Design and synthesis of aryl-substituted pyrrolidone derivatives as quorum sensing inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104376

Quorum sensing, a common cell-to-cell communication system, is considered to have promising application in antibacterial therapy since they are expected to induce lower bacterial resistance than conventional antibiotics. However, most of present quorum sensing inhibitors have potent cell toxicity, which limits their application. In this study we evaluated the diverse quorum sensing inhibition activities of different biaromatic furanones and brominated pyrrolones. On this basis, we further designed and synthesized a new series of aryl-substituted pyrrolones 12a-12f. In the quorum sensing inhibition assay, compound 12a showed improved characteristics and low toxicity against human hepatocellular carcinoma cell. In particular, it can inhibit the pyocyanin production and protease activity of Pseudomonas aeruginosa by 80.6 and 78.5%, respectively. Besides, in this series, some compounds exerted moderate biofilm inhibition activity. To sum up, all the findings indicate that aryl-substituted pyrrolidone derivatives are worth further investigation as quorum sensing inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.104376

Journal Pre-proofs
Design and synthesis of aryl-substituted pyrrolidone derivatives as quorum
sensing inhibitors
Zhiyang Liu, Panpan Zhang, Yinhui Qin, Nan Zhang, Yuetai Teng, Henrietta
Venter, Shutao Ma
PII:
S0045-2068(20)31674-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104376
YBIOO 104376
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 November 2019
4 September 2020
10 October 2020
Please cite this article as: Z. Liu, P. Zhang, Y. Qin, N. Zhang, Y. Teng, H. Venter, S. Ma, Design and synthesis of
aryl-substituted pyrrolidone derivatives as quorum sensing inhibitors, Bioorganic Chemistry (2020), doi: https://
doi.org/10.1016/j.bioorg.2020.104376
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Design and synthesis of aryl-substituted pyrrolidone
derivatives as quorum sensing inhibitors
Zhiyang Liu a,1, Panpan Zhang a,1, Yinhui Qin a, Nan Zhang a, Yuetai Teng a, Henrietta Venter b,
Shutao Ma a, *
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West
Wenhua Road, Jinan 250012, (P. R. China)
b School of Pharmacy and Medical Sciences, University of South Australia, SA 5000, Australia
1 The authors contributed equally.
* Corresponding author: Shutao Ma
Department of Medicinal Chemistry
School of Pharmaceutical Sciences, Shandong University
44, West Culture Road, Jinan 250012, P. R. China
Tel/Fax: +86 531 88382009; E-mail: mashutao@sdu.edu.cn

ABSTRACT
Quorum sensing, a common cell-to-cell communication system, is considered to have promising application
in antibacterial therapy since they are expected to induce lower bacterial resistance than conventional
antibiotics. However, most of present quorum sensing inhibitors have potent cell toxicity, which limits their
application. In this study we evaluated the diverse quorum sensing inhibition activities of different biaromatic
furanones and brominated pyrrolones. On this basis, we further designed and synthesized a new series of aryl-
substituted pyrrolones 12a-12f. In the quorum sensing inhibition assay, compound 12a showed improved
characteristics and low toxicity against human hepatocellular carcinoma cell. In particular, it can inhibit the
pyocyanin production and protease activity of Pseudomonas aeruginosa by 80.6 and 78.5%, respectively.
Besides, in this series, some compounds exerted moderate biofilm inhibition activity. To sum up, all the
findings indicate that aryl-substituted pyrrolidone derivatives is worth further investigation as quorum sensing
inhibitors.
KEYWORDS：Quorum sensing, Pseudomonas aeruginosa, Rubrolide, Pyocyanin
1

1 Introduction
Drug-resistance bacteria have become a growing concern in the world. Patients with infections caused
by drug-resistant bacteria are at increased risk of death. It is estimated that antimicrobial resistance induces
seven hundred thousand to several million deaths per year according to the WHO global antimicrobial
resistance surveillance report[1]. This trend will definitely continue to rise if no effective measures are carried
out.
Quorum sensing (QS) is a mechanism of bacterial communication (a cell-to-cell communication), which
regulates various phenotypes such as virulence factor expression and biofilm development [2, 3]. Up to now,
there are mainly three different QS systems: acylated homoserine lactones (AHLs), peptide signals and
autoinducer-2 (AI-2). Besides, there are also many other QS signals like Pseudomonas quinolone signal (PQS)
and diffusible signal factor (DSF). It is believed that more QS systems will be discovered in the future[2, 4].
Biofilms enable bacteria to create a microenvironment that allows bacteria to attach to the surface of the host
and evade its immune responses, even excluding standard antibiotic agent[5], which will result in the treatment
of bacteria associated with biofilms to require approximately 10- to 1000-fold higher doses of antibiotic than
that of planktonic bacteria[6]. In general, more than 80% of bacterial infections are related to biofilms. Since
QS is not an essential process in bacteria cells, QS mutants in general don’t show growth defects. Hence,
preventing the production of virulence factors or biofilm formation by blocking or interfering with the
communication between bacteria is an promising strategy to combat bacterial infections, thereby reducing the
possibility of causing bacterial resistance[7]. An ideal quorum sensing inhibitor (QSI) must regulate bacterial
virulence production without affect bacterial growth.
Br
Br
Br
Br
N
Br
O
Br
O
Br
O
O
Br
O
Br
O
O
4
Furanone 30 (3)
2
1
Figure 1. The structures of some quorum sensing inhibitors.
In 1993, compound 1 (Fig. 1) was firstly isolated from the marine red algae Delisea Pulchara[8], which
was then proved to be a potent QSI[9, 10]. Since then, much focus has been put on this molecule. The structure-
activity relationship study[11] shows that a conjugated exocyclic brominated vinyl group on the furanone ring
is the most essential structural element for the inhibition activity against the biofilm formation of Escherichia
coli. The mono-substituted bromine atom on the saturated carbon plays a negative role in biofilm
inhibition[11], while the biofilm formation can be regulated through QS in Pseudomonas aeruginosa[2].
Furthermore, those furanones with the brominated groups on the saturated carbons also appear to be toxic[12].
Compound 2 and furanone 30 (3) (Fig. 1), designed and synthesized eventually as an optimized molecules,
have been proved to be potent antagonists for LasR[13]. The predicted docking mode (Fig. 2) indicates that
the carbonyl group in the molecule can perform as a hydrogen bond receptor with Trp60, and the other oxygen
atom also forms a hydrogen bond with Arg61, thereby enhancing the binding affinity. However, all those
brominated furanones possess various toxicity, which limits their clinical application[12]. In 2015, Wai-Kean
et.al designed and synthesized various lactam analogues of furanones and tested for QS inhibition against the
E. coli AHL-monitor strain JB357 gfp (ASV). Among them, compound 4 (Fig. 1) showed the best activity,
which indicates that 1,5-dihydropyrrol-2-ones have the potential for a further development of new QS
inhibitors[14].
2

