Thioether-linked dihydropyrrol-2-one analogues as PqsR antagonists against antibiotic resistant Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.bmc.2020.115967

The Pseudomonas quinolone system (pqs) is one of the key quorum sensing systems in antibiotic-resistant P. aeruginosa and is responsible for the production of virulence factors and biofilm formation. Thus, synthetic small molecules that can target the PqsR (MvfR) receptor can be utilized as quorum sensing inhibitors to treat P. aeruginosa infections. In this study, we report the synthesis of novel thioether-linked dihydropyrrol-2-one (DHP) analogues as PqsR antagonists. Compound 7g containing a 2-mercaptopyridyl linkage effectively inhibited the pqs system with an IC₅₀ of 32 μM in P. aeruginosa PAO1. Additionally, these inhibitors significantly reduced bacterial aggregation and biofilm formation without affecting planktonic growth. The molecular docking study suggest that these inhibitors bind with the ligand binding domain of the MvfR as a competitive antagonist.

Full text of DOI:10.1016/j.bmc.2020.115967

Bioorg. Med. Chem. 31 (2021) 115967
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thioether-linked dihydropyrrol-2-one analogues as PqsR antagonists
against antibiotic resistant Pseudomonas aeruginosa
Shekh Sabira, Dittu Suresha, Sujatha Subramonib, Theerthankar Dasc, Mohan Bhadbhaded,
,
*
David StC. Blacka, Scott A. Riceb, Naresh Kumara
a School of Chemistry, The University of New South Wales, NSW 2052 Sydney, Australia
b Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, 637551 Singapore, Singapore
c Department of Infectious Diseases and Immunology, School of Medical Sciences, The University of Sydney, NSW 2006 Sydney, Australia
d Mark Wainwright Analytical Centre, UNSW Sydney, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
The Pseudomonas quinolone system (pqs) is one of the key quorum sensing systems in antibiotic-resistant
P. aeruginosa and is responsible for the production of virulence factors and biofilm formation. Thus, synthetic
small molecules that can target the PqsR (MvfR) receptor can be utilized as quorum sensing inhibitors to treat
P. aeruginosa infections. In this study, we report the synthesis of novel thioether-linked dihydropyrrol-2-one
(DHP) analogues as PqsR antagonists. Compound 7g containing a 2-mercaptopyridyl linkage effectively inhibited
the pqs system with an IC50 of 32 µM in P. aeruginosa PAO1. Additionally, these inhibitors significantly reduced
bacterial aggregation and biofilm formation without affecting planktonic growth. The molecular docking study
suggest that these inhibitors bind with the ligand binding domain of the MvfR as a competitive antagonist.
Pseudomonas quinolone system
Quorum sensing inhibitors
Pseudomonas aeruginosa
PqsR antagonist
Dihydropyrrol-2-one (DHP) analogues
1. Introduction
antibiotics than isolated bacterial cells.6,7
To overcome bacterial resistance, the development of anti-virulence
Pseudomonas aeruginosa is a highly opportunistic Gram-negative
bacterium that is responsible for a number of nosocomial infections
including pneumonia, cystic fibrosis, and urinary tract infection. These
infections can be life-threatening especially in immunocompromised
patients such as those battling cancer or HIV/AIDS.1,2 P. aeruginosa is
extremely resistant to several classes of antibiotics due to its highly
evolved mechanisms of resistance, including efflux pump expression and
biofilm formation.3 In its recent report, the World Health Organization
(WHO) listed P. aeruginosa as one of the critical priority pathogens
which require urgent attention for the development of new
therapeutics.4
agents could be an effective alternative strategy.8,9 Inhibitors that target
virulence factors without killing bacterial cells should diminish the se-
lective pressure on bacteria to develop drug resistance. In P. aeruginosa,
virulence factor production and biofilm formation are primarily regu-
lated by the quorum sensing (QS) system.10 Therefore, QS inhibitors
have the potential to reduce the severity of infections, allowing in-
fections to be cleared by either the host immune system or by conven-
tional antibiotic therapy.
QS is a bacterial cell density-dependent mechanism involving the
production and release of several different chemical classes of small
diffusible signalling molecules, known as autoinducers (AIs), which
mediate intercellular communication.11 As the bacterial cell number
grows, the concentration of these AIs in the surrounding medium in-
creases. Once the concentration of AIs reaches a certain threshold, the
bacteria initiate collective behaviours including the production of
virulence factors and biofilm formation.12
P. aeruginosa secretes a wide range of virulence factors such as
pyocyanin, rhamnolipids, exotoxins, elastases, which promote the in-
vasion and damage of host tissue.5 The ability to form biofilms is another
important virulence characteristic of P. aeruginosa. A biofilm is typically
an extracellular polymeric matrix made up of exopolysaccharides,
nucleic acids, proteins, and lipids produced by bacterial cells. Signifi-
cantly, bacterial cells in biofilm are 1000-fold more resistant to
In P. aeruginosa, there are three major interconnected QS systems,
namely the las, rhl and pqs systems. These QS systems are integrated and
* Corresponding author.
E-mail addresses: s.sabir@student.unsw.edu.au (S. Sabir), subramoni@ntu.edu.sg (S. Subramoni), das.ashishkumar@sydney.edu.au (T. Das), m.bhadbhade@
unsw.edu.au (M. Bhadbhade), d.black@unsw.edu.au (D.StC. Black), rscott@ntu.edu.sg (S.A. Rice), n.kumar@unsw.edu.au (N. Kumar).
https://doi.org/10.1016/j.bmc.2020.115967
Received 18 October 2020; Received in revised form 15 December 2020; Accepted 18 December 2020
Available online 1 January 2021
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
control the activity of each other.13 For example, the las system posi-
tively controls the rhl and pqs systems by activating the genes associated
with their cognate receptors (rhlR and PqsR). The las and rhl QS systems
utilize the N-acyl-L-homoserine lactone (AHL) class of signalling mole-
cules for the activation of their cognate receptors lasR and rhlR
respectively, while the pqs system utilizes alkyl quinolones, including 2-
heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy (4H) quino-
lone (PQS), to activate the receptor PqsR (MvfR) (Fig. 1).14,15 Upon
activation, PqsR upregulates the expression of several biosynthetic
genes including the pqsABCDE operon, which further exerts positive
feedback control of PQS biosynthesis and enhances virulence factor
production as well as biofilm formation.16 Synthetic PqsR receptor an-
tagonists have been reported to reduce virulence factor production and
biofilm formation in P. aeruginosa.17–20 Additionally, a P. aeruginosa
strain with mutant PqsR showed severely attenuated virulence factor
production and biofilm formation in an animal infection model.21
Therefore, targeting PqsR with synthetic small molecules could be a
promising strategy to combat infections caused by antibiotic-resistant
P. aeruginosa.
1,5-dihydro-2Hpyrrol-2-ones (5) (Scheme 1).24
To derivatize parent DHPs (5) containing an unsubstituted exocyclic
double bond, we began by investigating monobromination reactions at
the vinylic carbon. In initial attempts, treating compound (5) with 1
equiv. of N-bromo succinimide (NBS) in ACN gave the desired Z-
brominated compound (6) in 55% isolated yield within 1 h at room
temperature. Optimization experiments revealed that using 1,3-
dibromo-5,5-dimethylhydantoin (DBDMH) with triethylamine in
dichloromethane improved the yield, and Br-DHP compound (6) being
isolated in up to 77% yield after 30 min at 0 ◦C (Scheme 2, Table 1).
