Design, synthesis and evaluation of halogenated furanone derivatives as quorum sensing inhibitors in Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.ejps.2019.105058

The biofilm formation of Pseudomonas aeruginosa (P. aeruginosa) is regulated by a phenomenon of quorum sensing (QS). With 5-hydroxyl-3,4-halogenated-5H-furan-2-ones as beginning, analogs bearing alkyl chains, vinyl bromide, or aromatic rings were designed and synthesized. The minimum inhibitory concentration (MIC) of the compounds against P. aeruginosa was assayed and the biofilm inhibition ratio was determined at different concentrations lower than the MIC. C-5 aromatic substituted furanones showed remarkable biofilm formation as well as inhibition of virulence factor production in P. aeruginosa. Fluorescence report analysis identified the QS regulatory mechanism of the most active compound 29. This study provides us a novel candidate for combating drug resistant bacteria strains by merely inhibiting biofilm formation. Without suppressing the regular life cycle of the bacteria, bacterial resistance mechanisms may not be activated.

Full text of DOI:10.1016/j.ejps.2019.105058

Journal Pre-proof
Design, synthesis and evaluation of halogenated furanone
derivatives as quorum sensing inhibitors in Pseudomonas
aeruginosa
Yiqun Chang, Peng-Cheng Wang, Hong-Ming Ma, Si-Yu Chen,
Yu-Hang Fu, Yuan-Yuan Liu, Xuan Wang, Guang-Chao Yu, Tao
Huang, David E. Hibbs, Hai-Bo Zhou, Wei-Min Chen, Jing Lin,
Chao Wang, Jun-Xia Zheng, Ping-Hua Sun
PII:
S0928-0987(19)30329-X
DOI:
https://doi.org/10.1016/j.ejps.2019.105058
PHASCI 105058
Reference:
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
3 April 2019
23 August 2019
26 August 2019
Please cite this article as: Y. Chang, P.-C. Wang, H.-M. Ma, et al., Design, synthesis
and evaluation of halogenated furanone derivatives as quorum sensing inhibitors in
Pseudomonas aeruginosa, European Journal of Pharmaceutical Sciences(2019),
https://doi.org/10.1016/j.ejps.2019.105058
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2019 Published by Elsevier.

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Design, Synthesis and Evaluation of Halogenated Furanone
Derivatives as Quorum Sensing inhibitors
in Pseudomonas aeruginosa
1
,2#
1#
3#
1
1
Yiqun Chang , Peng-Cheng Wang , Hong-Ming Ma , Si-Yu Chen , Yu-Hang Fu ,
1
1
3
4
2
Yuan-Yuan Liu , Xuan Wang , Guang-Chao Yu , Tao Huang , David E. Hibbs ,
1
1
1
5,
4,
Hai-Bo Zhou , Wei-Min Chen , Jing Lin ,Chao Wang *, Jun-Xia Zheng *,
1
,
Ping-Hua Sun *
1
International Cooperative Laboratory of Traditional Chinese Medicine Modernization and
Innovative Drug Development of Chinese Ministry of Education, College of Pharmacy, Jinan
University, Guangzhou 510632; P. R. China.
2
Faculty of Medicine and Health, the University of Sydney, Sydney, NSW 2006, Australia.
3
Department of Respiratory, The first Affiliated Hospital of Jinan University, Guangzhou 510632;
P. R. China.
4
School of Chemical Engineering and Light Industry, Guangdong University of Technology,
Guangzhou 510006; P. R. China.
5
Department of Chemistry, Yale University, New Haven, Connecticut 06520; United States.
ABSTRACT: The biofilm formation of Pseudomonas aeruginosa (P. aeruginosa) is
regulated by a phenomenon of quorum sensing (QS). With 5-hydroxyl-3,4-
halogenated-5H-furan-2-ones as beginning, analogs bearing alkyl chains, vinyl
bromide, or aromatic rings were designed and synthesized. The minimum inhibitory
concentration (MIC) of the compounds against P. aeruginosa was assayed and the
biofilm inhibition ratio was determined at different concentrations lower than the MIC.
C-5 aromatic substituted furanones showed remarkable biofilm formation as well as
inhibition of virulence factor production in P. aeruginosa. Fluorescence report
analysis identified the QS regulatory mechanism of the most active compound 29.
This study provides us a novel candidate for combating drug resistant bacteria strains

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by merely inhibiting biofilm formation. Without suppressing the regular life cycle of
the bacteria, bacterial resistance mechanisms may not be activated.
KEYWORDS: Furanones; Quorum Sensing; Pseudomonas aeruginosa; Biofilm.
1
. Introduction
Since antibiotics were discovered in the early 20th century, many life-threatening
1
diseases to human beings had an effective cure. However, the indiscriminate and
excessive usage of antibiotics during these decades has led to drug resistance and
2
brought multiple drug resistant bacterial strains (MDR). With 16 million people
dying from infectious diseases caused by MDR strains every year, the urgent
3
,4
requirement for alternative therapies to fight against MDR admits no delay. Among
all infectious diseases, a noteworthy fact is that about 65% concern the proliferation
5
of forming bacterial biofilms. Biofilms are persistent and protective patterns of
structured microbial cell communities that allow bacteria to survive in hostile
6
circumstance by inserting in a matrix of extracellular polymeric substances. The
formation of bacterial biofilms is developed by a cell-to-cell signaling system of
quorum sensing (QS), through which bacteria synchronize their behaviors by a series
of signal molecules in a cell density dependent mode. In many species, this process
results in various pathogenic events like biofilm formation as well as virulence factor
7
,8
production.
Efforts have been made to disrupt biofilms via inhibiting the QS
system, many natural and synthetic molecules with QS inhibition have been identified
9
-11
as potential therapeutic approaches.
These quorum sensing inhibitors (QSIs) have
the ability to competitively quench QS signaling systems, providing a new way for
combating microbial infection.
P. aeruginosa, whose biofilm formation is based on quorum sensing system, is
1
2,13
the most common Gram-negative bacterium found in nosocomial infections.
With
14,15
a well-studied quorum sensing system regulating its biofilm formation
, P.
aeruginosa attracted many interest: two kinds of acyl homoserine lactones (AHLs)
produced by P. aeruginosa serve as the QS system signaling molecules:
N-3-oxododecanoyl homoserine lactone (3OC12) and N butanoyl homoserine lactone