Figure 2. The predicted binding mode of furanone 30 with LasR (PDB ID: 2UV0).
In 2012, antimicrobial rubrolides (Fig. 3) with the furanone ring were extracted and identified from a South
African Species of Synoicum Tunicate. Biological testing results indicated that they had moderate antibacterial
properties against methicillin resistant Staphylococcus epidermidis and diverse growth inhibition against
Enterococcus faecalis and E. coli[9]. Simultaneously, several antimicrobial cadiolide analogs (Fig. 3) were
firstly isolated from the Ascidian Synoicum sp. Most of them were found to be broad-spectrum antibacterial
agents. Besides, several compounds also exhibited significant inhibition of Candida albicans-derived
isocitrate lyase and Na+/K+-ATPase, and moderate activity against bacterial sortase A[15].
OH
Br
Br
O
Cl
O
Br
O
O
O
HO
H3C
Br
OH
Br
Br
OCH3
OCH3
Br
Cadiolide I (6)
Rubrolide I (5)
Figure 3. The structure of cadiolide I (MIC: 1.6 μg/mL against S. enterica) and rubrolide I (IC50 = 1.51 ± 0.116
μg/mL against E. faecalis while did not inhibit bacterial growth).
R1
O
O
R1
R1
Br
series 1
Br
R2
N
O
O
Br
O
R1
N
Br
O
series 3
Aryalkyl-substituted pyrrolidone
R2
series 2
Figure 4. The design strategy of aryl-substituted pyrrolidone.
This study focuses on the structural optimization of brominated furanones and pyrrolidones. Firstly, various
biaromatic furanone derivatives were designed, synthesized and evaluated to screen out suitable aromactic
substituents. Pyocyanin is one of the virulence factors secreted by P. aeruginosa, which can regulate the ion
transport of P. aeruginosa and interrupt the cell respiratory chain. Based on the various colors of different
states of pyocyanin, we can quantify the pyocyanin of P. aeruginosa[16, 17]. Besides, we also use a common
substrate “azocasein” to determine the activity of protease that can degrade immune components in P.
aeruginosa[16]. Secondly, brominated pyrrolidones with various N-substituents were synthesized and
evaluated to confirm suitable N-alkyl and N-aryl substituents. Finally, a new series of the aryl-substituted
pyrrolidone derivatives as QSIs were designed and synthesized on the basis of the optimization results from
the above two series of the furanones and pyrrolidones (Fig. 4).
3

2 Results and discussion
2.1 Synthesis of compounds 9-12
The biaromatic furanone derivatives were synthesized from commercially available levulinic acid as shown
in Scheme 1[18, 19]. In this procedure, compound 7 was dibrominated by 2.2 eq bromine to give compound
8 in a yield of 78%. After that, furanone 30 was prepared in a moderate yield (63%) through the dehydration
and cyclization of compound 8 under the condition of concentrated sulfuric acid. A subsequent Suzuki-
Miyaura cross-coupling between furanone 30 and arylboronic acids in the presence of Na2CO3,
tetrabutylammonium bromide and a catalytic amount of Pd[P(C6H5)3]4 resulted in the biaromatic furanone
derivatives (9a-9n) in different yields (38-89%)[19, 20]. Seen from the reaction mechanism, electron-
withdrawing groups arylboronic acids usually tend to be bad coupling partners, because they can be less
nucleophilic and transmetalate more slowly than arylboronic acids with electron neutral or electron-donating
groups[21]. In addition, the low conversions of the Suzuki cross-coupling reactions can be explained partly by
the formation of the biphenyls.
Furanone 30 reacted with various amines in dichloromethane (DCM) to yield the corresponding hydroxy-
lactams (10a-10i). Then the hydroxy-lactams were dehydrated by P2O5 to generate the brominated pyrrolidone
derivatives (11a-11i) in various yields[22].
1 = 2-Methoxy
1 = 2-methyl
1 = 2-fluoro
1 = 2-trifluoromethyl
1 = 3-methoxy
R
1 = 3-trifluoromethyl
R1 = 4-methoxy
R1 = 4-methyl
a:
b:
c:
d:
e:
f:
h:
i:
R
R
O
j:
R
1 = 4-fluoro
R1 = 4-trifluoromethyl
k:
l:
R
R
O
R
R1 = 3-methyl
R
1 = hydrogen
R1 = 4-phenyl
m:
n:
R1
1 = 3-fluoro
R1
g:
R
ⅲ
9a-9n
Br
Br
Br
ⅰ
ⅱ
O
O
COOH
O
COOH
O
Br
7
iv
Br
HO
Br
8
Furanone 30
Br
Br
v
N
N
2 = benzyl
R2
R2
a:
R
R
2 = n-butyl
2 = n-amyl
2 = n-hexyl
f:
R
R
2 = phenethyl
b:
c:
g:
h:
i:
O
O
R
R
2 = 4-fluorobenzyl
10a-10i
11a-11i
R
2 = n-heptyl
2 = n-octyl
R2 = tetrahydrofurfuryl
d:
e:
R
Scheme 1. Synthesis of rubrolide analogs 9a-9n and 11a-11i. Reagents and conditions: i: Br2, HBr in acetic acid,
petroleum ether (60-90 C); iii: 98%H2SO4, 115 C, 40 min; iii: arylboronic acid, Na2CO3, TBAB, Pd[P(C6H5)3]4,
toluene, H2O, reflux, 12 h; iv: amines, DCM, 0 C, 40 min; v: P2O5, reflux, 3 h.
After initial activity screening, we roughly determined that 2-methoxyphenyl and the 3-tolyl groups were
suitable aryl substituents for the biaromatic furanone derivatives. The compounds with the two substitutions
exhibited good inhibitory activity on protease, and moderate activity on pyocyanin production. Since
compounds 9 had a certain bacteriostatic effect, it was not certain what effect the compound would produce
on pyocyanin and protease activity. Thus we chose three different types of side chains to study the activity of
this series of compounds. On that basis, a new series of the aryl-substituted pyrrolidone derivatives 12a-12f
(Scheme 2) were designed and synthesized by further replacing the lactone of the furanone derivatives with a
lactam. The compounds 11 and the arylboronic acid underwent a Suzuki-Miyaura cross-coupling to finally
obtain the aryl-substituted pyrrolidone derivatives 12a-12f.
4