With the Z-brominated compounds (6) in hand, we were interested to
investigate their exchange reactions with different nucleophiles as re-
ported previously for bromo-thiolactone analogues.32 However, our
results suggested that bromo-DHPs were drastically less reactive than
bromo-thiolactones, as no reaction was observed when the Br-DHPs (6)
were reacted with aromatic or aliphatic amines at room temperature. On
the other hand, the exchange reaction with thiophenol was successful
and produced the thioether-linked DHP compound (7) in about 60%
yield. Different aromatic thiols were then investigated and the use of 4-
chlorothiophenol or 2-mercaptopyridine gave the desired thioether
compounds in high yields under mild reaction condition (Scheme 2).
However, the aliphatic thiol 1-propanethiol reacted very slowly and
gave lower yields of product, as the starting Br-DHP was not fully
consumed even after stirring for 2 days in the presence of excess thiol
and base. The structures of all new DHP thioether compounds were
characterised by 1H and 13C NMR spectroscopy and IR spectroscopy.
Additionally, X-ray crystal analysis of compound (7c) was carried out to
confirm the Z configuration of the final compounds (Supp. file).
Dihydropyrrol-2-one (DHP) and other structurally related com-
pounds which mimic the natural N-acyl homoserine lactones (AHLs)
have been reported by our group.22–24 These compounds competitively
inhibit the receptors of natural AHLs, leading to inhibition of QS and
consequently the production of virulence factors and biofilm formation.
Our group has also developed DHP analogues that showed QS inhibition
against P. aeruginosa with minimal or no effect on bacterial growth.25,26
Furthermore, attachment of DHPs to the surfaces of biomedical devices
significantly reduced biofilm formation in P. aeruginosa.22–23 DHPs have
also been reported as competitive antagonists of the PqsR receptor,
resulting in the inhibition of pqs signalling with micromolar potency.27
Additionally, small molecules containing substituted sulfur, thio-
ether or disulfide functional groups have been reported as QS inhibitors,
PqsR antagonists and/or biofilm inhibitors against P. aeruginosa.28–31 In
line with our continuous effort towards the development of DHP ana-
logues as inhibitors of P. aeruginosa QS, we report herein the synthesis of
novel thioether-containing DHP analogues by selective Z-bromination of
the exocyclic double bond of the lactam, followed by a subsequent ex-
change reaction using different aromatic and aliphatic thiols. All the
synthesized compounds were evaluated for the inhibition of pqs sig-
nalling, biofilm formation, pyocyanin production as well as tested for
the effect on bacterial growth.
2.2. pqs QS inhibition assay
All the synthesized DHP thioether compounds were screened for
PqsR inhibitory activity against the P. aeruginosa PAO1 pqsA-gfp reporter
strain. This assay determines the expression of the pqsABCDE operon
which is triggered by cognate receptor PqsR (MvfR). In the untreated
control experiment the expression of the reporter reached its peak be-
tween 6 and 8 h and then decrease to its basal level. Most of the com-
pounds inhibited the expression of the pqs-gfp reporter between 6 and 8
h in a dose-dependent manner. The thioether analogues (7a-7d) con-
taining an unsubstituted phenyl ring at the C-4 position displayed
moderate pqs inhibition ranges between 20% and 53% at 125 µg/mL
concentration. A significant increase in pqs inhibition activity was
observed with halogen-substituted (4-F phenyl) DHP analogue. The
compound 7g bearing a 4-fluorophenyl ring at C4 and a 2-mercaptopyr-
idyl group at the exocyclic position was the most potent pqs inhibitor
overall, with 63% pqs inhibition at 125 µg/mL and a calculated IC50 of
32 µM (Fig. 2, Table 2). However, the corresponding 4-Cl and 4-Br an-
alogues 7k and 7o only showed weak inhibition of 18% and 20%
respectively against pqs at 125 µg/mL. Compounds 7h and 7l having an
n-propyl side chain displayed 59% and 62% inhibition against pqs ac-
tivity respectively at 125 µg/mL, with calculated IC50 values of 90 µM
and 100 µM, respectively. Additionally, we found that the thiopyridine
analogues 7s and 7t having fluoro at 2 and 3 position of phenyl ring are
less active than the 4-fluoro analogue and displayed only 40% and 47%
pqs inhibition, respectively.
2. Results and discussion
2.1. Chemistry
The synthesis of DHP analogues involves condensation reaction of
the commercially available phenyl acetone derivatives (1) with glyox-
ylic acid (2) in phosphoric acid, which gives an intermediate lactone
compound (3). This lactone intermediate is subsequently converted into
the corresponding lactam analogue (4) in sequential steps by using
thionyl chloride followed by Aq⋅NH3. Finally, lactam compound dehy-
drated using phosphorous pentoxide which gives 5-methylene-4-phenyl-
2.3. Effect on bacterial aggregation and biofilm formation
We next investigated the effect of compounds 7g and 7l on bacterial
aggregation and biofilm formation. The phase contrast microscopic
images indicated that P. aeruginosa strains grown in presence of com-
pounds 7g and 7l are hindered in their coaggregation ability. The impact
of 7g and 7l in hindering P. aeruginosa ATCC and PAO1 aggregation in
24 h-old biofilm formation was clearly exhibited via microscopic images
(Fig. 3). P. aeruginosa ATCC and PAO1 strains grown in presence of 7g
and 7l showed less coaggregation (i.e. less decrease in its absorbance at
Fig. 1. Schematic diagram of the Pseudomonas quinolone quorum sensing (pqs)
system, involving biosynthesis 2-heptyl-3-hydroxy (4H) quinolone (PQS) from
anthranilic acid and activation of the PqsR (MvfR) receptor.
2

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
Scheme 1. General synthesis of dihydropyrrol-2-one (DHP) analogues (5).
2.4. Effect on bacterial growth
All the compounds were tested for their effect on the growth of
bacteria at concentrations up to 125 µg/mL. None of the compounds
inhibited the planktonic growth of bacteria up to the highest concen-
tration tested (Fig. 5). This indicates that these DHP analogues inhibit
pqs QS and biofilm formation of P. aeruginosa without interfering with
bacterial growth, which should make them less likely to induce
resistance.
3. Molecular docking study
To gain some understanding of the possible binding of 4-F phenyl
DHP thiopyridyl analogue (7g) with the PqsR (MvfR) receptor, we
performed an in silico molecular docking study using Discovery Studio
software. Using the GOLD algorithm, energy minimized ligand 7g was
docked into the active site of a model created from the crystal structure
of the MvfR protein (PDB: 6B8A) in complex with a previously reported
competitive inhibitor M-64 (IC50 < 1 µM) (Fig. 6).37
Scheme 2. Synthesis of dihydropyrrol-2-one (DHP) thioether analogues.
