.1
(M þ H), found m/z 334.2 (M þ H).
d
8.47 (s, 1H), 8.25 (d, J ¼ 8.56 Hz, 1H), 7.74 (d,
To a solution of 36 (1.60 g, 6.76 mmol) in anhydrous NMP was
added NaH (650 mg, 27.08 mmol) in small portions, forming a
white precipitate. After stirring for 30 min the solution returned to
a clear brown state and chloroacetone (2.4 mL, 29.28 mmol) was
added dropwise forming a deep red solution. The solution was
concentrated in vacuo and recrystallised in 3:1 hexane/ethyl ace-
tate, yielding 37 (1.80 g, 86%) as white needle crystals: 1H NMR
d
4.1.2.7. 7-Chloro-3-((2-pentylthiazol-4-yl)methyl)quinazolin-4(3H)-
(DMSO‑d6, 400 MHz)
(dd, J ¼ 8.73, 2.48 Hz, 1H), 7.74 (d, J ¼ 8.73 Hz, 1H), 4.98 (s, 2H), 2.25
(s, 3H) ppm; 13C NMR (CDCl3, 101 MHz)
199.7, 159.87, 146.7, 146.4,
135.0, 133.3, 129.3, 126.1, 122.9, 54.7, 27.5 ppm; LC-MS (þESI)
calculated for C11H9ClN2O2 m/z 237.0 (M þ H), found m/z 237.1
(M þ H).
d
8.25 (s, 1H), 8.07 (d, J ¼ 2.40 Hz, 1H), 7.89
one (33). Obtained 33 (2.0 mg, 11%) as a white solid: 1H NMR
(400 MHz, CDCl3)
d
8.20 (d, J ¼ 8.57 Hz, 1H), 7.99 (s, 1H), 7.76 (d,
d
J ¼ 2.00 Hz, 1H), 7.47 (dd, J ¼ 8.57, 2.00 Hz, 1H), 4.98 (s, 1H), 3.83 (s,
2H), 2.64 (t, J ¼ 7.55 Hz, 2H), 1.73e1.67 (m 2H), 1.36e1.28 (m, 4H),
0.94e0.86 (m, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d 199.0, 197.2,
160.1, 148.3, 147.9, 141.3, 128.5, 128.5, 126.9, 120.2, 53.8, 43.6, 36.5,
31.2, 25.3, 22.4, 14.0 ppm; LC-MS (þESI) calculated for
4.1.2.12. 3-(3-Bromo-2-oxopropyl)-6-chloroquinazolin-4(3H)-one
(38). To a solution of 37 (460 mg, 1.94 mmol) in acetic acid (10 mL)
was added bromine (125 mL, 62.42 mmol) dropwise, forming a deep
C
17H18ClN3OS m/z 348.1 (M þ H), found m/z 348.0 (M þ H).
4.1.2.8. 6-Chloro-3-((2-chlorothiazol-5-yl)methyl)quinazolin-4(3H)-
one (34). A solution of 36 (1.00 g, 5.50 mmol) in toluene (15 mL)
was treated with KOH (620 mg, 11.10 mmol) and TBAI (193 mg,
0.55 mmol) and heated at 70 ꢀC for 10 min. To this was added 2-
red solution. The reaction mixture was refluxed at 65 ꢀC for 16 h,
forming a red precipitate. The reaction mixture was concentrated in
vacuo, diluted with water and neutralised with 2 M NaOH. The
organic layer was extracted with ethyl acetate (3 ꢁ 20 mL) and
washed with water (2 ꢁ 20 mL) and brine (1 ꢁ 20 mL). The organic
layers were combined and concentrated in vacuo and purified
through flash column chromatography (1:1 ethyl acetate/petro-
leum ether), yielding 38 (790 mg, 66%) as a white solid: 1H NMR
chloro-5-(chloromethyl)thiazole (740
mL, 6.60 mmol) dropwise
and stirred for 1 h at 70 ꢀC forming a dark brown solution. The
reaction mixture was cooled to room temperature, diluted with
water and extracted with ethyl acetate (4 ꢁ 20 mL). The combined
organic layers were dried over Na2SO4 and filtered. The organic
layer was concentrated in vacuo and triturated with diethyl ether
(3 ꢁ 20 mL), yielding 34 (1.53 g, 89%) as a yellow solid: 1H NMR
(DMSO‑d6, 400 MHz)
(dd, J ¼ 8.69, 2.40 Hz, 1H), 7.75 (d, J ¼ 8.73 Hz, 1H), 5.13 (s, 2H), 4.57
(s, 2H) ppm; 13C NMR (CDCl3, 101 MHz)
194.6, 160.0, 146,7, 146.1,
d
8.27 (s, 1H), 8.08 (d, J ¼ 2.36 Hz, 1H), 7.90
d
(DMSO‑d6, 400 MHz)
J ¼ 8.89, 2.57 Hz, 1H), 7.81 (s, 1H), 7.73 (d, J ¼ 8.61 Hz, 1H), 5.36 (s,
2H) ppm; 13C NMR (CDCl3, 101 MHz)
160.1, 154.2, 146.6, 145.3,
d
8.62 (s,1H), 8.13 (d, J ¼ 2.57 Hz,1H), 7.89 (dd,
135.2, 133.6, 129.5, 126.1, 122.8, 52.4, 31.4 ppm; LC-MS (þESI)
calculated for C11H8BrClN2O2 m/z 314.9 (M þ H), found m/z 315.1
(M þ H).