O
R2
Br
N
Br
i
N
R2
R1
R1
12a-12f
1 = 3-methyl; R2 = phenethyl
O
11
a:
b:
c:
d:
e:
f:
R
R
1 = 2-methoxy; R2 = phenethyl
R
R
1 = 3-methyl;
R2 = n-hexyl
1 = 2-methoxy; R2 = n-hexyl
R
1 = 3-methyl;
R2 = tetrahydrofurfuryl
R1 = 2-methoxy; R2 = tetrahydrofurfuryl
Scheme 2. Synthesis of aryl-substituted pyrrolidone derivatives 12a-12f. Reagents and condition: i: arylboronic
acid, Na2CO3, TBAB, Pd[P(C6H5)3]4, toluene: H2O = 1:1, reflux for 12 h.
2.2 The result of the MIC assay
The results of the MIC assay[23, 24] (Table 1) indicate that the biaromatic furanone derivatives have no
antibacterial activity against common Gram-positive and Gram-negative bacteria, which further demonstrates
that this series of compounds do not interfere with the primary metabolism of bacterial cells and have little
effect on the growth or reproduction of bacteria. In contrast, the brominated pyrrolidone derivatives have
moderate inhibitory effects on Gram-positive bacteria such as Staphylococcus aureus ATCC25923 and S.
aureus ATCC43300, and Bacillus subtilis ATCC9372, but do not have any inhibitory activity against two
Gram-negative bacteria such as E. coli ATCC25922 and P. aeruginosa PAO1. Their inhibitory activity is
probably attributed to the presence of multiple electron-deficient sites in their molecules, which are easily
attacked by the protein’s nucleophilic groups such as sulfhydryl or amino group in the cell membrane, thereby
resulting in interaction of the molecules with the membrane proteins. Those pyrrolidone derivatives showed
little inhibition activity against Gram-negative bacteria E. coli and P. aeruginosa as well since the cell wall of
Gram-negative bacteria can protect the bacteria cells from antimicrobial agents.
Table 1. The result of minimum inhibitory concentration (μg/mL)
P.
B. subtilis
S. aureus
E. coli
S. aureus
S.
S.
Comp
aeruginosa
PAO1
128
ATCC9372
ATCC25923
ATCC25922
ATCC43300
pneumoniae
epidermidis
3
9a
9b
9c
32
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
64
32
>128
>128
>128
128
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
128
9d
9e
9f
9g
9h
9i
9j
>128
>128
>128
>128
128
128
64
9k
9l
64
9m
9n
11a
11b
>128
128
32
8
16
4
128
4
64
5

11c
11d
11e
11f
8
8
4
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
128
128
2
16
2
4
8
4
4
4
128
8
4
64
16
4
>128
128
16
16
16
11g
11h
11i
16
4
8
4
128
64
64
32
128
128
32
32
32
16
128
32
16
12a
12b
12c
12d
12e
12f
>128
>128
>128
>128
>128
>128
<0.25
2
>128
>128
>128
>128
>128
>128
<0.25
8
>128
>128
>128
>128
>128
>128
64
>128
128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
128
2
>128
>128
>128
>128
>128
>128
128
2
CLA
CIP
>128
4
4
CIP: ciprofloxacin; CLA: clarithromycin.
2.3 Growth Inhibition
For the growth inhibition assay[25] (Fig. S1-S6 in the supporting information), the biaromatic furanone
derivatives (1 mM) did not inhibit the growth of P. aeruginosa PAO1, while brominated pyrrolidone
derivatives had a similar activity as the positive control furanone 30 at 1 mM. This is potent growth inhibition
activity against P. aeruginosa PAO1 but does not kill the bacteria. As for the aryl-substituted pyrrolidone
derivatives, compounds 12a and 12e showed no inhibition activity against P. aeruginosa PAO1 at 1 mM,
while compounds 12b and 12f inhibited its growth slightly, and in particular, compounds 12c and 12d strongly
inhibited its growth.
As for E. coli ATCC25922, the biaromatic furanone derivatives (except compounds 9a and 9m) did not
inhibit the bacterial growth at 1 mM, while compounds 9a and 9m only exhibited moderate growth inhibition
without bactericidal activity. The brominated pyrrolidone derivatives (except compound 11f) did not also
inhibit the growth of E. coli ATCC25922 at 1 mM, while compound 11f showed sustained growth inhibition
without bactericidal activity. Among those aryl-substituted pyrrolidone derivatives, compounds 12a, 12d, 12e,
12b and 12f did not inhibit any growth of E. coli ATCC25922 at 1 mM. In contrast, compound 12c potently
inhibited the growth of E. coli ATCC25922 but did not kill the bacteria.
As for S. aureus ATCC25923, most biaromatic furanone derivatives showed no growth inhibition at 1 mM,
while compounds 9c, 9d and 9e only slightly inhibited its growth. The brominated pyrrolidone derivatives had
a significant growth inhibitory activity against S. aureus ATCC25923 at 1 mM, compound 11f exhibited a
moderate growth inhibitory activity, and compounds 11a, 11b, 11c, 11d and 11i exhibited bactericidal activity.
Although the aryl-substituted pyrrolidone derivatives did not have any bactericidal activity at 1 mM, they
exhibited varying degrees of inhibition effect, among which, compounds 12c, 12d and 12f had moderate
inhibitory effect on S. aureus.
As for S. pneumoniae, the biaromatic furanone derivatives did not have potent inhibitory activity of the
growth at 1 mM. For example, compounds 9a and 9f only inhibited the growth of bacteria without bactericidal
activity. In the brominated pyrrolidone derivatives, compound 11d had no inhibitory effect on the growth of
S. pneumoniae at 1 mM, while compounds 11e and 11h had moderate inhibition activity, and the other
brominated pyrrolidone derivatives were potent growth inhibitors of S. pneumoniae. The aryl-substituted
pyrrolidone derivatives (except 12d) did not inhibit the growth of S. pneumoniae at 1 mM, while compound
6