Table 1
Synthesized dihydropyrrol-2-one (DHP) thioether analogues.
Compounds
R1
R2
Yields (%)
The binding interactions of the highest-scoring pose (Gold score
52.27, pose 27) of 7g present in the largest cluster (63%) were analysed.
7a
7b
7c
7d
7e
7f
H
n-Propyl
Phenyl
44
60
64
60
92
92
73
35
80
50
63
37
78
70
67
51
87
75
70
84
H
The phenyl moiety of compound 7g showed π-alkyl hydrophobic in-
H
4-Cl-Phenyl
2-Pyridyl
Phenyl
teractions with Ala168, Leu197, Leu208, Val211, and Ile236. The ni-
trogen atom of the pyridine ring interacted via hydrogen bonding with
Gln194, which was reported as one of the key amino acids for the
competitive inhibition of PqsR.37 Additionally, the carbonyl oxygen of
the lactam interacts with Ser196 via a hydrogen bond (Fig. 7).
Besides the central ligand binding site of MvfR, there is an adjacent
hydrophobic pocket lined by residues Leu183, Ile186, Leu189 and
Tyr258 connected via a narrow channel to the main site. M-64 occupies
this additional hydrophobic pocket in MvfRLBD via hydrophobic in-
teractions between its phenoxy group with the aforementioned amino
acid residues. However, compound 7g is unable to occupy this site due
to its shorter side chain, which might be the reason for the lower PqsR
antagonistic activity of 7g compared to M-64.
H
4-F
4-F
4-F
4-Cl
4-Cl
4-Cl
4-Cl
4-Br
4-Br
4-Br
4-Br
2-F
2-F
2-F
2-F
3-F
4-Cl-Phenyl
2-Pyridyl
n-Propyl
Phenyl
7g
7h
7i
7j
4-Cl-Phenyl
2-Pyridyl
n-Propyl
Phenyl
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
4-Cl-Phenyl
2-Pyridyl
n-Propyl
Phenyl
4-Cl-Phenyl
2-Pyridyl
2-Pyridyl
4. Conclusion
In conclusion, a library of novel DHP thioether analogues has been
synthesized and investigated for the inhibition of PqsR (MvfR) signaling
and biofilm formation in P. aeruginosa PAO1. Most of the derivatives
effectively blocked pqs QS with micromolar IC50 values of these com-
pounds. Dihydropyrrol-2-one analogue bearing a 4-fluorophenyl ring
and a 2-thiopyridyl linkage (7g) showed most potent activity with an
IC50 value of 32 µM. In addition, these analogues also disrupted bacterial
aggregation and biofilm formation without affecting bacterial growth.
The molecular docking study revealed that compound 7g bound within
the ligand-binding domain of PqsR, with similar interactions with a
previously reported potent PqsR antagonist. Our future efforts will be
focused on improving the potency of these inhibitors by lead optimi-
zation and structure–activity relationship analysis, including extension
of the hydrophobic side chain to facilitate binding to the additional
hydrophobic pocket of PqsR.
OD600nm) only about 11–13% and 13–18% respectively in two hours.
Whereas its control exhibited significantly higher coaggregation rate
about 23% for ATCC and 26% for PAO1. 7g and 7l showed radical
decrease in P. aeruginosa aggregation in comparison to the control. In-
hibition of pqs molecules in P. aeruginosa cell population plays a critical
role in impeding biosynthesis of various essential molecules such as
extracellular DNA (eDNA) which are responsible for coaggregation,
colonization and biofilm formation. Most importantly earlier studies
proved that pqs inhibition associated with debilitating phage mediated
release of DNA in P. aeruginosa cultures and biofilms and eDNA en-
courages physio-chemical interactions that favours bacterial adhesion to
the surface, coaggregation and scaffold for biofilm matrix.34–36 Further,
biomass quantification of 48 hr grown mature biofilm results suggest
overall reduction of 20–25% in biofilm biomass in comparison to the
control (Fig. 4). These results signify that these novel PqsR inhibitors
would facilitate inhibition of pathogen colonization and biofilm
formation.
3

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
Fig. 2. Dose response curve and % Inhibition data of compound 7g and 7l in pqsA-gfp reporter assay in µg/mL. Negative control refers to 1.25% DMSO only and
positive control refers to assay with a known inhibitor of pqs.33.
acetate in hexane or methanol in dichloromethane used as mobile phase.
Table 2
Yields refer to the pure compounds isolated after flash column chro-
PqsR inhibition activity of the compounds using PAO1-pqsA-gfp reporter assay.
matography unless otherwise stated. Melting points of the new com-
Entry
Compounds
(%) Inhibition (at 125 µg/mL)
pounds were evaluated by using SRS MPA100 OptiMelt instrument and
are reported without correction. IR spectra were recorded using Cary
630 ATR FTIR spectrophotometer. High-resolution mass spectrometry
(HRMS) was performed by electrospray (ESI) ionization using a Thermo
LTQ Orbitrap XL instrument at Bioanalytical Mass Spectrometry Facility
(BMSF) of Mark Wainwright Analytical Centre (MWAC), UNSW Sydney.
1H and 13C NMR were recorded in deuterated solvents (CDCl3 and
DMSO‑d6) using Bruker Avance III 300 and Bruker Avance III 400 MHz
instruments (Bruker Pty Ltd, Preston, Australia) at 24 ◦C. Chemical shifts
(δ) are reported as relative to the corresponding solvent peak, with
tetramethylsilane as the internal standard and quoted in parts per
million (ppm). The coupling constants (J) are reported in hertz (Hz). X-
ray crystallography was carried out using single crystal XRD analysis
and data have been deposited in the Cambridge Crystallographic Data
Centre: Compound 7c- Deposition Number 2027653.
1
7a
7b
7c
7d
7e
7f
20
52
43
53
45
46
63
59
45
36
18
62
52
33
20
7
2
3
4
5
6
7
7g
7h
7i
8
9
10
11
12
13
14
15
16
17
18
19
20
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
42
55
40
47
General procedure (A) for bromination of DHP: DBDMH (1.8
mmol) was added in portions to a solution of DHP 1 (3.0 mmol) and Et3N
(3.0 mmol) in dry DCM (25 mL) at 0 ◦C. The reaction was stirred at 0 ◦C
for 30 min, and the reaction progress was monitored by TLC. After
complete conversion, a saturated solution of NH4Cl (10 mL) was added.
The mixture was extracted with DCM (3 × 10 mL), the extract dried with
anhydrous Na2SO4 and concentrated. The crude product was purified
with silica gel flash column chromatography eluting with 30–40%
EtOAc:Hex, and the desired compound (6) was isolated as a white/off-
white solid in 60–77% yield.