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8
,16
(C4). These AHLs are received by the following receptors: QscR, LasR, and RhlR.
QscR and LasR normally sensed 3OC12 but regulate different regulons. RhlR
operates its own regulon by recognizing C4 as well. QS systems control numerous
genes expression, besides biofilm formation and virulence factor production; the
1
7
production of extra cytoplasmic substances and other metabolites are also regulated.
A lot of works have been made to search for novel natural QS inhibitors (QSI)
1
5-17
from natural products.
Different categories of natural QSI of gram-negative
1
8
bacteria have been identified, such as cyclic sulfur compounds from garlic,
1
9
halogenated furanones from Delisea pulchra, patulin and penicillic acid from
2
0
fungi, and so forth. Among the above molecules, the QS inhibitory activities of
furanone derivatives draw most attentions. Natural and synthetic furanone derivatives,
as well as unsaturated lactones, have been shown to exhibit a wide range of biological
activities including anticonvulsant, anti-inflammatory, analgesic, antitumor, antiviral,
2
1,22
and anticancer activities.
Patulin can down-regulate QS genes in P. aeruginosa
and halogenated furanones extracted from some marine plants also have antifouling
1
9-20
23
activities.
Based on the furanone derivatives and our previous work , we
designed a new series of halogenated furanone derivatives to investigate the structural
requirements in inhibition of biofilm formation. Taking the natural products as
promising lead compounds, 5-hydroxyl-3,4-halogenated-5H-furan-2-ones were
obtained from 2-furaldehyde as the beginning of our chemical exploration (Figure 1).
[Figure 1]
2
. Materials and methods
Bacterial strains
Pseudomonas aeruginosa strains ATCC27853, ATCC9027, and PAOA (clinical
isolates) were used to study the effects of new compounds to biofilm assay. A
o
bacterial suspension was made from fresh culture and aliquots were stored at -20 C in
glycerol and used within 2 weeks. Before used, bacterial suspensions were spread
o
onto Mueller-Hinton solid medium and incubated at 37 C for 18h.
2
.1 Synthesis of halogenated furanone derivatives

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2
.1.1 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-carbaldehyde (1a)
In a 500 mL three-necked flask equipped with a condensed and stirred HCl exhaust
gas absorption device, fresh distilled furfural (8 mL, 0.088 mol) and 320 mL of
concentrated hydrochloric acid were added, 40 g of MnO was added in portions with
2
vigorous stirring (finished in two hours). After that, the mixture was heated to reflux
for about 1 h, and another 8 g of MnO was added until the black color did not fade,
2
and the mixture was stirred at room temperature overnight. Directly add about 4 g of
active charcoal to boil and decolorize, then filtered while hot. When the filtrate is
cooled, a large amount of white fish scaly crystals are precipitated, then filtered and
washed with ice water, dried to obtain 3.9 g of product. The filtrate was further
concentrated twice to obtain 4.0 g and 2.1 g crystals respectively, the total yield was
1
0.0 g, yield 66.7%, and the mp was 124-125 ° C.
2
.1.2 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl 4-chlorobutanoate (2)
5
-hydroxy-3,4-dichloro-2(5H)-furanone (0.168 g, 1.0 mmol) was added into a 50
mL three-neck flask with N protection, then 7 mL of anhydrous CH Cl was added to
2
2
2
dissolve below 0 °C. Then 0.15 mL of 4-chlorobutyryl chloride was slowly aadded.
After 30 minutes, 0.5 mL of anhydrous Et3N was added, slowly increase to room
temperature for 4 h, and monitored the reaction by TLC. Quenched with 4~5 mL of
water, and extracted with CH Cl (15 mL×3), washed with saturated brine and dried
2
2
over anhydrous Na SO4. After filtration, the solvent was evaporated to dryness.
2
Purified with flash column (eluting with 1:8 v/v ethyl acetate/petroleum ether)
afforded yellow oil compound 2 for 0.232g, and yield was 85.3%. HRMS found m/z:
+
1
2
95.2 [M+Na] . H NMR (300 MHz, CDCl ): δ6.92 (s, 1H, Furan-H), 3.62 (t, J= 6.3
3
1
3
Hz, 2H, CH Cl), 2.63 (m, 2H, COCH ), 2.14 (m, 2H, CH ). C NMR (75 MHz,
2
2
2
CDCl ): δ170.4, 162.6, 146.9, 124.6, 91.3, 43.5, 30.7, 27.0.
3
2
.1.3 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yloctanoate (3)
Same method as compound 2. White solid 0.262 g was obtained in a yield of 89.2%.
+
1
HRMS found m/z: 317.3 [M+Na] . H NMR (300 MHz, CDCl ): δ6.92 (s, 1H,
3

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Furan-H), 2.33 (t, J = 7.5 Hz, 2H, COCH ), 1.64 (m, 2H, CH ), 1.28 (m, 8H, CH ),
2
2
2
1
3
0
1
.87 (t, J = 6.6 Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ171.3, 162.6, 147.1,
3
3
24.4, 91.2, 33.7, 31.5, 28.9, 28.8, 24.5, 22.5, 14.0.
2
.1.4 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yldecanoate (4)
Same method as compound 2. White solid 0.266 g was obtained in a yield of 82.7%,
and purity was 97.0% (HPLC analysis conditions: octylsilane-bonded silica gel as a
packed column; acetonitrile-water (40:60) as a mobile phase; detection wavelength of
+
1
2
1
54 nm). HRMS found m/z: 345.4 [M+Na] . H NMR(300 MHz, CDCl ): δ6.91 (s,
3
H, Furan-H), 2.44 (t, J = 7.5 Hz, 2H, COCH ), 1.66 (m, 2H, CH ), 1.24 (m, 12H,
2
2
13
CH ), 0.86 (t, J = 6.3 Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ171.2, 162.5,
2
3
3
1
47.1, 124.4, 91.1, 33.7, 31.8, 29.3, 29.2, 29.1, 28.8, 24.4, 22.6, 14.1.
.1.5 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yldodecanoate (5)
Same method as compound 2. White solid 0.159 g was obtained in a yield of 79.4%,
2
and purity was 93.8% (HPLC analysis, detection wavelength of 230 nm). HRMS
+
1
found m/z: 373.2 [M+Na] . H NMR(300 MHz, CDCl ): δ6.92 (s, 1H, Furan-H), 2.44
3
(
t, J = 7.5 Hz, 2H, COCH ), 1.65 (m, 2H, CH ), 1.28 (m, 16H, CH ), 0.86 (t, J = 6.6
2 2 2
13
Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ171.2, 162.5, 147.1, 124.3, 91.1, 33.7,
3
3
3
1.9, 29.5, 29.5, 29.3, 29.3, 29.1, 28.8, 24.4, 22.6, 14.1.
2
.1.6 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl undec-10-enoate (6)
Same method as compound 2. White oil 0.236g was obtained in a yield of 70.7%,
and purity was 97.1% (HPLC analysis, detection wavelength of 254 nm). HRMS
+
1
found m/z: 357.3 [M+Na] . H NMR(300 MHz, CDCl ): δ6.92 (s, 1H, Furan-H), 5.81
3
(
m, 1H, =CH), 4.97 (m, 2H, CH =), 2.43 (t, J = 7.5 Hz, 2H, COCH ), 2.07 (m, 2H,
2
2
13
=
C-CH ), 1.65 (m, 2H, CH ), 1.22 (m, 10H, CH ). C NMR (75 MHz, CDCl3):
2 2 2
δ179.4, 163.1, 156.1, 139.1, 114.1, 101.7, 91.1, 33.7, 29.2, 29.0, 28.9, 27.5, 26.3, 24.7,
2
4.4.
2
.1.7 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl tetradecanoate (7)