d

12
S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
4.1.2.13. 2-Isopropylthiazole-5-carbaldehyde
oxime
(39).
(3 ꢁ 20 mL). The combined organic layers were dried over brine,
concentrated in vacuo and purified through flash column chroma-
tography in petroleum ether/ethyl acetate (7:3 to 3:7) yielding 44
as a white solid (30 mg, 11%): 1H NMR (400 MHz, Acetic Acid-d4)
Bromomalonaldehyde (150 mg, 1.00 mmol) and 2-methylpro
panethioamide (103 mg, 1.00 mmol) were dissolved in EtOH
(5 mL) and stirred at room temperature for 4 h. Hydroxylamine
hydrochloride (90 mg, 1.30 mmol) and TEA (1 mL) was added and
stirred for a further 2 h at room temperature. The reaction mixture
was concentrated in vacuo and purified through flash column
chromatography in petroleum ether/ethyl acetate (4:1 to 3:2)
yielding 39 (70 mg, 34%) as a yellow solid: 1H NMR (DMSO‑d6,
d
8.43 (s, 1H), 8.26 (d, J ¼ 8.59 Hz, 1H), 7.81 (d, J ¼ 2.05 Hz, 1H), 7.59
(dd, J ¼ 8.63, 2.01 Hz, 1H), 5.23 (s, 2H), 4.52 (s, 2H) ppm; 13C NMR
(101 MHz, Acetic Acid-d4)
d 195.7, 160.4, 149.8, 147.7, 141.2, 128.5,
128.4, 125.5, 119.8, 52.7, 46.4 ppm; LC-MS (þESI) calculated for
C11H8Cl2N2O2 m/z 271.0 (M þ H), found m/z 270.6 (M þ H).
400 MHz) d 12.29 (s, 1H), 8.28 (s, 1H), 8.02e8.00 (m, 3H), 7.54e7.50
(m, 3H) ppm;
4.2. PqsR bioreporter assay
4.1.2.14. (2-Isopropylthiazol-5-yl)methanamine (40). To a solution
of 39 (59 mg, 0.29 mmol) in EtOH (4 mL) was added zinc powder
(47 mg, 0.72 mmol) and hydrochloric acid (130 mL,13 N,1.72 mmol).
4.2.1. Bacterial growth conditions
P. aeruginosa strains PAO1-L and PA14 were grown in lysogeny
broth (LB). All strains were incubated at 37 ꢀC for 16 h, prior to use
when OD600 was approximately 2.5. Strains PAO1-L and PA14
contained a chromosomal mCTX:PpqsA-lux fusion such that light is
emitted following activation of the pqsA promoter. Consequently, a
reduction in bioluminescence is indicative of pqs system inhibition.