5. Experimental section
5.1. Synthesis
All the chemicals (reagents and solvents) were obtained from com-
mercial sources (Chem-Impex Combi-Blocks, and Sigma-Aldrich) and
used without further purification. Reactions were performed using oven-
dried glassware under Argon atmosphere (if required). Room tempera-
ture (rt) refers to the ambient temperature. Reaction progress were
monitored by thin layer chromatography (TLC) using precoated Merck
silica gel 60 F254 plates and visualization using UV light (254 nm). Flash
column chromatography was performed using Grace Davison LC60A
40–63 µm silica gel as the stationary phase and solvent gradients of ethyl
(Z)-5-(bromomethylene)-4-phenyl-1,5-dihydro-2H-pyrrol-2-one (6a):
Following the general procedure A, the title product was obtained as an
off-white solid (451 mg, 60% yield); mp 118–120 ◦C; 1H NMR (300
MHz, CDCl3) δ 8.00 (s, 1H), 7.50 – 7.44 (m, 3H), 7.40 (m, 2H), 6.28 (d, J
= 1.8 Hz, 1H), 6.11 (s, 1H). 13C NMR (76 MHz, CDCl3) δ 169.94, 150.52,
141.66, 131.10, 130.07, 129.31, 129.13, 128.88, 128.58, 128.47,
122.02, 92.86; IR (ATR): νmax 3154, 3109, 3029, 2762, 1886, 1687,
4

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
Fig. 3. Phase-contrast microscopy images showing aggregation of P. aeruginosa PAO1 grown in the presence of (A) Dimethyl sulfoxide (DMSO) only; (B) 250 µM 7g
(C) 250 µM 7l. Scale bar = 20 µm.
Fig. 4. Example of P. aeruginosa settling indicated through decrease in absorbance over time (A). Co-aggregation rate of P. aeruginosa PAO1 and ATCC grown in
presence of 7g and 7l. 7g and 7l showed significant decrease in co-aggregation percentage in comparison to the control (B). Quantification of P. aeruginosa biofilm
biomass grown in presence of 7g and 7l (C).
Fig. 5. Growth curve of PAO1 P. aeruginosa, treated with different concentrations of compound 7g or 7l. Negative controls refers to DMSO (1.25%) only. Positive
controls refer to blank experiment.
5

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
Fig. 6. Molecular docking study. Orientation (A) of ligand M-64 and (B) compound 7g in the pocket of MvfRLBD
.
Fig. 7. (A) 3D Ligand-receptor interactions of compound 7g with MvfRLBD (B) 2D Ligand-receptor interactions of compound 7g with MvfRLBD (C) Overlay poses of 7g
(sticks) and M-64 (blue line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
1632, 1324, 1124, 940, 850, 766, 732, 698; ESI-HRMS m/z: calcd for
C11H8BrNO [M+Na]+: 271.9681; found 271.9683.
(m, 2H), 6.26 (d, J = 1.8 Hz, 1H), 6.06 (s, 1H); 13C NMR (101 MHz,
CDCl3) δ 169.71, 165.12, 162.62, 149.39, 141.68, 130.51, 130.42,
127.19, 127.15, 122.15, 116.46, 116.25, 92.67; IR (ATR): νmax 3114,
3029, 1956, 1892, 1681, 1601, 1498, 1322, 1211, 1157, 943, 835, 700;
ESI-HRMS m/z: calcd for C11H7BrFNO [M+ Na]+: 289.9587; found
289.9588.
(Z)-5-(bromomethylene)-4-(4-fluorophenyl)-1,5-dihydro-2H-pyrrol-2-
one (6b): Following the general procedure A, the title product was ob-
1
◦
tained as an off-white solid (610 mg, 76% yield); mp 128–130 C; H
NMR (300 MHz, CDCl3) δ 7.72 (s, 1H), 7.45 – 7.32 (m, 2H), 7.23 – 7.05
6

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
(Z)-5-(bromomethylene)-4-(4-chlorophenyl)-1,5-dihydro-2H-pyrrol-2-
one (6c): Following the general procedure A, the title product was ob-
129.06, 128.72, 127.99, 120.52, 110.33; IR (ATR): νmax 766, 858, 988,
1125, 1333, 1476, 1577, 1613, 1675, 3018, 3139; ESI-HRMS m/z: calcd
for C17H13NOS [M+Na]+: 302.0610; found 302.0609.
1
◦
tained as an off-white solid (520 mg, 60% yield); mp 142–144 C; H
NMR (300 MHz, CDCl3) δ 8.00 (s, 1H), 7.46 – 7.40 (m, 2H), 7.39 – 7.32
(m, 2H), 6.27 (d, J = 1.8 Hz, 1H), 6.06 (s, 1H); 13C NMR (76 MHz,
CDCl3) δ 169.60, 149.21, 141.48, 136.39, 130.29, 129.83, 129.46,
128.81, 122.40, 92.74; IR (ATR): νmax 3160, 3112, 3031, 2178, 1681,
1628, 1593, 1482, 1369, 1323, 1086, 989, 829, 700; ESI-HRMS m/z:
calcd for C11H7BrClNO [M+Na]+: 305.9292; found 305.9293.
(Z)-5-(((4-chlorophenyl)thio)methylene)-4-phenyl-1,5-dihydro-2H-pyr-
rol-2-one (7c): Following the general procedure B, the title product was
obtained as a yellow solid (38 mg, 64% yield); mp 171–173 ◦C; 1H NMR
(400 MHz, CDCl3) δ 8.06 (s, 1H), 7.48 – 7.37 (m, 5H), 7.31 (s, 4H), 6.24
(s, 1H), 6.08 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 150.32, 138.79,
134.13, 132.34, 131.62, 131.20, 129.81, 129.78, 129.09, 128.69,
122.37, 108.91; IR (ATR): νmax 790, 805, 1088, 1333, 1473, 1672, 2111,
3014, 3144; ESI-HRMS m/z: calcd for C17H12ClNOS [M+Na]+:
336.0220; found 336.0221.
(Z)-5-(bromomethylene)-4-(4-bromophenyl)-1,5-dihydro-2H-pyrrol-2-
one (6d): Following the general procedure A, the title product was ob-
1
◦
tained asan off-white solid (630 mg, 64% yield); mp 144–146 C; H
NMR (300 MHz, CDCl3) δ 8.14 (s, 1H), 7.69 – 7.50 (m, 2H), 7.41 – 7.12
(m, 2H), 6.29 (s, 1H), 6.07 (s, 1H); 13C NMR (76 MHz, CDCl3) δ 169.68,
164.54, 161.25, 149.07, 149.03, 141.39, 133.09, 132.98, 130.94,
130.83, 124.38, 124.34, 122.73, 117.18, 116.90, 115.81, 115.51, 93.05;
IR (ATR): νmax 3158, 3109, 3032, 1937, 1685, 1628, 1589, 1372, 1227,
1067, 991, 825, 700; ESI-HRMS m/z: calcd for C11H7Br2NO [M+H]+:
329.8947; found 329.8949.