12d inhibited its growth moderately. In particular, compound 12c inhibited the growth of S. pneumoniae
potently, retaining the density of S. pneumoniae at a low range (OD620 < 0.075).
As for S. epidermidis, the biaromatic furanone derivatives (1 mM) significantly inhibited bacterial growth
without bactericidal activity. In the brominated pyrrolidone derivatives, compounds 11a, 11b, 11f and 11h
had a bactericidal activity, while the other biaromatic furanone derivatives had an inhibitory effect on the
growth of S. epidermidis, but the bactericidal activity was not obvious. All of the aryl-substituted pyrrolidone
derivatives had potent and significant growth inhibitory activity, but only compound 12b had a bactericidal
activity.
As for B. subtilis ATCC9372, the biaromatic furanone 9g (1 mM) had a potent growth inhibitory effect,
while compounds 9a, 9b, 9k and 9n did not display obvious inhibition activity. Additionally, the other
compounds showed slight effects on promoting bacterial growth. The brominated pyrrolidone derivatives were
potent growth inhibitors except 11f and 11h. Besides, compounds 11a, 11b, 11d and 11e and 11i were even
bactericidal agents. Among the aryl-substituted pyrrolidone derivatives, compounds 12a 12b and 12d were
potent inhibitors, while the other compounds had no growth inhibition activity.
In general, the biaromatic furanone derivatives had little growth inhibition activity against the tested
bacterial strains. The brominated pyrrolidone derivatives possessed less potent inhibitory effect on Gram-
negative bacteria than Gram-positive strains, in which some brominated pyrrolidone derivatives had
bactericidal activity. As for the aryl-substituted pyrrolidone derivatives, compounds 12c and 12d had potent
growth inhibitory activity against some tested Gram-positive strains without bactericidal activity.
Pyocyanin production
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
Protease activity
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
7

Figure 5. Pyocyanin production and protease inhibition activities of compounds 9 and 11 (1 mM). (P. aeruginosa
PAO1, BC: blank control, DMSO: DMSO only).
2.4 Pyocyanin production and protease inhibition of compounds 9 and 11
In the pyocyanin expression inhibition assay of P. aeruginosa PAO1[16] (Fig. 5), compound 9g showed the
strongest inhibition of pyocyanin production with an inhibition ratio of 66.4%, which is narrowly followed by
compound 9a with an inhibition ratio of 61.7%. However, all of the other biaromatic furanone derivatives
were proved to be less effective. Among them, compounds 9c, 9e, 9f, 9l, 9m and 9n had similar activity and
inhibited the production of pyocyanin by more than 40%. Compounds with a fluorine in the ortho or meta
positions of the benzene ring possessed considerable activity, but the compounds possessing a fluorine in the
para position displayed extremely poor activity and hardly inhibited the production of pyocyanin. Excitingly,
the results of the protease activity assay [16] indicated that compounds 9a, 9f and 9g also showed good
inhibition of protease activity, the inhibition percentages of which reached 76.3, 77.4 and 74.1%, respectively.
In addition, the other compounds in this series displayed varying degrees of protease inhibition. Given the fact
that the above compounds have little effects on bacterial growth, it can be concluded that their protease
inhibition and pyocyanin production inhibition effects do not interfere with the growth and reproduction of
bacteria. Therefore, they can be used as ideal QSIs. In the series of the brominated pyrrolidone derivatives,
most of them can inhibit pyocyanin production moderately, such as compounds 11a, 11c, 11d, 11f and 11i
with the inhibition ratios of pyocyanin production by more than 60%. However, compounds 11b, 11e and 11g
seem to be ineffective with inhibition ratios of 16.8, 30.1 and 25.4%, respectively. It appears that the length
of the alkyl substituents on the nitrogen atom should not exceed 7 atoms. The protease activity assay results
show similar pattern as well. The most active compound 11c is inhibition percentage of 66.68%. Octyl-
substituted compound 11e has less potent protease inhibition activity than heptyl-substituted compound 11d.
Besides, benzyl-substituted compound 11f and tetrahydroindenyl-substituted compound 11i exhibit excellent
protease inhibitory activity with inhibition percentages of 63.4 and 66.3%, respectively. Overall, the inhibitory
effect of the compound on protease activity is a little better than that on the production of pyocyanin. Thus we
selected the compounds 9f and 11c with the best protease activity in their respective series to determine their
IC50 values. The result indicated that compound 11c showed better activity on pyocyanin production with an
IC50 value of 0.3918 mM than compound 9f. Moreover, both compounds had moderate inhibitory activity
against proteases activity (Table 2).
Table 2. The IC50 values against protease inhibition and pyocyanin production (P. aeruginosa PAO1) of
compounds 9f and 11c.
Pyocyanin production
Protease activity
Comp
IC50 (mM)
0.9710
R2
IC50 (mM)
R2
9f
0.9333
0.9651
0.5344
0.6645
0.9305
0.9784
11c
0.3918
R2: coefficient of determination.
2.5 evaluation of compounds 12a-12f
Seen from the MIC results shown in Table 1, all of the above six compounds don’t have any antibacterial
activity against either Gram-positive or Gram-negative bacteria. Similarly, they have little inhibition effect on
8