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Same method as compound 2. White solid 0.288g was obtained in a yield of 76.3%,
and purity was 95.0% (HPLC analysis, detection wavelength of 230 nm). HRMS
+
1
found m/z: 401.4 [M+Na] . H NMR(300 MHz, CDCl ): δ6.93 (s, 1H, Furan-H), 2.45
3
(
t, J = 7.2 Hz, 2H, COCH ), 1.68 (m, 2H, CH ), 1.28 (m, 20H, CH ), 0.88 (t, J = 6.9
2 2 2
13
Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ171.3, 162.4, 147.1, 124.3, 91.2, 33.7,
3
3
3
1.9, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.1, 28.9, 26.2, 24.4, 22.7, 14.1.
2
.1.8 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl palmitate (8)
Same method as compound 2. Yellow oil 0.304g was obtained in a yield of 74.9%.
+
1
HRMS found m/z: 429.5 [M+Na] . H NMR(300 MHz, CDCl ): δ6.93 (s, 1H,
3
Furan-H), 2.46 (t, J = 7.5 Hz, 2H, COCH ), 1.74 (m, 2H, CH ), 1.28 (m, 24H, CH ),
2
2
2
1
3
0
1
2
.88 (t, J = 6.9 Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ170.2, 163.9, 145.5,
3 3
26.4, 101.5, 31.9, 29.7, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 28.9, 27.5, 26.3, 24.6,
4.5, 22.7, 14.1.
2
.1.9 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl (E)-undec-2-enoate (9)
Same method as compound 2. Yellow liquid 0.276g was obtained in a yield of
3.9%, and purity was 92.6% (HPLC analysis, detection wavelength of 240 nm).
6
+
1
HRMS found m/z: 455.4 [M+Na] . H NMR(300 MHz, CDCl ): δ6.92 (s, 1H,
3
Furan-H), 5.32 (m, 2H, CH=CH), 2.44 (t, J = 7.2 Hz, 2H, COCH ), 1.99 (m, 4H,
2
13
=
C-CH ), 1.67 (m, 2H, CH ), 1.24 (m, 20H, CH ), 0.86 (t, J = 6.6 Hz, 3H, CH ). C
2 2 2 3
NMR (75 MHz, CDCl ): δ171.3, 162.7, 147.1, 130.1, 129.6, 124.4, 91.2, 33.9, 33.7,
3
3
1.9, 29.7, 29.7, 29.5, 29.3, 29.1, 29.0, 28.8, 27.2, 27.1, 24.4, 22.7, 14.1.
2
.1.10 3-acetylphenyl 4-chlorobutanoate (10)
7
mL of anhydrous CH Cl was added into 50 mL three-necked flask concluding
2 2
M-hydroxyacetophenone (0.136 g, 1.0 mmol) with N protection below -2 °C, after
2
dissolved, 0.15 mL of 4-chloroprene was slowly added. After 30 min, 0.5 mL of
anhydrous Et N was added and slowly increase to room temperature for 4 h. Then
3
monitored the reaction by TLC. Quenched with 4~5 mL of water, extracted with
CH Cl (20 mL×3), washed with saturated brine and dried over anhydrous Na SO4.
2
2
2

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The mixture was filtered and the solvent was evaporated to dryness. Purified with
flash column (eluting with 1:6 v/v ethyl acetate/petroleum ether) afforded white solid
+
1
compound 10 for 0.21g, yield 87.6%. HRMS found m/z: 263.0 [M+Na] . H
NMR(300 MHz, CDCl ): δ7.83 (ddd, J = 7.8 Hz, 1.8 Hz, 1.2 Hz, 1H, ArH), 7.67 (t, J
3
=
7.8 Hz, 1H, ArH), 7.49 (t, J = 8.4 Hz, 1H, ArH), 7.30 (ddd, J = 8.4 Hz, 2.4 Hz, 1.2
Hz, 1H, ArH), 3.69 (t, J = 6.3 Hz, 2H, ClCH ), 2.81 (m, 2H, COCH ), 2.60 (s, 3H,
2
2
1
3
CH ), 2.23 (m, 2H, CH ). C NMR (75 MHz, CDCl ): δ196.9, 171.1, 150.8, 138.6,
3
2
3
1
29.7, 127.0, 125.9, 121.3, 43.9, 31.2, 27.4, 26.7.
2
.1.11 3-acetylphenyl octanoate (11)
Same method as compound 10. White solid 0.239g was obtained in a yield of
+
1
9
1.4%. HRMS found m/z: 285.0 [M+Na] . H NMR(300 MHz, CDCl ): δ7.81 (ddd, J
3
=
7.8 Hz, 1.5 Hz, 1.2 Hz, 1H, ArH), 7.66 (t, J = 1.8 Hz, 1H, ArH), 7.47 (t, J = 8.1 Hz,
1
H, ArH), 7.29 (ddd, J = 8.1 Hz, 2.4 Hz, 1.2 Hz, 1H, ArH), 2.59 (s, 3H, CH ), 2.57 (t,
3
J = 7.5 Hz, 2H, COCH ), 1.74 (m, 2H, CH ), 1.28 (m, 8H, CH ), 0.86 (t, J = 6.6 Hz,
2
2
2
1
3
3
1
H, CH ). C NMR (75 MHz, CDCl ): δ197.0, 172.1, 151.0, 138.5, 129.6, 126.5,
3 3
25.7, 121.4, 34.3, 31.6, 29.1, 28.9, 26.7, 24.9, 22.6, 14.1.
2
.1.12 3-acetylphenyl decanoate (12)
Same method as compound 10. White solid 0.261g was obtained in a yield of
+
1
9
0.1%. HRMS found m/z:313.4 [M+Na] . H NMR(300 MHz, CDCl ): δ7.81 (ddd, J
3
=
7.8 Hz, 1.2 Hz, 0.9 Hz, 1H, ArH), 7.66 (t, J = 2.1 Hz, 1H, ArH), 7.47 (t, J = 8.1
Hz, 1H, ArH), 7.29 (ddd, J = 8.1 Hz, 2.1 Hz, 0.9 Hz, 1H, ArH), 2.59 (s, 3H, CH3),
2
6
1
.35 (t, J = 7.5 Hz, 2H, COCH ), 1.74 (m, 2H, CH ), 1.27 (m, 12H, CH ), 0.88 (t, J =
2 2 2
1
3
.6 Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ197.1, 172.2, 151.0, 138.5, 129.6,
3
3
26.5, 125.7, 121.4, 34.3, 34.0, 31.9, 29.4, 29.3, 29.1, 26.7, 24.9, 22.7, 14.1.
2
.1.13 3-acetylphenyl dodecanoate (13)
Same method as compound 10. White solid 0.284g was obtained in a yield of
+
1
8
9.4%. HRMS found m/z:341.5 [M+Na] . H NMR(300 MHz, CDCl ): δ7.84 (ddd, J
3
=
7.8 Hz, 1.5 Hz, 0.9 Hz, 1H, ArH), 7.68 (t, J = 2.1 Hz, 1H, ArH), 7.50 (t, J = 7.8