(Z)-4-phenyl-5-((pyridin-2-ylthio)methylene)-1,5-dihydro-2H-pyrrol-2-
one (7d): Following the general procedure B, the title product was ob-
tained as a yellow solid (34 mg, 60% yield); mp 226–227 ◦C; 1H NMR
(400 MHz, DMSO- d6) δ 10.45 (s, 1H), 8.46 (ddd, J = 4.9, 1.9, 1.0 Hz,
1H), 7.76 (ddd, J = 8.1, 7.4, 1.9 Hz, 1H), 7.59 – 7.44 (m, 6H), 7.24 (ddd,
J = 7.4, 4.9, 1.0 Hz, 1H), 6.99 (s, 1H), 6.33 – 6.18 (m, 1H); 13C NMR
(101 MHz, DMSO‑d6) δ 170.03, 153.69, 149.91, 149.39, 137.68, 135.95,
131.63, 129.45, 128.93, 128 0.62, 122.78, 121.51, 120.06, 104.42; IR
(ATR): νmax 787, 811, 1118, 1332, 1415, 1575, 1675, 2794, 3013, 3135;
ESI-HRMS m/z: calcd for C16H12N2OS [M+Na]+: 303.0562; found
303.0563.
(Z)-5-(bromomethylene)-4-(2-fluorophenyl)-1,5-dihydro-2H-pyrrol-2-
one (6e): Following the general procedure A, the title product was ob-
tained as an white solid (620 mg, 77% yield); mp 125–127 ◦C; 1H NMR
(400 MHz, CDCl3) δ 7.89 (s, 1H), 7.49 – 7.39 (m, 1H), 7.34 (dd, J = 7.5,
1.8 Hz, 1H), 7.17–7.25 (m, 2H), 6.37 (s, 1H), 5.99 (s, 1H); 13C NMR
(101 MHz, CDCl3) δ 169.52, 160.91, 160.74, 156.63, 143.64, 141.23,
131.68, 131.60, 130.69, 124.75, 124.45, 124.41, 116.64, 116.42, 92.48,
92.45; IR (ATR): νmax 3154, 3033, 2754, 2114, 1894, 1700, 1626, 1482,
1447, 945, 824, 702; ESI-HRMS m/z: calcd for C11H7BrFNO [M+H]+:
289.9587; found 289.9588.
(Z)-4-(4-fluorophenyl)-5-((phenylthio)methylene)-1,5-dihydro-2H-pyr-
rol-2-one (7e): Following the general procedure B, the title product was
obtained as a yellow solid (55 mg, 92% yield); mp 202–204 ◦C; 1H NMR
(300 MHz, CDCl3) δ 7.90 (s, 1H), 7.46 – 7.28 (m, 6H), 7.14 (t, J = 8.6 Hz,
1H), 6.20 (d, J = 1.8 Hz, 1H), 6.12 (s, 1H); 13C NMR (76 MHz, CDCl3) δ
169.91, 163.59, 149.16, 137.90, 133.57, 130.59, 130.48, 130.09,
129.69, 128.09, 120.62, 116.39, 116.10, 110.25; IR (ATR): νmax 3148,
3066, 2938, 2764, 1680, 1607, 1330, 1222, 834, 797, 678; ESI-HRMS
m/z: calcd for C17H12FNOS [M+H]+: 298.0696; found 298.0697.
(Z)-5-(((4-chlorophenyl)thio)methylene)-4-(4-fluorophenyl)-1,5-dihy-
dro-2H-pyrrol-2-one (7f): Following the general procedure B, the title
product was obtained as a yellow solid (61 mg, 92% yield); mp
225–226 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.98 (bs, 1H), 7.44 – 7.35 (m,
2H), 7.31 (s, 4H), 7.14 (dd, J = 9.5, 7.7 Hz, 2H), 6.21 (d, J = 1.9 Hz, 1H),
6.03 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 169.92, 164.98, 162.49,
149.19, 138.70, 134.28, 132.17, 131.29, 130.56, 130.48, 129.84,
127.67, 127.64, 121.02, 116.41, 116.19, 108.91, 108.89; IR (ATR): νmax
3143, 3056, 2961, 2768, 1901, 1669, 1607, 1500, 1088, 1007, 790; ESI-
HRMS m/z: calcd for C17H11ClFNOS [M+H]+: 332.0307; found
332.0306.
(Z)-5-(bromomethylene)-4-(3-fluorophenyl)-1,5-dihydro-2H-pyrrol-2-
one (6f): Following the general procedure B, the title product was ob-
tained as a white solid (520 mg, 64% yield); mp 129–131 ◦C; 1H NMR
(300 MHz, CDCl3) δ 8.18 (s, 1H), 7.44 (m, J = 8.0, 5.7 Hz, 1H), 7.23 –
7.02 (m, 3H), 6.31 (d, J = 1.8 Hz, 1H), 6.12 (s, 1H). 13C NMR (76 MHz,
CDCl3) δ 169.68, 164.54, 161.25, 149.07, 149.03, 141.39, 133.09,
132.98, 130.94, 130.83, 124.38, 124.34, 122.73, 117.18, 116.90,
115.81, 115.51, 93.05; IR (ATR): νmax 3123, 3004, 2756, 2052, 1930,
1703, 1632, 1565, 1333, 1245, 958, 886, 843, 785, 701; ESI-HRMS m/z:
calcd for C11H7BrFNO [M+Na]+: 289.9587; found 289.9588.
General procedure (B) for synthesis of thioether compounds: A
mixture of thiol (0.24 mmol) and Et3N (0.4 mmol) in dry CH2Cl2 (2 mL)
was added dropwise to a solution of bromo-DHP 6 (0.2 mmol) in dry
DCM (5 mL) at 0 ◦C. The reaction was stirred at room temperature for 2
to 24 h. Progress of the reaction was monitored by TLC. After completion
of the reaction, water was added. The mixture was extracted with DCM
(3 × 10 mL), and the combined organic extracts were washed with brine,
dried with anhydrous Na2SO4 and concentrated. The crude product was
purified with basic aluminium oxide column chromatography eluting
with 1–2% MeOH:CH2Cl2, and the desired compound (7) was isolated as
a yellow solid.
(Z)-4-(4-fluorophenyl)-5-((pyridin-2-ylthio)methylene)-1,5-dihydro-
2H-pyrrol-2-one (7g): Following the general procedure B, the title
product was obtained as a yellowish solid (44 mg, 73% yield); mp
214–215 ◦C; 1H NMR (300 MHz, DMSO- d6) δ 10.46 (s, 1H), 8.47 (ddd, J
= 4.9, 1.9, 0.9 Hz, 1H), 7.82 – 7.69 (m, 1H), 7.64 – 7.55 (m, 2H), 7.48
(dt, J = 8.1, 1.0 Hz, 1H), 7.37 (t, J = 8.8 Hz, 2H), 7.24 (ddd, J = 7.4, 4.9,
1.0 Hz, 1H), 6.96 (s, 1H), 6.28 (d, J = 1.6 Hz, 1H); 13C NMR (76 MHz,
DMSO‑d6) δ 169.95, 153.67, 149.94, 148.26, 137.71, 135.96, 130.93,
130.82, 128.09, 122.79, 121.55, 120.24, 116.11, 115.82, 104.47; IR
(ATR): νmax 3135, 2970, 2864, 2782, 1897, 1683, 1625, 1500, 1116,
808, 767; ESI-HRMS m/z: calcd for C16H11FN2OS [M+H]+: 299.0649;
found 299.0649.