P. aeruginosa growth. This indicates that those compounds do not participate in the primary metabolic process
of common bacteria and do not affect the growth and reproduction of P. aeruginosa as well.
Protease activity
Pyocyanin production
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
140.00%
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
Figure 6. Pyocyanin production and protease inhibition activities of compounds 9 and 11 (1 mM). (P. aeruginosa
PAO1, BC: blank control, DMSO: DMSO only).
In the inhibition of pyocyanin production assay of P. aeruginosa PAO1 (Fig. 6), compound 12a, with an
OD520 value of 0.0082, showed the most potent inhibition activity among the six compounds, which could
inhibit over 80% of pyocyanin production in P. aeruginosa compared with blank control. Besides, compounds
12b, 12c and 12f also exerted potent inhibition activity with inhibition rates of 43.3, 52.6 and 43.1%,
respectively. However, the other two compounds 12d and 12e had no activity in the pyocyanin production
assay. The protease activity assay results indicate that all the six compounds possess inhibition effects on the
protease activity of P. aeruginosa. Among them, compound 12a has the most potent inhibitor with an
inhibition ratio of 78.5%, which is narrowly followed by compound 12f with an inhibition ratio of 75.6%. The
other four compounds display similar inhibition activity with inhibition ratios of around 65% against protease
activity. Then, we determined the IC50 valuesof compounds 12a and 12c, the inhibitory activity of which
reached more than 50% against pyocyanin production and protease activity (Table 3). For instance, compound
12a could inhibit the production of pyocyanin by 50% at a concentration of 0.2897 mM, but it needed a
concentration of 0.4364 mM to inhibit 50% of protease activity. In contrast, compound 12c exhibited weak
activity with the IC50 values of 0.7492 and 0.7060 mM on pyocyanin production and protease activity,
respectively.
Table 3. The IC50 values on protease inhibition and pyocyanin production (P. aeruginosa PAO1) of compounds
12a and 12c.
Pyocyanin production
Protease activity
Comp
IC50 (mM)
R2
IC50 (mM)
R2
12a
12c
0.2897
0.7492
0.9464
0.9020
0.4364
0.7060
0.9540
0.9390
R2: coefficient of determination.
According to the dose-inhibition curve （ Fig. 7 ） of compound 12a, its inhibition rate on pyocyanin
production and protease activity were found to increase with the rise of concentration, but there were also
obvious differences between their inhibition rates. The inhibition rate of the compound on the protease activity
9

increased significantly at high concentration while the inhibition rate of the compound on pyocyanin
production increased rapidly at low concentration. For example, at a concentration of 0.2909 mM, compound
12a suppressed the expression of pyocyanin and the protease activity by 80.20 and 58.11%, respectively.
100
80
60
40
20
0
100
80
60
40
20
0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
(mM)
(mM)
Figure 7. Protease inhibition and pyocyanin production inhibition activity (P. aeruginosa PAO1, 18 h) of
compound 12a in different concentration.
Furthermore, in vitro cytotoxicity of compound 12a to Hela human cancer cell was measured. The result
indicated that no toxicity was observed for 12a up to 100 μM while furanone 30 was evaluated as a high-toxic
substance (up to 20 μM on HeLa human cancer cells) in previous study[26, 27]. Further, the degree of
inhibition of this series of compounds on the biofilms were determined to confirm whether they were biofilm
inhibitors[28]. The results showed that this series had different degrees of biofilm inhibition activity (Table
4). In particular, compound 12a exhibited an IC50 value of 0.2599 mM with R2 value of 0.885, which implied
this compound inhibited the biosynthesis of biofilms in P. aeruginosa to some extent. What’s more, the other
compounds such as 12b and 12c displayed better biofilm inhibition activity with the IC50 values of 0.219 and
0.242 mM than compound 12a, which can be used as potential biofilm inhibitors. In remarkable contrast,
compounds 12e and 12f were found to have no inhibition activity against biofilm formation in P. aeruginosa.
Table 4. The biofilm inhibition activity of the aryl-substituted pyrrolidone derivatives 12a-12f.
Comp
12a
12b
12c
IC50 (mM)
0.260 ± 0.035
0.219 ± 0.008
0.242 ± 0.032
0.281 ± 0.012
NI
R2
0.885
0.7983
0.7292
0.6931
-
12d
12e
12f
NI
-
NI: no inhibition activity found (IC50≥1mM). R2: coefficient of determination.
Therefore, it is reasonable that compound 12a is a promising QSI. Then we explored the inhibition activity
of compound 12a combined with antibiotics against P. aeruginosa PAO1. The results indicated that this
compound at 10 μM improved the antibacterial activity of ciprofloxacin and clarithromycin (Table 5). When
compound 12a (10 μM) were added to ciprofloxacin (0.60 μM) or clarithromycin (4.28 μM), their inhibition
rates against P. aeruginosa were 60.4 and 50.6%, respectively, while ciprofloxacin and clarithromycin had
only inhibition rates of 26.1 and 21.6% against P. aeruginosa when used alone in the same concentrations. On
basis of the fact that it has no inhibition activity against P. aeruginosa, we speculate that compound 12a
interferes with bacterial biosynthesis process of bacterial biofilm, which increases the proportion of the above
antibiotics into the bacterial cells, thereby resulting in the enhanced effectiveness of the antibiotics.
Table 5. The drug combination results.
10

Drug combination
Inhibition rate (%)
32.4 ± 4.4
CIP (0.60 μM)
CLA (4.28 μM)
12a (10 μM)
24.6 ± 4.7
NI
12a (10 μM) + CIP (0.60 μM)
12a (10 μM) + CLA (4.28 μM)
60.4 ± 1.9
50.6 ± 2.6
NI: no inhibition activity found.
The crystal structure (PDB code: 2uv0) of LasR with the auto-inducer N-(3-oxododecanoyl)-L-homoserine
(OdDHL) was used to analyze the interaction mode of those compound with the quorum sensing LasR
receptor. The proposed binding mode of compound 12a indicates that the oxygen atom in the molecule can
form two different hydrogen bonds with Trp60 and Arg61 in protein LasR which is similar with furanone 30.
Compared with the results of previous studies on the interaction between the autoinducer OdDHL and
LasR[29], the amide and ketone functional groups in OdDHL can form multiple hydrogen bonds with the
residues in the LasR ligand binding site (for example, Arg 61, Trp 60 and Ser129). Thus, we speculate that
compound 12a may inteact the same binding site on LasR as OdDHL. The formation of hydrogen bonds
between compounds with Trp60 and Arg61 seem to be the key to the formation of antagonistic activity. In
addition, there are also hydrophobic interactions between compound12a and LasR as shown in Fig. 8.
(A)
(B)
11