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Hz, 1H, ArH), 7.31 (ddd, J = 8.1 Hz, 2.4 Hz, 0.9 Hz, 1H, ArH), 2.62 (s, 3H, CH3),
2
6
1
1
.58 (t, J = 7.5 Hz, 2H, COCH ), 1.77 (m, 2H, CH ), 1.28 (m, 16H, CH ), 0.90 (t, J =
2 2 2
1
3
.3 Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ193.0, 172.2, 150.6, 137.6, 129.5,
3
3
26.7, 125.4, 121.4, 34.3, 32.0, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 26.7, 24.9, 22.7,
4.1.
2
.1.14 3-acetylphenyl undec-10-enoate (14)
Same method as compound 10. White solid 0.262g was obtained in a yield of
+
1
8
6.7%. HRMS found m/z: 325.0 [M+Na] . H NMR(300 MHz, CDCl ): δ7.78 (dd, J
3
=
7.8 Hz, 1.2 Hz, 1H, ArH), 7.65 (s, 1H, ArH), 7.43 (td, J = 7.8 Hz, 1.2Hz, 1H, ArH),
7
.26 (m, 1H, ArH), 5.79 (m, 1H, =CH), 4.94 (m, 2H, CH =), 2.55 (m, 5H, CH ), 2.03
2
3
1
3
(
t, J = 7.5 Hz, 2H, COCH ), 1.72 (m, 2H, CH ), 1.30 (m, 10H, CH ). C NMR (75
2 2 2
MHz, CDCl ): δ193.9, 172.0, 151.0, 139.1, 138.5, 129.6, 126.4, 125.7, 121.4, 114.2,
3
3
4.3, 33.8, 29.3, 29.2, 29.1, 29.0, 28.9, 26.6, 24.8.
2
.1.15 3-acetylphenyl tetradecanoate (15)
Same method as compound 10. White solid 0.315g was obtained in a yield of
+
1
9
7
1.1%. HRMS found m/z: 369.5 [M+Na] . H NMR(300 MHz, CDCl ): δ7.84 (dt, J =
3
.8 Hz, 1.5 Hz, 1H, ArH), 7.68 (m, 1H, ArH), 7.50 (t, J = 7.8 Hz, 1H, ArH), 7.31 (ddd,
J = 8.1 Hz, 2.4 Hz, 0.9 Hz, 1H, ArH), 2.62 (s, 3H, CH ), 2.58 (t, J = 7.5 Hz, 2H,
3
13
COCH ), 1.78 (m, 2H, CH ), 1.37 (m, 16H, CH ), 0.90 (t, J = 6.3 Hz, 3H, CH ). C
2
2
2
3
NMR (75 MHz, CDCl ): δ197.1, 172.2, 151.0, 138.5, 129.6, 126.5, 125.7, 121.5, 34.3,
3
3
2.0, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.1, 26.7, 24.9, 22.7, 14.1.
2
.1.16 3-acetylphenyl palmitate (16)
Same method as compound 10. White solid 0.331g was obtained in a yield of
+
1
8
7
8.4%. HRMS found m/z: 397.6 [M+Na] . H NMR(300 MHz, CDCl ): δ7.84 (dt, J =
3
.8 Hz, 1.2 Hz, 1H, ArH), 7.68 (m, J = 2.1 Hz, 1H, ArH), 7.50 (t, J = 7.8 Hz, 1H,
ArH), 7.31 (ddd, J = 8.1 Hz, 2.4 Hz, 0.9 Hz, 1H, ArH), 2.62 (s, 3H, CH ), 2.58 (t, J =
3
7
.5 Hz, 2H, COCH ), 1.78 (m, 2H, CH ), 1.28 (m, 24H, CH ), 0.90 (t, J = 6.3 Hz, 3H,
2 2 2
13
CH ). C NMR (75 MHz, CDCl ): δ197.0, 172.3, 151.0, 138.7, 129.6, 125.7, 121.6,
3
3

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1
21.5, 34.3, 31.9, 29.7, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 29.1, 26.7, 24.9, 22.7, 14.1.
2
.1.17 3-acetylphenyl (E)-octadec-9-enoate (17)
Same method as compound 10. White solid 0.336g was obtained in a yield of
+
1
8
7
3.9%. HRMS found m/z: 423.6 [M+Na] . H NMR(300 MHz, CDCl ): δ7.81 (dt, J =
3
.8 Hz, 1.2 Hz, 1H, ArH), 7.66 (m, 1H, ArH), 7.47 (t, J = 8.1 Hz, 1H, ArH), 7.29 (ddd,
J = 8.1 Hz, 2.4 Hz, 0.9 Hz, 1H, ArH), 5.36 (m, 2H, CH=CH), 2.59 (s, 3H, CH ), 2.56
3
(
t, J = 7.8 Hz, 2H, COCH ), 2.03 (m, 4H, =C-CH2), 1.75 (m, 2H, CH ), 1.28 (m, 20H,
2
2
13
CH ), 0.87 (t, J = 6.3 Hz, 3H, CH ). C NMR (75 MHz, CDCl ): δ197.0, 172.1,
2
3
3
1
2
51.0, 138.5, 129.6, 126.5, 125.7, 121.4, 34.3, 31.9, 31.9, 31.5, 29.7, 29.5, 29.4, 29.3,
9.2, 29.1, 27.2, 26.6, 24.9, 22.7, 22.6, 14.1.
2
.1.18 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl-4-(2-chloroethyl)benzoate (18)
7
mL of anhydrous CH Cl was added into 50 mL three-necked flask concluding
2 2
5
-hydroxy-3,4-dichloro-2(5H)-furanone (0.168 g, 1.0 mmol) with N2 protection
below -2 °C, after dissolved, 0.21mg of p-chloromethylbenzoyl chloride was slowly
added. After 30 min, 0.5 mL of anhydrous Et N was added and slowly increase to
3
room temperature for 6 h. Then monitored the reaction by TLC. Quenched with 4~5
mL of water, extracted with CH Cl (20 mL×3), washed with saturated brine and
2
2
dried over anhydrous Na SO . The mixture was filtered and the solvent was
2
4
evaporated to dryness. Purified with flash column (eluting with 1:5 v/v ethyl
acetate/petroleum ether) afforded white solid compound 18 for 0.271g, and yield was
+
1
8
4.8%. HRMS found m/z: 343.3 [M+Na] . H NMR(300 MHz, CDCl ): δ8.08 (dd, J
3
=
6.6 Hz, 1.8 Hz, 2H, ArH), 7.52 (dd, J = 6.6 Hz, 1.8 Hz, 2H, ArH), 7.17 (s, 1H,
13
Furan-H), 4.63 (s, 2H, CH Cl). C NMR (75 MHz, CDCl ): δ163.6, 162.4, 146.9,
2
3
1
44.3, 130.9, 129.1, 127.2, 123.3, 91.7, 44.9.
2
.1.19 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl thiophene-2-carboxylate (19)
mL of anhydrous CH Cl was added into 50 mL three-necked flask concluding
7
2
2
5
-hydroxy-3,4-dichloro-2(5H)-furanone (0.168 g, 1.0 mmol) with N protection below
2
-2 °C, after dissolved, 0.13 ml of 2-thiophenecarbonyl chloride was slowly added.