Strain BL21 (DE3) contained a pET28:PqsR plasmid, allowing for
overexpression of the protein PqsR, and containing a kanamycin
The reaction mixture was stirred at 60 ꢀC for 2 h, then cooled to
room temperature. The mixture was diluted with water and neu-
tralised with saturated NaHCO3, then extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic layers were dried over brine and
concentrated in vacuo yielding 40 (55 mg, 100%) as a yellow solid:
1H NMR (DMSO‑d6, 400 MHz)
7.53e7.47 (m, 3H), 5.02 (s, br, 2H), 4.10 (s, 2H) ppm;
d 7.92e7.90 (m, 2H), 7.79 (1H, s),
resistance cassette. To these cultures was added 100
mycin for selection.
mg/mL kana-
4.1.2.15. Methyl
5-chloro-2-(((dimethylamino)methylene)amino)
benzoate (41). A solution of 2-amino-5-chlorobenzoic acid (50 mg,
0.29 mmol) in acetonitrile (5 mL) was treated with 1,1-dimethoxy-
N,N- dimethylmethanamine and stirred at 100 ꢀC for 2 h. The in-
termediate 41 was confirmed through LC-MS and carried through
without purification: LC-MS (þESI) calculated for C11H13ClN2O2 m/z
241.1 (M þ H), found m/z 240.7 (M þ H).
4.2.2. Bioluminescence reporter gene IC50 and 10
assays
m
M spot test
Two 1.5 mL Eppendorf tubes were filled with 500
To one was added 3.16 L of a 10 mM stock of a given inhibitor, and
to the other 1 L, resulting in overall concentrations of 63.2 M and
20 M respectively. A one in 10 serial dilution of both stock solu-
tions into four further Eppendorf tubes each containing 450 L of LB
broth gave 10 concentrations of the compound from 63.2 M to
2 nM. Using only the central 60 wells of a Grenier 96 well flat black
plate, 100 L of each concentration was added to three wells for
mL of LB broth.
m
m
m
m
m
4.1.2.16. 7-Chloroquinazolin-4(3H)-one (42).
m
4-chloroanthranilic acid (1.00 g, 5.82 mmol) was refluxed in
formamide (10 mL) at 150 ꢀC for 16 h, forming a brown precipitate.
The reaction mixture was cooled to room temperature and 20 g ice
was added. The mixture was left to stand for 1 h, then the precip-
itate filtered and concentrated in vacuo, yielding 42 (850 mg, 81%)
m
results in triplicate.
Concurrently, an overnight culture of either PAO1-L or PA14
with the relevant antibiotic, grown to an OD600 of between 2.0 and
3.0, was diluted in LB broth to OD600 ¼ 0.02. To each of the wells
containing a given concentration of compound in LB, was added
as a brown solid: 1H NMR (DMSO‑d6, 400 MHz)
d 12.39 (s, 1H), 8.14
(s, 1H), 8.11 (d, J ¼ 8.56 Hz, 1H), 7.72 (d, J ¼ 2.09 Hz, 1H), 7.55 (dd,
J ¼ 8.52, 2.11 Hz, 1H) ppm; 13C NMR (DMSO‑d6, 101 MHz)
d
160.1,
100
31.6
m
m
L of the diluted P. aeruginosa, giving overall concentrations of
M to 1 nM. The outer wells were filled with 200 L of LB broth,
149.9, 146.9, 138.9, 128.0, 127.0, 126.4, 121.5 ppm; LC-MS (þESI)
calculated for C8H5ClN2O m/z 181.0 (M þ H), found m/z 181.2
(M þ H).
m
as well as any unfilled inner wells, and the plate placed into a
luminometer-spectrometer (Tecan GENios Pro), running a script at
37 ꢀC over 24 h, with a kinetic cycle measuring OD600 and lumi-
nescence every 30 min.
4.1.2.17. 7-Chloro-3-(2-oxopropyl)quinazolin-4(3H)-one
(43).
Compound 42 (850 mg, 4.70 mmol) was dissolved in NMP (5 mL)