Figure 8. (A) Proposed hydrogen bonds and hydrophobic interactions between compound 12a and LasR (PDB ID:
2UV0); (B) The crystal structure of LasR with autoinducer N-(3-oxododecanoyl)-L-homoserine (OdDHL) (PDB
code: 2UV0)
2.6 Conclusion and discussion
In summary, we explored the appropriate substituents on the benzene ring of biaromatic furanones and
found that the compounds with electron-donating groups had the better inhibition activity against pyocyanin
production and protease activity in P. aeruginosa. The bioevaluation results of the brominated pyrrolones
indicated the compounds with benzyl and n-hexyl substituents had good quorum sensing inhibition activity.
Based on the above findings and the computational docking result, a new series of aryl-substituted pyrrolidone
derivatives were designed and synthesized. Further investigation indicated that compound 12a, as a potent
quorum sensing inhibitor, effectively inhibited pyocyanin production (by 80.6%) and protease activity (by
78.5%) in P. aeruginosa without affecting the growth and reproduction of the tested bacteria. Moreover, it
blocked the biofilm formation of P. aeruginosa to some extent, which makes it possible to improve the effects
of antibiotics. In the cytotoxicity assay this compound showed little toxicity against human hepatocellular
carcinoma cell. Finally, molecular docking method was used to visualize possible binding mode of compound
12a and protein LasR. Taken together, compound 12a is believed to serve as a novel structure with low toxicity
for further investigation as a quorum sensing inhibitor. Moreover, researches on aryl-substituted pyrrolidone
derivatives can be carried out for enhanced their activity.
Supporting informations
Growth inhibition results; Growth curves; Experimental methods; 1H NMR, 13C NMR, HRMS and MS spectra of
representative compounds can be found in supporting informations.
Acknowledgements
This research was supported financially by the National Natural Science Foundation of China (81973179 and
81673284), and Key Research and Development Project of Shandong Province (2017CXGC1401) and the China-
Australia Centre for Health Sciences Research (CACHSR no. 2019GJ05).
Conflicts of interest
The authors declare no competing financial interest.
ABBREVIATIONS
QSIs: quorum sensing inhibitors
QS: quorum sensing
BC: blank control
CIP: ciprofloxacin
CLA: clarithromycin
DCM: dichloromethane
REFERENCES
[1] G. Humphreys, F. Fleck, United Nations meeting on antimicrobial resistance, Bull. World Health Organ.
94(9) (2016) 638-639.
[2] B. LaSarre, M.J. Federle, Exploiting quorum sensing to confuse bacterial pathogens, Microbiol. Mol. Biol.
Rev. 77(1) (2013) 73-111.
[3] N.N. Biswas, S.K. Kutty, G.M. Iskander, M. Mielczarek, N. Kumar, Synthesis of brominated novel N-
heterocycles: New scaffolds for antimicrobial discovery, Tetrahedron 72(4) (2015) 539-546.
12

[4] E.P. Greenberg, M. Schuster, C. Lostroh, Quorum sensing signaling in bacteria, EP, 2002.
[5] X. Chen, L. Zhang, M. Zhang, H. Liu, P. Lu, K. Lin, Quorum sensing inhibitors: a patent review (2014-
2018), Expert Opin. Ther. Pat. 28(7-12) (2018) 849-865.
[6] Reto, Frei, Anthony, S., Breitbach, Helen, E., Blackwell, 2‐Aminobenzimidazole derivatives strongly
inhibit and disperse Pseudomonas aeruginosa biofilms, Angew. Chem. Int. Ed. 51(21) (2012) 5226-5229.
[7] Nripendra, Nath, Biswas, Samuel, Kutty, Nicolas, Barraud, George, Indole-based novel small molecules
for the modulation of bacterial signalling pathways, Org. Biomol. Chem. 13(3) (2015) 925-937.
[8] M. Manefield, R. de Nys, N. Kumar, R. Read, M. Givskov, P. Steinberg, S.A. Kjelleberg, Evidence that
halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene
expression by displacing the AHL signal from its receptor protein, Microbiology 145(Pt 2) (1999) 283-291.
[9] J. Sikorska, S. Parker-Nance, M.T. Davies-Coleman, O.B. Vining, A.E. Sikora, K.L. McPhail,
Antimicrobial rubrolides from a South African Species of synoicum tunicate, J. Nat. Prod. 75(10) (2012) 1824-
1827.
[10] M. Manefield, T.B. Rasmussen, M. Henzter, J.B. Andersen, M. Givskov, Halogenated furanones inhibit
quorum sensing through accelerated LuxR turnover, Microbiology 148(Pt 4) (2002) 1119-1127.
[11] Y. Han, S. Hou, K.A. Simon, D. Ren, Y.Y. Luk, Identifying the important structural elements of
brominated furanones for inhibiting biofilm formation by Escherichia coli, Bioorg. Med. Chem. Lett. 18(3)
(2008) 1006-1010.
[12] J. Lonn-Stensrud, M.A. Landin, T. Benneche, F.C. Petersen, A.A. Scheie, Furanones, potential agents for
preventing Staphylococcus epidermidis biofilm infections?, J. Antimicrob. Chemother. 63(2) (2009) 309-316.
[13] C. Costas, V. Lopez-Puente, G. Bodelon, C. Gonzalez-Bello, J. Perez-Juste, I. Pastoriza-Santos, L.M.
Liz-Marzan, Using surface enhanced raman scattering to analyze the interactions of protein receptors with
bacterial quorum sensing modulators, ACS Nano. 9(5) (2015) 5567-5576.
[14] W.K. Goh, C.R. Gardner, K. Sekhar, N.N. Biswas, S. Nizalapur, S.A. Rice, M. Willcox, D.S. Black, N.
Kumar, Synthesis, quorum sensing inhibition and docking studies of 1,5-dihydropyrrol-2-ones, Bioorg. Med.
Chem. 23(23) (2015) 7366-7377.
[15] T.H. Won, J.E. Jeon, S.H. Kim, S.H. Lee, B.J. Rho, D.C. Oh, K.B. Oh, J. Shin, Brominated aromatic
furanones and related esters from the Ascidian Synoicum sp, J. Nat. Prod. 75(12) (2012) 2055-2061.
[16] M. Hentzer, K. Riedel, T.B. Rasmussen, A. Heydorn, J.B. Andersen, M.R. Parsek, S.A. Rice, L. Eberl,
S. Molin, N. Hoiby, S. Kjelleberg, M. Givskov, Inhibition of quorum sensing in Pseudomonas aeruginosa
biofilm bacteria by a halogenated furanone compound, Microbiology 148 (2002) 87-102.
[17] A. Latifi, M.K. Winson, M. Foglino, B.W. Bycroft, G. Stewart, A. Lazdunski, P. Williams, Multiple
homologues of luxR and luxI control expression of virulence determinants and secondary metabolites through
quorum sensing in Pseudomonas aeruginosa PAO1, Mol. Microbiol. 17(2) (1995) 333-343.
[18] A.J. Manny, S. Kjelleberg, N. Kumar, R. deNys, R.W. Read, P. Steinberg, Reinvestigation of the sulfuric
acid-catalysed cyclisation of brominated 2-alkyllevulinic acids to 3-alkyl-5-methylene-2(5H)-furanones,
Tetrahedron 53(46) (1997) 15813-15826.
[19] E. Paunescu, N. Matuszak, P. Melnyk, Suzuki-Miyaura cross-coupling reaction as the key step for the
synthesis of some new 4 '-aryl and alkyl substituted analogues of amodiaquine and amopyroquine, Tetrahedron
63(51) (2007) 12791-12810.
[20] R. Zhang, G. Iskander, P.D. Silva, D. Chan, V. Vignevich, V. Nguyen, M.M. Bhadbhade, D.S. Black, N.
Kumar, Synthesis of new aryl-substituted furan-2(5H)-ones using the Suzuki-Miyaura reaction, Tetrahedron
13