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After 30 min, 0.5 mL of anhydrous Et N was added and slowly increase to room
3
temperature for 6 h. Then monitored the reaction by TLC. Quenched with 9~10 mL of
water, extracted with CH Cl (20 mL×3), washed with saturated brine and dried over
2
2
anhydrous Na SO4. The mixture was filtered and the solvent was evaporated to
2
dryness. Purified with flash column (eluting with 1:3 v/v ethyl acetate/petroleum ether)
afforded white solid compound 19 for 0.250g, yield 90.1%. HRMS found m/z: 317.1
+
1
[
M+K] . H NMR(300 MHz, CDCl ): δ7.27 (d, J = 3.3 Hz, 1H, Thiophene-H), 7.00 (s,
3
1
3
1
H, Thiophene-H), 6.99 (d, J = 3.0 Hz, 1H, Thiophene-H), 6.93 (s, 1H, Furan-H). C
NMR (75 MHz, CDCl ): δ168.3, 161.0, 146.9, 132.3, 127.6, 127.1, 125.8, 124.9,
3
9
1.4.
2
.1.20 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl 2-(pyridin-2-yl)acetate (20)
2
-pyridine acetic acid (0.521 g, 3.0 mmol) was added into a 50 mL three-necked
flask, evacuated and filled with N for 3 to 4 times, 7 mL of anhydrous CH Cl and
2
2
2
0
.05 mL of DMF were added below -5 °C. After dissolved, 0.5 mL of anhydrous Et3N
was added, 0.6 mL of PivCl was added dropwise. After 30 minutes,
-hydroxy-3,4-dichloro-2(5H)-furanone dissolved in CH2Cl2 was added (0.168 g, 1.0
5
mmol). after that, DMAP (0.086 g, 0.05 mmol) was added, the reaction was
gradually returned to room temperature for 8 h. The reaction was monitored by TLC.
Quenched with 10 mL of water, extract with CH Cl (20 mL×3), wash with saturated
2
2
NaHCO3 solution and saturated brine and dry with anhydrous Na SO . The mixture
2
4
was filtered and the solvent was evaporated to dryness. Purified with flash column
(eluting with 1:3 v/v ethyl acetate/petroleum ether) afforded pale yellow solid 20 for
+
1
0
.146g, and yield was 50.9%. HRMS found m/z: 598.8 [2M+Na] . H NMR(300
MHz, CDCl ): δ8.40 (ddd, J = 5.4 Hz, 1.8 Hz, 0.9 Hz, 1H, Pyridine-H), 7.93 (ddd, J =
3
8
.7 Hz, 5.7 Hz, 1.8 Hz, 1H, Pyridine-H), 7.24 (m, 1H, Pyridine-H), 7.23 (m, 1H,
13
Pyridine-H), 6.38 (s, 1H, Furan-H), 3.72 (dd, J = 14.1 Hz, 6.9 Hz, 2H, CH ). C
2
NMR (75 MHz, CDCl ): δ170.6, 157.1, 142.4, 138.6, 130.7, 127.6, 123.7, 120.4,
3
112.3, 95.6, 47.3.
2
.1.21 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl 2-(3,4-dimethoxyphenyl)acetate

Journal Pre-proof
(21)
3
0 mL of anhydrous CH Cl was added into 100 mL round bottom flask concluding
2
2
3
(
,4-dimethoxyphenylacetic acid (0.588 g, 3.0 mmol), 0.1 mL of DMF and 0.6 mL of
COCl) were added. At this moment, the color turned yellow. After 4 h, the
2
reaction was stopped and the solvent was evaporated to give a yellow oil. After the oil
was dissolved in CH Cl , 1.5 mL of anhydrous Et 3 N was added, followed by
2
2
5
-hydroxy-3,4-dichloro-2(5H)-furanone (0.168 g, 1.0 mmol), and the reaction was
stirred at room temperature overnight. The reaction was monitored by TLC, then the
solvent was evaporated to dryness. The product was dissolved in ethyl acetate (10
mL), 10 ml Water was added. Then extracted with ethyl acetate (20 mL×3), washed
with saturated NaHCO saturated brine, dried over anhydrous Na SO . The mixture
3
2
4
was filtered and the solvent was evaporated to dryness. Purified with flash column
(eluting with 1:5 v/v ethyl acetate/petroleum ether) afforded yellow solid 21 for
+
1
0
.238g, and yield was 68.7%. HRMS foundm/z: 348.2 [M+H] . H NMR(300 MHz,
CDCl ): δ7.61 (s, 1H, Furan-H), 6.80 (d, J = 8.1 Hz, 1H, ArH), 6.70 (dd, J = 8.4 Hz,
3
2
.1 Hz, 1H, ArH), 6.63 (d, J = 2.1 Hz, 1H, ArH), 3.87 (s, 3H, OCH ), 3.83 (s, 3H,
3
1
3
OCH ), 3.65 (s, 2H, CH ). C NMR (75 MHz, CDCl ): δ173.3, 162.4, 152.6, 149.1,
3
2
3
1
48.1, 126.5, 125.0, 121.7, 112.5, 112.3, 103.4, 55.9, 55.8, 48.5.
2
.1.22 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl-4-nitrobenzoate (22)
Same method as compound 21. Yellow solid 0.159g was obtained in a yield of
+
1
5
9
0.3%. HRMS found m/z: 356.4 [M+K] . H NMR(300 MHz, CDCl ): δ8.32 (dd, J =
3
.0 Hz, 1.2 Hz, 2H, ArH), 8.23 (dd, J = 9.3 Hz, 1.8 Hz, 2H, ArH), 6.54 (s, 1H,
1
3
Furan-H). C NMR (75 MHz, CDCl ): δ164.8, 163.2, 150.5, 146.0, 135.8, 130.7,
3
1
23.5, 117.7, 88.5.
2
.1.23 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl 2-(4-nitrophenyl)acetate (23)
Same method as compound 21. Yellow solid 0.161g was obtained in a yield of
+
1
5
3.4%. HRMS found m/z: 370.1 [M+K] . H NMR(300 MHz, CDCl ): δ8.19 (m, 2H,
3
ArH), 7.48 (m, 2H, ArH), 6.10 (s, 1H, Furan-H), 3.70 (dd, J = 14.1 Hz, 7.2 Hz, 2H,

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13
CH ). C NMR (75 MHz, CDCl ): δ175.5, 164.1, 150.5, 143.9, 139.6, 130.3, 129.2,
2
3
1
23.6, 117.2, 40.5.
2
.1.24 3,4-dichloro-5-oxo-2,5-dihydrofuran-2-yl cinnamate(24)
mL of anhydrous CH Cl was added into 50 mL three-necked flask concluding
7
2
2
5
-hydroxy-3,4-dichloro-2(5H)-furanone (0.168 g, 1.0 mmol) with N protection below
2
-2 °C, after dissolved, 0.21mg of cinnamoyl chloride was slowly added. After 30
min, 0.5 mL of anhydrous Et N was added and slowly increase to room temperature
3
for 6 h. Then monitored the reaction by TLC. Quenched with 4~5 mL of water,
extracted with CH Cl (20 mL×3), washed with saturated brine and dried over
2
2
anhydrous Na SO . The mixture was filtered and the solvent was evaporated to
2
4
dryness. Purified with flash column (eluting with 1:5 v/v ethyl acetate/petroleum ether)
afforded yellow solid compound for 0.24g, and yield was 78.1%. HRMS found m/z:
+
1
3
7
6
1
37.3 [M+K] . H NMR(300 MHz, CDCl ): δ7.85 (m, 1H, =CH), 7.59 (m, 2H, ArH),
3
.46 (m, 2H, ArH), 7.43 (dd, J = 6.6 Hz, 3.0 Hz, 1H, ArH), 7.09 (s, 1H, Furan-H),
13
.52 (m, 1H, CH=). C NMR (75 MHz, CDCl ): δ172.0, 162.6, 148.8, 147.1, 131.4,
3
30.8, 129.2, 129.0, 128.7, 128.4, 117.3, 116.8, 106.4.
2
.1.25 3,4-dibromo-5-hydroxyfuran-2(5H)-one (1b)
In a 250 mL three-necked flask equipped with a condensed and stirred HBr tail gas
absorbing apparatus, fresh distilled furfural (8.1 mL, 0.1 mmol) and 96 mL of water
were added, and 25.2 mL of liquid bromine was added dropwise at -5 °C. After that,
the reaction turned to room temperature and then slowly heated to reflux for about 30
minutes. Then, it was changed to a vacuum distillation system. At this time, a large
amount of white solid was precipitated. Then, 0.9 g of NaHSO dissolved in ice water
3
was added to remove excess bromine, which was filtered with suction and washed
with cold water and dried to give a white solid of 18.6 g. 73.4%, mp is 125~126 °C.
2
.1.26 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl 4-(2-chloroethyl)benzoate (25)
Same method as compound 18. Yellow solid 0.382g was obtained in a yield of
+
1
7
9.6%. HRMS found m/z: 431.4 [M+Na] . H NMR(300 MHz, CDCl ): δ8.15 (dd, J
3