(Z)-4-phenyl-5-((propylthio)methylene)-1,5-dihydro-2H-pyrrol-2-one
(7a): Following the general procedure B, the title product was obtained
1
◦
as a yellow solid (22 mg, 44% yield); mp 128–132 C; H NMR (400
MHz, CDCl3) δ 7.71 (bs, 1H), 7.50 – 7.42 (m, 3H), 7.42 – 7.35 (m, 2H),
6.14 (dd, J = 1.9, 0.6 Hz, 1H), 5.98 (s, 1H), 2.94 – 2.59 (t, 2H), 1.76 –
1.64 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
170.12, 149.70, 135.89, 132.10, 129.53, 128.98, 128.72, 119.51,
113.61, 37.10, 23.80, 13.17; IR (ATR): νmax 790, 833, 1073, 1129, 1335,
1619, 1677, 2959, 3143; ESI-HRMS m/z: calcd for C14H15NOS
[M+Na]+: 268.0767; found 268.0766.
(Z)-4-(4-chlorophenyl)-5-((propylthio)methylene)-1,5-dihydro-2H-pyr-
rol-2-one (7h): Following the general procedure B, the title product was
obtained as a yellow solid (20 mg, 35% yield); m.p. 146–148 ◦C; 1H NMR
(300 MHz, CDCl3) δ 8.05 (s, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.33 (d, J =
8.2 Hz, 2H), 6.13 (s, 1H), 5.95 (s, 1H), 2.81 (t, J = 7.2 Hz, 2H), 1.69 (h, J
= 7.3 Hz, 2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (76 MHz, CDCl3) δ
172.61, 148.36, 135.76, 135.50, 130.49, 129.99, 129.28, 119.74,
114.24, 37.17, 23.82, 13.18; IR (ATR): νmax 3571, 3138, 2958, 2922,
1954, 1676, 1616, 1332, 1090, 864, 826, 661; ESI-HRMS m/z: calcd for
(Z)-4-phenyl-5-((phenylthio)methylene)-1,5-dihydro-2H-pyrrol-2-one
(7b): Following the general procedure B, the title product was obtained
1
◦
as a yellow solid (36 mg, 60% yield) ; mp 171–172 C; H NMR (400
MHz, CDCl3) δ 7.87 (s, 1H), 7.52 – 7.41 (m, 5H), 7.41 – 7.28 (m, 5H),
6.23 (d, J = 1.8 Hz, 1H), 6.20 (s, 1H); 13C NMR (101 MHz, CDCl3) δ
170.05, 150.31, 138.01, 133.66, 131.72, 130.02, 129.74, 129.65,
C
14H14ClNOS [M+Na]+: 302.0377; found 302.0377.
(Z)-4-(4-chlorophenyl)-5-((phenylthio)methylene)-1,5-dihydro-2H-
7

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
pyrrol-2-one (7i): Following the general procedure B, the title product
was obtained as a yellow solid (50 mg, 80% yield); mp 209–210 ◦C; 1H
NMR (400 MHz, DMSO- d6) δ 10.73 – 10.16 (m, 1H), 7.56 – 7.51 (m,
4H), 7.47 – 7.27 (m, 5H), 6.31 (d, J = 1.6 Hz, 1H), 6.08 (s, 1H); 13C NMR
(101 MHz, DMSO‑d6) δ 169.79, 147.88, 136.81, 134.19, 133.91, 130.43,
130.40, 130.29, 129.68, 128.96, 128.94, 128.91, 127.50, 120.64,
108.82; IR (ATR): νmax 3140, 3053, 3013, 2112, 1899, 1672, 1598,
1478, 1329, 1091, 826, 788; ESI-HRMS m/z: calcd for C17H12ClNOS
[M+H]+: 314.0401; found 314.0401.
135.61, 131.93, 130.81, 130.69, 123.00, 122.80, 121.56, 120.51,
104.61; IR (ATR): νmax 3748, 3617, 3121, 3121, 2993, 1688, 1573,
1453, 1121, 807, 748; ESI-HRMS m/z: calcd for C16H11BrN2OS [M+H]+:
358.9648; found 358.9649.
(Z)-4-(2-fluorophenyl)-5-((propylthio)methylene)-1,5-dihydro-2H-pyr-
rol-2-one (7p): Following the general procedure B, the title product was
obtained as a yellow solid (27 mg, 51% yield); mp 175–176 ◦C; 1H NMR
(400 MHz, CDCl3) δ 7.83 (s, 1H), 7.42 (m, J = 8.2, 7.2, 5.1, 1.9 Hz, 1H),
7.32 (td, J = 7.4, 1.9 Hz, 1H), 7.27 – 7.09 (m, 3H), 6.22 (s, 1H), 5.89 –
5.84 (m, 1H), 2.79 (t, J = 7.2 Hz, 2H), 1.68 (q, J = 7.3 Hz, 2H), 1.00 (t, J
= 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 170.05, 161.05, 158.56,
143.02, 135.62, 131.24, 131.16, 131.11, 131.09, 124.43, 124.39,
122.13, 119.82, 119.68, 116.64, 116.42, 113.57, 37.11, 23.78, 13.14; IR
(ATR): νmax 3752, 3132, 2961, 2924, 2861, 1651, 1488, 1333, 1221,
818, 790; ESI-HRMS m/z: calcd for C14H14FNOS [M+H]+: 264.0853;
found 264.0854.
(Z)-4-(4-chlorophenyl)-5-(((4-chlorophenyl)thio)methylene)-1,5-dihy-
dro-2H-pyrrol-2-one (7j): Following the general procedure B, the title
product was obtained as a yellow solid (35 mg, 50% yield); mp
243–246 ◦C; 1H NMR (300 MHz, DMSO- d6) δ 10.49 (s, 1H), 7.53 (s, 4H),
7.45 (d, J = 2.2 Hz, 4H), 6.33 (s, 1H), 6.07 (s, 1H); 13C NMR (76 MHz,
DMSO- d6) δ 169.82, 147.95, 137.54, 134.23, 133.07, 132.16, 130.63,
130.44, 130.20, 129.53, 128.95, 120.93, 107.75; IR (ATR): νmax 3150,
3019, 2296, 1901, 1678, 1612, 1475, 1330, 1100, 1007, 795, 739; ESI-
HRMS m/z: calcd for C17H11Cl2NOS [M+Na]+: 369.9831; found
369.9831.