and treated with NaH (380 mg, 9.40 mmol). The reaction mixture
was stirred at room temperature for 30 min, then chloroacetone
(1.1 mL, 14.10 mmol) was added dropwise. The reaction was stirred
for 30 min at room temperature, the diluted with water (20 mL),
and extracted with ethyl acetate (4 ꢁ 15 mL). The combined organic
layers were dried over brine, concentrated in vacuo and recrystal-
lised in 2:1 hexane/ethyl acetate, yielding compound 43 (827 mg,
A peak in luminescence was observed always between 8 and 9 h,
after which deterioration of the growth curve led to a drop in
bioluminescence. As such, readouts of the OD600 and biolumines-
cence were taken from the highpoint of the luminescence readout,
and the results plotted in Graphpad Prism 8, measuring log [con-
centration] against luminescence (Relative Light Units)/OD600
A similar methodology was applied for the 10 M spot test as-
says, but all compounds were diluted to 10 M from the 10 mM
stock by adding 1 L of the stock to 500 L of LB broth, yielding a
20 M solution. As with the IC50 assays, 100 L was then added to at
least three wells and diluted twofold by the addition of 100 L of
P. aeruginosa at OD600 ¼ 0.02. As all compounds were tested at
10 M, no serial dilutions of the stock solutions were performed.
.
m
m
74%) as a silver crystal: 1H NMR (CDCl3, 400 MHz)
d
8.21 (d,
m
m
J ¼ 8.52 Hz, 1H), 7.88 (s, 1H), 7.73 (d, J ¼ 2.02 Hz, 1H), 7.47 (dd,
m
m
J ¼ 8.57, 1.98 Hz, 1H), 4.79 (s, 2H), 2.35 (s, 3H) ppm; 13C NMR (CDCl3,
m
101 MHz) d 199.8, 160.4, 149.3, 147.5, 141.0, 128.4, 127.4, 120.5, 77.2,
76.8, 54.8, 27.7 ppm; LC-MS (þESI) calculated for C11H9ClN2O2 m/z
m
237.0 (M þ H), found m/z 237.1 (M þ H).
Data was displayed as a column graph, with all results normalised
against the negative control containing 0.1% DMSO (100%).
4.1.2.18. 7-Chloro-3-(3-chloro-2-oxopropyl)quinazolin-4(3H)-one
(44). Compound 43 (235 mg, 1.00 mmol) and NCS (133 mg,
1.00 mmol) were stirred in DCM (5 mL) and H2SO4 (1 mL) at 40 ꢀC
for 6 h. The reaction mixture was cooled to room temperature,
neutralised with NaOH and extracted with ethyl acetate
4.2.3. Crystallography and protein determination
Protein was prepared as previously described by Ilangovan et al.
[32] Crystals were grown in 24 well sitting and hanging drop Cry-
schem plates. Crystallisation reservoir consisted of 100 mM

S. Grossman et al. / European Journal of Medicinal Chemistry 208 (2020) 112778
13
trisodium citrate, 200 mM ammonium acetate and 2-methyl-2,4-
pentanediol (MPD). Citrate pH (5.5e6.5) and MPD concentration
(3%e10%) were varied along the plate’s X and Y axes respectively,
and optimal conditions for crystal soaking of 6, 12, 18 and 19 were
found to be a pH of 6.25 containing 6% MPD. Ligands were dissolved
in DMSO or a multi-component solvent mixture and allowed to
incubate in the crystallisation drop for 24 h.
Diffraction images were integrated with DIALS [47] (via Xia2 or
DUI) and scaled in the CCP4 suite. Structures were solved by Mo-
lecular Replacement with PHASER [48] using 6Q7W as a search
model. Model was refined with REFAC5 [49] with Jellybody re-
strains and ligand fitted into the Fo-Fc density map using COOT
[50]. Ligand restrains were generated in AceDrg [51]. Omit and
POLDER maps were generated using Phenix [52]. Finished struc-
tures have been deposited in the PDB as 6Z17 (6), 6Z07 (12), 6Z5K
(18) and 6YZ3 (19). Ligand description (CIF), is available in sup-
plementary information.
and PW. FS, MS, MC, PW and JE contributed to the study and
experimental design. (Synthesis: SG, FS; Biological Assays: SG and
FS; X-ray crystallography WR with supervision from JE.)
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Wellcome Trust doctoral
training programme in antimicrobials and antimicrobial resistance
(SG, WR ref: 108876/B/15/Z) and the JPI-AMR/MRC funded SEN-
BIOTAR program (Ref. MR/N501852/1). FS, JE, PW, MC and MS are
funded by the National Biofilms Innovation Centre (NBIC) which is
an Innovation and Knowledge Centre funded by the Biotechnology
and Biological Sciences Research Council, InnovateUK and Hartree
Centre [Award Number BB/R012415/1. We would like to thank our
colleagues at Diamond Light Source for the provision of Synchro-
tron radiation at Beamline I04 under session MX19880-21.