67(16) (2011) 3010-3016.
[21] W.J. Tang, A.G. Capacci, X.D. Wei, W.J. Li, A. White, N.D. Patel, J. Savoie, J.J. Gao, S. Rodriguez, B.
Qu, N. Haddad, B.Z. Lu, D. Krishnamurthy, N.K. Yee, C.H. Senanayake, A general and special catalyst for
Suzuki-Miyaura coupling processes, Angew. Chem.-Int. Edit. 49(34) (2010) 5879-5883.
[22] W.K. Goh, G. Iskander, D.S. Black, N. Kumar, An efficient lactamization of fimbrolides to novel 1,5-
dihydropyrrol-2-ones, Tetrahedron Lett. 48(13) (2007) 2287-2290.
[23] N.C.f.C.L. Standard, Methods for dilution antimicrobial susceptibility tests for bacteria that grow
aerobically, Aproveded standard-5th ed, (2000).
[24] M. Ferraro, Methods for dilution antimicrobial susceptibility de test for bacteria that grow aerobically,
Approved Standard, (2003).
[25] X.J. Jiang, P. Yu, J. Jiang, Z.J. Zhang, Z.L. Wang, Z.Q. Yang, Z.M. Tian, S.C. Wright, J.W. Larrick, Y.Q.
Wang, Synthesis and evaluation of antibacterial activities of andrographolide analogues, Eur. J. Med. Chem.
44(7) (2009) 2936-2943.
[26] M. Rasch, C. Buch, B. Austin, W.J. Slierendrecht, K.S. Ekmann, J.L. Larsen, C. Johansen, K. Riedel, L.
Eberl, M. Givskov, L. Gram, An inhibitor of bacterial quorum sensing reduces mortalities caused by vibriosis
in rainbow trout (Oncorhynchus mykiss, Walbaum), Syst. App. Microbiol. 27(3) (2003) 350-359.
[27] S. Ekins, R.C. Reynolds, H. Kim, M.S. Koo, M. Ekonomidis, M. Talaue, S.D. Paget, L.K. Woolhiser,
A.J. Lenaerts, B.A. Bunin, N. Connell, J.S. Freundlich, Bayesian models leveraging bioactivity and
cytotoxicity information for drug discovery, Chem. Biol. 20(3) (2013) 370-378.
[28] A.D. Pimenta, P. Di Martino, E. Le Bouder, C. Hulen, M.A. Blight, In vitro identification of two
adherence factors required for in vivo virulence of Pseudomonas fluorescens, Microbes Infect. 5(13) (2003)
1177-1187.
[29] D.E. Manson, M.C. O'Reilly, K.E. Nyffeler, H.E. Blackwell, Design, synthesis, and biochemical
characterization of non-native antagonists of the Pseudomonas aeruginosa quorum sensing receptor LasR with
nanomolar IC50 values, ACS Infect. Dis. 6(4) (2020) 649-661.
14

The captions for Figures and Schemes
Figure 1. The structures of some quorum sensing inhibitors.
Figure 2. The predicted binding mode of furanone 30 with LasR (PDB ID: 2UV0).
Figure 3. The structure of cadiolide I (MIC: 1.6 μg/mL against S. enterica) and rubrolide I (IC50 = 1.51 ± 0.116
μg/mL against E. faecalis while did not inhibit bacterial growth).
Figure 4. The design strategy of aryl-substituted pyrrolidone.
Figure 5. Pyocyanin production and protease inhibition activities of compounds 9 and 11 (1 mM). (P. aeruginosa
PAO1, BC: blank control, DMSO: DMSO only).
Figure 6. Pyocyanin production and protease inhibition activities of compounds 9 and 11 (1 mM). (P. aeruginosa
PAO1, BC: blank control, DMSO: DMSO only).
Figure 7. Protease inhibition and pyocyanin production inhibition activity (P. aeruginosa PAO1, 18 h) of compound
12a in different concentration.
Figure 8. (A) Proposed hydrogen bonds and hydrophobic interactions between compound 12a and LasR (PDB ID:
2UV0); (B) The crystal structure of LasR with autoinducer N-(3-oxododecanoyl)-L-homoserine (OdDHL) (PDB
code: 2UV0)
Scheme 1. Synthesis of rubrolide analogs 9a-9n and 11a-11i. Reagents and conditions: i: Br2, HBr in acetic acid,
petroleum ether (60-90 C); iii: 98%H2SO4, 115 C, 40 min; iii: arylboronic acid, Na2CO3, TBAB, Pd[P(C6H5)3]4,
toluene, H2O, reflux, 12 h; iv: amines, DCM, 0 C, 40 min; v: P2O5, reflux, 3 h.
Scheme 2. Synthesis of aryl-substituted pyrrolidone derivatives 12a-12f. Reagents and condition: i: arylboronic acid,
Na2CO3, TBAB, Pd[P(C6H5)3]4, toluene: H2O = 1:1, reflux for 12 h.
15

Figure 1
Br
Br
Br
Br
N
Br
O
Br
O
Br
O
O
Br
O
Br
O
O
4
Furanone 30 (3)
2
1
Figure 2
Figure 3
OH
Br
Br
O
Cl
O
Br
HO
O
O
O
H3C
Br
OH
Br
Br
OCH3
OCH3
Br
Cadiolide I (6)
Rubrolide I (5)
Figure 4
R1
O
O
R1
R1
Br
series 1
R2
N
O
O
Br
O
Br
R1
N
Br
O
series 3
Aryalkyl-substituted pyrrolidone
R2
series 2
16