(Z)-4-(2-fluorophenyl)-5-((phenylthio)methylene)-1,5-dihydro-2H-pyr-
rol-2-one (7q): Following the general procedure B, the title product was
obtained as a yellow solid (52 mg, 87% yield); mp 170–171 ◦C; 1H NMR
(400 MHz, CDCl3) δ 7.80 (s, 1H), 7.53 – 7.29 (m, 7H), 7.26 – 7.10 (m,
3H), 6.30 (s, 1H), 6.05 (d, J = 1.3 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ
169.87, 160.26, 158.37, 143.75, 137.91, 133.73, 131.47, 131.39,
131.08, 131.05, 129.91, 129.64, 129.21, 127.93, 124.56, 124.52,
123.28, 116.69, 116.48, 109.77; IR (ATR): νmax 3126, 3061, 3018, 2762,
1909, 1677, 1476, 1327, 1102, 851, 826, 766, 736; ESI-HRMS m/z:
calcd for C17H12FNOS [M+H]+: 298.0696; found 298.0696.
(Z)-4-(4-chlorophenyl)-5-((pyridin-2-ylthio)methylene)-1,5-dihydro-
2H-pyrrol-2-one (7k): Following the general procedure B, the title
product was obtained as a yellow solid (40 mg, 63% yield); mp
209–210 ◦C; 1H NMR (300 MHz, DMSO‑d6) δ 10.55 – 10.40 (s, 1H), 8.49
(ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 7.77 (td, J = 7.7, 1.9 Hz, 1H), 7.64 – 7.54
(m, 4H), 7.49 (dt, J = 8.0, 1.0 Hz, 1H), 7.25 (ddd, J = 7.4, 4.8, 1.0 Hz,
1H), 6.98 (s, 1H), 6.32 (d, J = 1.6 Hz, 1H); 13C NMR (76 MHz, DMSO‑d6)
δ 169.88, 153.62, 149.97, 148.03, 137.74, 135.70, 134.31, 130.46,
129.02, 122.81, 121.58, 120.54, 104.63; IR (ATR): νmax 3132, 2996,
2780, 1686, 1574, 1452, 1120, 1090, 862, 805; ESI-HRMS m/z: calcd for
(Z)-5-(((4-chlorophenyl)thio)methylene)-4-(2-fluorophenyl)-1,5-dihy-
dro-2H-pyrrol-2-one (7r): Following the general procedure B, the title
product was obtained as a yellow solid (50 mg, 75% yield); mp
208–210 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.42 (m, J = 8.3,
7.1, 5.2, 1.8 Hz, 1H), 7.37 – 7.28 (m, 5H), 7.24 – 7.15 (m, 2H), 6.31 (s,
1H), 5.96 (d, J = 1.4 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 172.66,
160.50, 158.48, 143.88, 138.69, 134.09, 132.33, 131.59, 131.51,
131.05, 129.79, 124.62, 124.59, 121.80, 116.73, 116.51, 108.43; IR
(ATR): νmax 3145, 3016, 2922, 1906, 1674, 1617, 1474, 1090, 816, 765;
ESI-HRMS m/z: calcd for C17H11ClFNOS [M+H]+: 332.0307; found
332.0307.
C
16H11ClN2OS [M+H]+: 315.0353; found 315.0354.
(Z)-4-(4-bromophenyl)-5-((propylthio)methylene)-1,5-dihydro-2H-pyr-
rol-2-one (7l): Following the general procedure B, the title product was
obtained as a yellow solid (24 mg, 37% yield); mp 141–144 ◦C; 1H NMR
(400 MHz, CDCl3) δ 7.65 (bs, 1H), 7.64 – 7.54 (m, 2H), 7.31 – 7.22 (m,
2H), 6.13 (dd, J = 1.9, 0.6 Hz, 1H), 5.93 (s, 1H), 2.85 – 2.77 (t, 2H), 1.76
– 1.62 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
169.76, 148.43, 135.48, 132.25, 130.90, 130.21, 124.01, 119.69, 37.15,
23.81, 13.16; IR (ATR): νmax 3133, 3014, 2957, 2922, 1670, 1612, 1480,
1009, 820; ESI-HRMS m/z: calcd for C14H14BrNOS [M+H]+: 324.0052;
found 324.0053.
(Z)-4-(2-fluorophenyl)-5-((pyridin-2-ylthio)methylene)-1,5-dihydro-
2H-pyrrol-2-one (7s): Following the general procedure B, the title
product was obtained as a yellow solid (42 mg, 70% yield); mp
218–219 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 10.58 (s, 1H), 8.44 (ddd, J
= 4.9, 1.9, 1.0 Hz, 1H), 7.75 (ddd, J = 8.0, 7.3, 1.8 Hz, 1H), 7.61 – 7.32
(m, 5H), 7.23 (ddd, J = 7.4, 4.9, 1.1 Hz, 1H), 6.83 (s, 1H), 6.31 (s, 1H);
13C NMR (101 MHz, DMSO‑d6) δ 169.93, 160.28, 157.82, 153.46,
149.90, 142.92, 137.70, 135.86, 131.63, 131.55, 131.33, 131.30,
124.82, 124.79, 122.75, 122.58, 121.57, 119.21, 119.07, 116.36,
116.14, 104.29; IR (ATR): νmax 3127, 2993, 2783, 2320, 1907, 1686,
1624, 1575, 1416, 1121, 825, 770; ESI-HRMS m/z: calcd for
(Z)-4-(4-bromophenyl)-5-((phenylthio)methylene)-1,5-dihydro-2H-pyr-
rol-2-one (7m): Following the general procedure B, the title product was
obtained as a yellow solid (56 mg, 78% yield); mp 202–204 ◦C; 1H NMR
(400 MHz, CDCl3) δ 7.96 (bs, 1H), 7.63 – 7.53 (m, 2H), 7.41 – 7.27 (m,
7H), 6.22 (s, 1H), 6.12 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 13C NMR
(76 MHz, DMSO) δ 169.78, 147.93, 136.73, 133.89, 131.86, 130.65,
129.68, 128.96, 127.49, 122.89, 120.61, 108.84.; IR (ATR): νmax 3140,
3056, 3013, 2923, 2111, 1897, 1672, 1611, 1072, 823, 788; ESI-HRMS
m/z: calcd for C17H12BrNOS [M+H]+: 357.9896; found 357.9897.
(Z)-4-(4-bromophenyl)-5-(((4-chlorophenyl)thio)methylene)-1,5-dihy-
dro-2H-pyrrol-2-one (7n): Following the general procedure B, the title
product was obtained as a yellow solid (55 mg, 70% yield); mp
240–241 ◦C; 1H NMR (300 MHz, CDCl3) δ 7.81 (s, 1H), 7.63 – 7.49 (m,
2H), 7.32 – 7.18 (m, 6H), 6.17 (d, J = 1.7 Hz, 1H), 5.97 (s, 1H); 13C NMR
(76 MHz, CDCl3) δ 170.34, 159.74, 149.05, 138.27, 135.48, 135.27,
132.38, 132.01, 131.34, 130.43, 130.17, 129.87, 124.33, 121.20,
109.07; IR (ATR): νmax 3138, 3060, 3017, 2299, 1678, 1612, 1475,
1091, 1009, 805, 738; ESI-HRMS m/z: calcd for C17H11BrClNOS
[M+H]+: 391.9506; found 391.9506.