Journal Pre-proof
=
8.4 Hz, 1.8 Hz, 2H, ArH), 7.56 (dd, J = 8.7 Hz, 1.8 Hz, 2H, ArH), 6.91 (s, 1H,
13
Furan-H), 4.65 (s, 2H, CH Cl). C NMR (75 MHz, CDCl ): δ167.8, 161.7, 144.1,
2
3
1
31.0, 130.6, 129.0, 128.6, 114.2, 93.8, 45.1.
2
.1.27 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl thiophene-2-carboxylate (26)
Same method as compound 19. Yellow solid 0.317g was obtained in a yield of
+
1
8
6.8%. HRMS found m/z: 404.9 [M+K] . H NMR(300 MHz, d6-DMSO): δ7.46 (dd,
J = 4.8 Hz, 1.2 Hz, 1H, Thiophene-H), 7.12(s, 1H, Furan-H), δ7.02 (m, 2H,
1
3
Thiophene-H). C NMR (75 MHz, d6-DMSO): δ169.2, 164.5, 144.3, 134.2, 128.2,
1
27.3, 126.5, 118.3, 94.9.
2
.1.28 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl cinnamate (27)
Same method as compound 24. Yellow solid 0.30g was obtained in a yield of
+
1
7
6.3%. HRMS found m/z: 408.9 [M+Na] . H NMR(300 MHz, CDCl ): δ7.83 (m, 1H,
3
=CH), 7.57 (m, 2H, ArH), 7.44 (m, 2H, ArH), 7.40 (dd, J = 7.2 Hz, 3.0 Hz, 1H, ArH),
1
3
7
1
.07 (s, 1H, Furan-H), 6.50 (m, 1H, CH=). C NMR (75 MHz, CDCl ): δ171.9, 162.5,
3
48.7, 147.1, 134.1, 130.8, 129.1, 129.0, 128.6, 128.4, 117.3, 116.8, 108.6.
2
.1.29 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl 2-(4-nitrophenyl)acetate (28)
Same method as compound 21. Yellow solid 0.24g was obtained in a yield of
+
1
5
7.3%. HRMS found m/z: 441.4 [M+Na] . H NMR(300 MHz, CDCl ): δ8.19 (m, 2H,
3
ArH), 7.46 (m, 2H, ArH), 6.61 (s, 1H, Furan-H), 4.18 (dd, J = 14.4 Hz, 7.2 Hz, 2H,
13
CH ). C NMR (75 MHz, CDCl ): δ170.2, 163.3, 147.4, 144.5, 141.4, 130.3, 123.7,
2
3
112.8, 107.2, 41.1.
2
.1.30 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl 2-(3,4-dimethoxyphenyl)acetate
(29)
Same method as compound 21. Yellow solid 0.242g was obtained in a yield of
+
1
5
5.7%. HRMS found m/z: 473.3 [M+K] . H NMR(300 MHz, CDCl ): δ7.08 (s, 1H,
3
Furan-H), 6.83 (d, J = 8.4 Hz, 1H, ArH), 6.71 (dd, J = 8.1 Hz, 2.1 Hz, 1H, ArH), 6.64
d, J = 1.8 Hz, 1H, ArH), 3.88 (s, 3H, OCH ), 3.86 (s, 3H, OCH ), 3.67 (s, 2H, CH ).
(
3
3
2

Journal Pre-proof
1
3
C NMR (75 MHz, CDCl ): δ172.2, 168.2, 149.1, 148.1, 131.6, 126.5, 121.7, 116.6,
3
112.5, 111.3, 100.8, 55.9, 55.8, 48.5.
2
.1.31 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl 4-nitrobenzoate(30)
Same method as compound 21. Yellow solid 0.193g was obtained in a yield of
+
1
4
7.7%. HRMS found m/z: 428.3 [M+Na] . H NMR(300 MHz, CDCl ): δ8.31 (dd, J
3
=
9.0 Hz, 1.8 Hz, 2H, ArH), 8.24 (dd, J = 9.3 Hz, 2.1 Hz, 2H, ArH), 6.54 (s, 1H,
1
3
Furan-H). C NMR (75 MHz, CDCl ): δ164.8, 163.2, 146.0, 135.8, 131.3, 130.7,
3
1
23.5, 117.7, 88.5.
2
.1.32 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl nicotinate (31)
Same method as compound 21. Yellow solid 0.167g was obtained in a yield of
+
1
4
6.3%. HRMS found m/z: 722.8 [2M+H] . H NMR(300 MHz, CDCl ): δ8.78 (ddd, J
3
=
4.5 Hz, 1.5 Hz, 0.6 Hz, 1H, Pyridine-H), 8.17(dt, J =7.8 Hz, 1.2 Hz, 1H,
Pyridine-H), 7.88 (td, J =6.6 Hz, 1.5 Hz 1H, Pyridine-H), 7.51 (ddd, J = 6.0 Hz, 4.8
1
3
Hz, 1.2 Hz, 1H, Pyridine-H), 6.93 (s, 1H, Furan-H). C NMR (75 MHz, CDCl ):
3
δ168.1, 165.7, 149.8, 147.9, 137.1, 134.0, 127.0, 125.1, 117.8, 103.4.
2
.1.33 3,4-dibromo-5-oxo-2,5-dihydrofuran-2-yl 2-(pyridin-2-yl)acetate (32)
Same method as compound 21. Yellow solid 0.197g was obtained in a yield of
+
1
5
8
2.5%. HRMS found m/z: 373.7 [M-H] . H NMR(300 MHz, CDCl ): δ9.36 (ddd, J =
3
.7 Hz, 6.2 Hz, 1.5 Hz, 1H, Pyridine-H), 9.02 (ddd, J = 7.5 Hz, 6.3 Hz, 1.8 Hz, 1H,
Pyridine-H), 8.40 (m, 1H, Pyridine-H), 7.93 (m, 1H, Pyridine-H), 6.38 (s, 1H,
13
Furan-H), 3.72 (dd, J = 14.4 Hz, 7.2 Hz, 2H, CH ). C NMR (75 MHz, CDCl ):
2
3
δ170.7, 163.9, 142.6, 138.5, 130.7, 127.6, 123.7, 120.4, 112.3, 95.5, 39.7.
2
.2 MIC determination
To define the biofilm inhibitory test concentration under MIC and make sure the
effects of new compounds were via QS inhibition not that of inhibiting the bacteria
themselves. The MICs of all compounds were to measure. It was performed on
planktonic cultures using the two-fold dilution method according to clinical and