The construct pET28a:pqsR is available upon request.
4.2.4. Pyocyanin quantification
A 5 mL liquid culture of PAO1-L wildtype in LB medium was
grown overnight for over 16 h with shaking at 200 rpm. Flasks
containing 15 mL overnight PAO1-L culture diluted in LB to an
OD600 of 0.05, and either an inhibitor compound at 3 ꢁ IC50 value,
or an equivalent volume of DMSO control was added and incubated
for a further 16 h with shaking at 200 rpm.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112778.
An OD600 measurement was taken for each culture after 16 h for
normalisation, before each sample was centrifuged for 10 min at
10,000ꢁg and 24 ꢀC. The supernatants were filtered through a
Abbreviations
0.22 mm filter, and 7.5 mL collected. To each 7.5 mL filtered super-
natant was added 4.5 mL chloroform, and each sample vortexed at
3000 rpm for 10 s. The samples were centrifuged at 10,000 rcf for
10 min at 4 ꢀC, and the aqueous layer discarded. To 3 mL of each
sample was added to 1.5 mL 0.2 M HCl and each vortexed for 10 s,
followed by centrifugation at 10,000ꢁg for 2 min at 4 ꢀC. Mea-
surement of the OD520 of the aqueous layer of each sample against a
0.2 M HCl control provided the raw data which could subsequently
be normalised against the OD600 readings taken after incubation.
Adapted from Essar et al. [53].
AI
AQ
DCM
DMEM
auto inducer
alkyl quinolone
dichloromethane
Dulbecco’s modified eagle medium
DMFDMA N,N-dimethylformamide dimethyl acetate
DMSO
EDTA
EtOH
FBS
dimethyl sulfoxide
ethylenediaminetetraacetic acid
ethanol
foetal bovine serum
HHQ
HPLC
IC50
IPTG
LB
2-heptyl-4-hydroxyquinoline
high performance liquid chromatography
half maximal inhibitory concentration
isopropyl b-D-1-thiogalactopyranoside
lysogeny broth
4.2.5. Cytotoxicity
A549 lung epithelial cells were cultured in Gibco’s DMEM media
containing 10% FBS and 1% penicillin-streptomycin at 37 ꢀC with 5%
CO2. When 80% confluent, the medium was removed and the cells
washed with 2 ꢁ 10 mL PBS and 1 ꢁ 7 mL trypsin-EDTA. To the flask
was added 7 mL trypsin-EDTA and incubated for 5 min at 37 ꢀC, 5%
CO2. 7 mL FBS was added to quench trypsinization, and the cells
centrifuged at 300 g for 5 min.
The supernatant was removed and the cell pellet washed with
DMEM-F12 medium and centrifuged again at 300 g for 5 min. The
pellet was then resuspended in 3 mL DMEM-F12 media and diluted
LBD
ligand-binding domain
LC-MS
LD50
MCCC
MPD
NBS
NCS
liquid chromatography-mass spectrometry
half maximal lethal dose
managed chemical compound collection
2-methyl-2,4-pentanediol
N-bromosuccinimide
N-chlorosuccinimide
further to allow loading of 100
10,000 cells per well into 96 well plates. Cells were incubated for
16 h, at which point 100 L of each compound was added to the
relevant wells to give a total volume of 200 L. Cells were incubated
for a further 16 h, and 20 L of Alamar blue added to each well. After
5 h incubation, fluorescence was measured with excitation at
510 nm and emission at 590 nm. Values were then normalised
against the untreated cell control. Adapted from O’Brien et al. [54].
m
L of suspension containing
NHQ
NMP
NMR
OD600
PA
PQS
QS
QSSM
rcf
SAR
2-nonyl-4-hydroxyquinoline
N-methyl pyrrolidinone
m
nuclear magnetic resonance
optical density at 600 nm
Pseudomonas aeruginosa
2-heptyl-3-hydroxy-4(1H)-quinolone
quorum sensing
quorum sensing signal molecule
relative centrifugal force
structure-activity relationship
standard deviation
m
m
Author contributions
SD
SG wrote the manuscript with supervision from MS, MC and PW
with input from all co-authors. The junior authors performed the
experiments and analysed the data with supervision from MS, MC
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
TBAI
tetrabutylammonium iodide

14
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