C
16H11FN2OS [M+Na]+: 321.0468; found 321.0468.
(Z)-4-(3-fluorophenyl)-5-((pyridin-2-ylthio)methylene)-1,5-dihydro-
2H-pyrrol-2-one (7t): Following the general procedure B, the title prod-
uct was obtained as a yellow solid (50 mg, 84% yield); mp 215–217 ◦C;
1H NMR (300 MHz, DMSO- d6) δ 10.53 (s, 1H), 8.48 (ddd, J = 4.9, 1.9,
0.9 Hz, 1H), 7.77 (ddd, J = 8.1, 7.4, 1.9 Hz, 1H), 7.59 (td, J = 8.0, 6.0
Hz, 1H), 7.50 (dt, J = 8.1, 1.0 Hz, 1H), 7.44 – 7.32 (m, 3H), 7.25 (ddd, J
= 7.4, 4.9, 1.1 Hz, 1H), 7.02 (s, 1H), 6.43 – 6.14 (m, 1H); 13C NMR (76
MHz, DMSO‑d6) δ 169.81, 163.85, 160.79, 153.61, 149.93, 147.87,
137.72, 135.57, 133.89, 131.07, 130.96, 124.86, 122.83, 121.58,
120.90, 116.42, 116.14, 115.69, 115.39, 104.72; IR (ATR): νmax 3139,
3006, 2787, 2343, 1913, 1688, 1574, 1451, 1119, 957, 842, 778; ESI-
HRMS m/z: calcd for C16H11FN2OS [M+Na]+: 321.0468; found
321.0468.
(Z)-4-(4-bromophenyl)-5-((pyridin-2-ylthio)methylene)-1,5-dihydro-
2H-pyrrol-2-one (7o): Following the general procedure B, the title
product was obtained as a yellow solid (48 mg, 67% yield); mp
235–236 ◦C; 1H NMR (300 MHz, DMSO- d6) δ 10.50 (s, 1H), 8.49 (ddd, J
= 4.9, 1.9, 0.9 Hz, 1H), 7.84 – 7.65 (m, 3H), 7.61 – 7.44 (m, 3H), 7.25
5.2. GFP reporter (pqs:gfp) strain assay
(ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 6.97 (s, 1H), 6.32 (d, J = 1.6 Hz, 1H); 13
C
NMR (101 MHz, DMSO‑d6) δ 169.86, 153.60, 149.96, 148.07, 137.72,
The assay for PqsR inhibition activity was performed using the PAO1
8

S. Sabir et al.
Bioorganic & Medicinal Chemistry 31 (2021) 115967
P. aeruginosa strain carrying the PqsR-regulated pqsA promoter fused to
gfp.38 The compounds were dissolved in 100% DMSO to make 40 mM
stock solutions. The test compounds (serially diluted with medium) were
then incubated with overnight cultures of PAO1-pasA-gfp using MHB
(Mueller Hinton Broth) in 96-well plates at 37 ◦C with intermittent
shaking. Readings were taken at 15 min intervals for at least 6–8 h and
both GFP fluorescence and OD600 were recorded. The fluorescence
reader (Infinite M1000 pro). DMSO-treated biofilm was used as a control
and normalized to 100% growth, while the percentage decrease in
biofilm biomass grown in presence of compounds 7g and 7l was ana-
lysed with respect to the DMSO control.
5.6. Planktonic growth assay
values shown in the graph were normalized with respect to OD600
.
An overnight culture of P. aeruginosa PAO1 at a concentration of 5 ×
105 CFU/mL in MHB was transferred to a 96-well plate. The test com-
pounds at a concentration of 10 mg/mL in DMSO were added to each
well at a maximum concentration of 125 µg/mL and incubated for 24 h
at 37 ◦C with intermittent agitation. The OD600 was recorded for each
well at 15 min intervals to measure bacterial growth. The experiment
was carried out once for each compound with three technical replicates
per experiment.
Negative control refers to medium containing DMSO (1.25%) as the
highest concentration of the test compound. The pqs inhibition assay
was carried out in triplicate manner.
5.3. Co-aggregation of P. aeruginosa strains
Planktonic cultures of P. aeruginosa ATCC 25619 and PAO1 were
grown in tryptone soya broth (TSB, Oxoid, Thermo Scientific, Australia)
overnight at 37 ◦C and 150 rpm. The cultures were then re-suspended in
5 mL TSB media at a bacterial density of OD600nm = 0.1 ± 0.02 in
presence of compounds 7g and 7l (150, 250 and 400 µM) and DMSO
(control) for 24 h, at 37 ◦C and 150 rpm. After 24 h growth, bacterial
pellet was collected via centrifugation (5000g, 10 min, 25 ◦C), the su-
pernatant was removed and the pellet were washed twice with PBS with
centrifugation (5000g, 5 min, 25 ◦C). The pellet was finally resuspended
in PBS at a density of 0.1 ± 0.02 (OD600nm) and 1 mL of the bacterial
suspension was transferred into a 1 mL plastic cuvette (SARSTEDT) and
its absorbance at OD600nm was measured every 15 min for up to 120 min
at room temperature without agitation. The decrease in absorbance, as a
result of coaggregation of bacteria and settling down due to gravity, was
determined as a percentage reduction in OD after 120 min.39
5.7. Pan assay interference compounds (PAINS) screening
PAINS are the compounds which gives false and misleading results in
the biological assays mainly false positive. To determine whether our
new compounds belong to these categories or not we have screened
these analogues in a PAIN filter (SwissADME) and the predicted results
shows that these analogues validate most of the characteristics of drug
likeness and cannot be categorised as PAINS.41
Funding
This work was supported by a Discovery Project from Australian
Research Council grant (DP180100845).
OD0m in ꢀ OD
120m in × 100%
(1)
Declaration of Competing Interest
OD0m in
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.
where OD0min is the initial OD at the beginning of an experiment and
OD120min is the OD after 120 min. 1 mL of PBS without bacteria was used
as the blank.
Acknowledgements
5.4. Imaging of P. aeruginosa biofilms
We thank the NMR, X-ray and BMSF facility, at Mark Wainwright
Analytical Centre (MWAC), UNSW Sydney. S.Sabir would like to
acknowledge UNSW TFS scholarship.
Planktonic cultures of P. aeruginosa ATCC 25619 and PAO1 were
grown as described above. The cultures were then resuspended in TSB
media at a bacterial density of OD600nm = 0.1 ± 0.02. 1 mL of the
resuspended bacterial cells were then added into 12-well plates (Corning
Corp. USA) and incubated for at 37 ◦C and 100 rpm to initiate bacterial
adhesion and biofilm growth. Where indicated, biofilm growth was also
initiated in the presence of compounds 7g and 7l at 250 µM. As a control,
P. aeruginosa was also grown in presence of DMSO. After 24 h, the
biofilms were washed twice with PBS to remove any loosely adhered
bacteria and imaged using phase contrast microscopy (Zeiss, Axio,
Germany) to assess biofilm morphology as described previously.40
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115967.
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