Journal Pre-proof
2
4
laboratory standards institute guidelines. MICs were performed in 96-well sterile
o
microplates (cosmo) and the results were recorded after 18h of incubation at 37 C.
5
-(dibromomethylene)furan-2(5H)-one was used as positive control.
2
.3 In vitro determination of biofilm formation
All the synthetic compounds above were assayed for their in vitro bacterial biofilm
formation inhibitory activities against P. aeruginosa ATCC27853, ATCC9027 and
PAOA. Method of crystal violet staining was used. P. aeruginosa was diluted with LB
broth at OD =0.05. A concentrated compound solution was transferred into each
6
00
well, except those used as controls, to achieve a final test concentration of 64 μg/mL,
o
and each well was filled to a final volume of 200μL. After 20h of incubation at 35 C
and being washed twice with PBS (Phosphate Buffered Saline) solution, the biofilm
remained on the wells was fixed with 200μL of 99% methanol for 15 min. The
solution was discarded and the microplate was put in super clean bench. When the
wells were dried, each of them was added with 250 μL of 1% solution of crystal violet
(CV). After 15 min staining at room temperature, the wells were washed twice
carefully with distilled solution. Then 250 μL 95% ethanol was added to each well, as
to dissolve the stain and biofilm. 15 min later, the absorbance of plates was
determined at 570 nm in a spectrophotometer. The percentage of biofilm inhibition
was calculated using the following formula: inhibition percentage=[(ODnegative control
–
2
5-30
OD ) /ODnegative control]×100, where x refers to the tested halogenated compounds.
x
To find out the rule of concentration affecting QS system, some of the synthesized
compounds were chose to determine the biofilm inhibition percentages at
concentrations of MIC, 1/2MIC, 1/4MIC, 1/8MIC and 1/16MIC using the same
method described above.
2
.4 Observation of P. aeruginosa biofilm formation with scanning electron
microscopy (SEM)
The P. aeruginosa inoculums were centrifuged (10000 rpm, 5 min) and washed with
saline twice to acquire planktonic cultures. It was diluted with sterile saline at
0
.5MCF (Mcfarland standard), and then diluted 25 fold with sterile saline. 1mLof the
prepared cultures was added to each test tube, as well as 1mL compound solution

Journal Pre-proof
(dissolved by LB broth, 128 μg/mL). The final concentration of compounds was
6
4μg/mL. After the polyvinyl chloride catheter (0.5 cm×0.5 cm) was set on the
o
bottom of each tube, cultures were incubated in shaking bath at 37 C for 7 days.
Following washed twice with PBS solution, the bacteria on the catheter was fixed by
placing in 2.5% glutaraldehyde/cacodylate (v/v) buffer for 5h. The samples were then
dried with ethanol steps—50% aqueous ethanol (v/v) for 10min, 75% aqueous ethanol
(v/v) for 10min, 85% aqueous ethanol (v/v) for 10min, 95% aqueous ethanol (v/v) for
1
0min, 100% aqueous ethanol (v/v) for 10min, and isoamyl acetate for 10 min. They
o
were frozen in a freezer at -65 C and dried at the critical point of vacuum pressure at
o
-
53 C, and then covered with gold. Biofilms on the catheter were then observed with a
31,32
SEM.
2
.5 Pyocyanin Quantification Assay
The OD600 value of overnight culture of P. aeruginosa PAOA was adjusted to 0.1 and
diluted 100 times with fresh King’s medium B with a final volume of 5 mL. Then,
compounds were added to the bacteria suspensions with a final concentration of 64
o
μg/mL and cultured for 48 h at 37 C in a shaking-bath (200 rpm). After that, bacteria
cultures were centrifuged for 10 min and the supernatants were collected. Pyocyanin
in the supernatants were extracted using 1 mL chloroform and 0.3 mL HCl (0.2 M).
The content of the extracted pyocyanin was quantified by measuring the OD value at
5
20 nm. The absorbance of this solution was measured at 520 nm. Concentrations,
expressed as micrograms of pyocyanin produced per milliliter of culture supernatant,
3
3
were determined by multiplying the optical density at 520 nm (OD ) by 17.072.
5
20
2
.6 P. aeruginosa QS Inhibition Assays.
Test compounds were mixed with ABTGC medium and serial diluted to give a final
concentration of 20 μM in the first well. An overnight culture of PAO1-lasB-gfp strain
(grown in LB medium at 37°C, 200 rpm) was diluted in ABTGC medium to a final
optical density at 600 nm (OD ) of 0.02 (2.5 × 108 CFU/mL). An equal amount of
6
00
the bacterial suspension was added to the wells to reach final inhibitor concentration

Journal Pre-proof
of 10 μM. DMSO control (0.1% final concentration) and blank control were used. The
microtiter plate was incubated at 37°C in Tecan Infinate 200 Pro plate reader (Tecan
Group Ltd., Mꢀnnedorf, Switzerland) to measure the cell density (OD ) and GFP
600
fluorescence (excitation at 485 nm, emission at 535 nm) with 15min intervals for at
least 15 h. The inhibition assay for all test compounds and controls were done in
triplicate manner. For P. aeruginosa Rhl and Pqs inhibition assay, a similar method
3
4
was performed as the LasB inhibition assay.
.7 Cytotoxicity and plasma stability study
2
1
The cell viability was measured as described in a previous report . Briefly, RAW264.7
cells were grown in DMEM media supplements with 10% FBS, counted using a
hemocytometer. Cells were then serially diluted in a clear cell culture plate and
incubated for 4 hours with MTT reagent at 37°C. After incubation, cells were treated
with MTT solvent for 15 minutes at room temperature. Absorbance was measured at
35
OD .
5
90
Plasma and compound 29 (100 µM in DMSO) are added to the individual wells of a
6-well microtiter plate. The plate is incubated at 37 °C with gentle agitation. During
9
the incubation, aliquots are withdrawn at 0, 15, 30, 60, and 120 minutes time points
and quenching solution (25/50 ng/mL terfenadine/tolbutamide in ACN/MeOH (1:1,
v/v)) is added. After mixing, the quenched aliquots are centrifuged and supernatant is
3
6
withdrawn for analysis by HPLC-UV.
3
3
. Results
.1 Synthesis of Furanone Derivatives
Alkyl chains with different length were first considered as a potent key structure in
QS and biofilm inhibition according to AHLs and 3OC12. Referring to the length of
alkyl chains in native signal molecule, a range of C3 to C15 alkyl acids were acylated
at C-5 hydroxyl in 5-hydroxyl-3,4-halogenated-5H-furan-2-ones. Meanwhile,
molecules with aromatic ring substituted furanone rings drew our attention for their
reported activity in inhibiting LasR at low micromolar concentrations and reducing
the production of various virulence factors. Thus, a series of compounds with an