Figure 5
Pyocyanin production
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
Protease activity
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
Figure 6
Protease activity
Pyocyanin production
140.00%
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
Figure 7
17

100
80
60
40
20
0
100
80
60
40
20
0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
(mM)
(mM)
Figure 8
(A)
(B)
18

Scheme 1
a:
b:
c:
d:
e:
f:
h:
i:
R
1 = 2-Methoxy
1 = 2-methyl
1 = 2-fluoro
1 = 2-trifluoromethyl
1 = 3-methoxy
R
1 = 3-trifluoromethyl
R1 = 4-methoxy
R1 = 4-methyl
R
O
j:
R
1 = 4-fluoro
R1 = 4-trifluoromethyl
k:
l:
R
R
O
R
R1 = 3-methyl
R
1 = hydrogen
R1 = 4-phenyl
m:
n:
R1
1 = 3-fluoro
R1
g:
R
ⅲ
9a-9n
Br
Br
Br
ⅰ
ⅱ
O
O
COOH
O
COOH
O
Br
7
iv
Br
HO
Br
8
Furanone 30
Br
Br
v
N
N
2 = benzyl
R2
R2
a:
R
R
2 = n-butyl
2 = n-amyl
2 = n-hexyl
f:
R
R
2 = phenethyl
b:
c:
g:
h:
i:
O
O
R
R
2 = 4-fluorobenzyl
10a-10i
11a-11i
R
2 = n-heptyl
R2 = tetrahydrofurfuryl
d:
e:
R
2 = n-octyl
Scheme 2
O
R2
Br
N
Br
i
N
R2
R1
R1
12a-12f
1 = 3-methyl; R2 = phenethyl
O
11
a:
b:
c:
d:
e:
f:
R
R
1 = 2-methoxy; R2 = phenethyl
R
R
1 = 3-methyl;
R2 = n-hexyl
1 = 2-methoxy; R2 = n-hexyl
R
1 = 3-methyl;
R2 = tetrahydrofurfuryl
R1 = 2-methoxy; R2 = tetrahydrofurfuryl
Table 1. The result of minimum inhibitory concentration (μg/mL)
B. subtilis
S. aureus
E. coli
P. aeruginosa
S. aureus
S.
S.
ATCC9372
ATCC25923
ATCC25922
PAO1
ATCC43300
pneumoniae
epidermidis
Comp
3
9a
9b
9c
32
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
64
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
128
8
32
>128
>128
>128
128
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
9d
9e
9f
9g
9h
9i
9j
128
64
9k
9l
64
9m
9n
11a
11b
11c
11d
>128
128
32
16
4
128
4
64
8
4
128
2
2
8
8
4
128
16
4
4
19

11e
11f
4
4
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
128
>128
128
8
4
64
16
16
16
16
11g
11h
11i
16
4
8
4
128
64
64
32
128
128
32
32
32
16
128
32
16
12a
12b
12c
12d
12e
12f
>128
>128
>128
>128
>128
>128
<0.25
2
>128
>128
>128
>128
>128
>128
<0.25
8
>128
>128
>128
>128
>128
>128
64
>128
128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
128
2
>128
>128
>128
>128
>128
>128
128
2
CLA
CIP
>128
4
4
CIP: ciprofloxacin; CLA: clarithromycin.
Table 2. The IC50 values against protease inhibition and pyocyanin production (P. aeruginosa PAO1) of
compounds 9f and 11c.
Pyocyanin production
Protease activity
Comp
IC50 (mM)
0.9710
R2
IC50 (mM)
R2
9f
0.9333
0.9651
0.5344
0.6645
0.9305
0.9784
11c
0.3918
R2: coefficient of determination.
Table 3. The IC50 values on protease inhibition and pyocyanin production (P. aeruginosa PAO1) of compounds
12a and 12c.
Pyocyanin production
Protease activity
Comp
IC50 (mM)
R2
IC50 (mM)
R2
12a
12c
0.2897
0.7492
0.9464
0.9020
0.4364
0.7060
0.9540
0.9390
R2: coefficient of determination.
20

Table 4. The biofilm inhibition activity of the aryl-substituted pyrrolidone derivatives 12a-12f.
Comp
12a
12b
12c
IC50 (mM)
0.260 ± 0.035
0.219 ± 0.008
0.242 ± 0.032
0.281 ± 0.012
NI
R2
0.885
0.7983
0.7292
0.6931
-
12d
12e
12f
NI
-
NI: no inhibition activity found (IC50≥1mM). R2: coefficient of determination.
Table 5. The drug combination results.
Drug combination
Inhibition rate (%)
32.4 ± 4.4
CIP (0.60 μM)
CLA (4.28 μM)
12a (10 μM)
24.6 ± 4.7
NI
12a (10 μM) + CIP (0.60 μM)
12a (10 μM) + CLA (4.28 μM)
60.4 ± 1.9
50.6 ± 2.6
NI: no inhibition activity found.
21

Graphical Abstract：
Design and synthesis of aryl-substituted pyrrolidone derivatives as
quorum sensing inhibitors
Zhiyang Liu a,1, Panpan Zhang a,1, Yinhui Qin a, Nan Zhang a, Yuetai Teng a, Henrietta Venter b, Shutao Ma a, *
A novel series of aryl-substituted pyrrolones were confirmed to be quorum sensing
inhibitors against P. aeruginosa. In particular, compound 12a was found to inhibit the
pyocyanin production and protease activity of P. aeruginosa by 80.6 and 78.5%,
respectively.
optimization
Low toxicity and moderate
biofilm inhibition activity
Toxic
22

Research Highlights
˃ A novel series of aryl-substituted pyrrolidone were designed and synthesized. ˃ They were evaluated
as quorum sensing inhibitors against P. aeruginosa. ˃ Compound 12a inhibited the pyocyanin production
and protease activity by 80.6 and 78.5%, respectively. ˃ Compound 12a is worth further investigation as
a quorum sensing inhibitor.
Conflicts of interest
The authors declare that this study was carried out only with public funding. There is no funding or no
agreement with commercial for profit firms.
23