Journal Pre-proof
aromatic core structure instead of furanone rings were also synthesized. The same
alkyl chains were acylated to 3-hydroxy acetophenone to obtain compounds 10-17. A
hypothesis of a key π-π interaction driven us thinking to replace the alkyl chain by an
aromatic substituent, hence different aromatic acids with electron-withdrawing or
electron-donating substituents were linked to chloro or bromo 5H-furan-2-ones to
obtain compounds 24-32. The detailed procedures and yields are shown in the method
section.
[
Scheme 1]
Scheme 2]
[
3
.2 MIC of inhibitors
The results (Table 1) showed that most of compounds we synthesized have MICs
higher than 1024 µg/mL except compounds 20 and 21, whose MIC against strain
ATCC 9027 is 32 µg/mL.
[Table 1]
3
.3 Biofilm inhibition
Biofilm inhibition ratio was evaluated at concentrations lower than the MIC by
crystal violet staining quantitative determination. The assays were carried out at 1/2,
/4, 1/8 and 1/16 the concentration of MIC to investigate the effect on biofilm
1
inhibition under a safe dose for the P. aeruginosa life cycle. The results were shown
in Table 2, 3, 4, 5 and indicated a concentration-dependent relation between the
compounds and biofilm inhibition.
[Table 2]
[Table 3]
[Table 4]
[Table 5]
3
.4 SEM Observation of compound 29
The effect of compound 29 on biofilm formation was further confirmed by scanning
electron microscopy (SEM). As shown in Figure 2, compared with the blank,

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incubation of P. aeruginosa PAOA with compounds 29 at different sub-MIC resulted
in obvious reduction of the biofilm.
[Figure 2]
3
.5 Pyocyanin Quantification Assay
P. aeruginosa also produces a blue-green phenazine pigment called pyocyanin, a
secondary metabolite whose expression is under the control of QS. To verify the
anti-biofilm and anti-bacteria activity of the obtained compounds, pyocyanin
production assays were established. As shown in Figure 3, a significant decrease in
pyocyanin production of P. aeruginosa was observed after treatment with phenolic
compounds and their derivatives. Among which compound 29 showed obvious
reduction in pyocyanin formation, which is consistent with the biofilm formation
inhibitory activity.
[
Figure 3]
3
.6 P. aeruginosa QS Inhibition Assays
To further confirm the QS inhibitory activity of compound 29, reporter strain
3
7
PAO1-lasB-gfp was introduced to perform the screening. As shown in Figure 4, the
decrease of GFP fluorescence indicated the presence of 3-oxo-C12-HSL antagonist
and the inhibition of the lasB gene. The GFP expression was normalized by dividing
the GFP values with the OD600 measured at the corresponding time points. Compound
2
9 were found to be effective in inhibiting the lasB expression in a dose-dependent
manner.
[Figure 4]
3
.7 Cytotoxicity and plasma stability of compound 29
The cytotoxicity of compound 29 was determined using MTT assay in RAW264.7
cells. As shown in Figure 5, the cell viability remained basically at 100% after
treatment with the compounds, indicating the low toxicity of compound 29.
Additionally, compound 29 in Table 6 showed relatively stable in the plasma stability
assay.
[Figure 5]

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[Table 6]
4
. Discussion
As mentioned above, our goal is to achieve a new approach to combat P.
aeruginosa without disturbing bacteria's life cycle. A safe concentration must be
determined before the evaluation of biofilm inhibition. The MIC assay results suggest
that the compounds do not disturb the normal life cycle of P. aeruginosa at high
3
8
concentrations as most antibiotics do. For the drug resistance are caused on occasion
in eliminating bacteria, those compounds might coexist with P. aeruginosa and do not
trigger the mechanism of resistance. On the basis of the MIC data, biofilm inhibition
ratio was evaluated at concentrations lower than the MIC. The assays were carried out
at 1/2, 1/4, 1/8 and 1/16 the concentration of MIC to investigate the effect on biofilm
inhibition under a safe dose for the P. aeruginosa life cycle. And the results showed a
concentration-dependent relation between the compounds and biofilm inhibition. At
1
/2 and 1/4 MIC most of the furanone compounds inhibit biofilm formation well in
drug resistant strains, including P. aeruginosa strains ATCC27853, ATCC9027, and
PAOA (clinical isolates). At 1/8 MIC, the inhibition declined but most chlorofuranone
compounds still exhibit considerable inhibition. However, at 1/16 MIC the inhibition
ratios declined to a quite low level, and only compound 9 showed a promising effect
with 45.4% inhibition on PAOA. Some compounds even demonstrated a promotion
effect on biofilm formation. Although the biofilm inhibitory effects at low
concentrations are not as promising as compound 9, compounds with activity superior
to positive control still account for a large proportion of those tested. The overall
analysis revealed that compounds with furanone as core structure are in general more
potent than those with a 3-hydroxyacetophenone core. The hydrophobic alkyl chain of
a length above C-10 is desirable. Chloro-substitutuents seem more active at high
concentration, whereas at low concentrations they resemble bromo substituted
furanone derivatives. This structure information emphasizes the halogenate furanone
and the hydrophobic side chain.
After comparing the furanone and acetophenone derivatives, we returned our

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focus to the alkyl chain. As we know, the long alkyl chain normally contributes no
polar interaction in binding with acceptors, we wondered if this alkyl chain could be
replaced by other hydrophobic groups of less flexibility. Fortunately, the AHLs
bearing such a long alkyl chain have been well studied by X-ray crystallography. The
interaction mode between AHLs and LasR was depicted from an X-ray
3
9-43
crystallography 4NG2.
As shown in Figure 6, the two key contacts with LasR are
a) hydrogen bonding interactions with protein at residues Asp73, Ser 129 and Tyr56,
b) van der Waals contact deep in the hydrophobic pocket via the alkyl group.
[Figure 6]
Having investigated the conformation of alkyl group and hydrophobic pocket
consisted of Tyr64, Val76 and Tyr47, we noted that the pocket is not a narrow
channel but a cavity surrounded by residues with aromatic rings. The pocket wraps
curly alkyl chains. A hypothesis occurred to us: if the alkyl chain was replaced by an
aromatic substituent, the π-π interaction would help improve binding affinity and
biofilm inhibition. To verify this hypothesis compounds 24-32 were synthesized and
evaluated too.
Among compounds 24-32, the MIC values are not that stagnant for a multitude
of compounds can inhibit strain ATCC 9027 at 32 µg/mL, except for compounds 25
and 29 which are of desired tolerance. The biofilm inhibition ratio was still evaluated
in concentrations at 1/2, 1/4, 1/8 and 1/16 MIC. We are overjoyed by the excellent
performance of compound 29 in biofilm inhibition against ATCC 9027 whose
inhibitory rate at 1/16 MIC was 63%. With other strains and concentrations
compound 29 also demonstrated adorable activities. The effect of compound 29 on
biofilm formation was further confirmed by scanning electron microscopy (SEM).
These assays exhibited the outstanding biofilm inhibitory effects of compound 29 at
every level of concentration. The pyocyanin assay also indicated compound 29 with
obvious reduction in pyocyanin formation, which is consistent with the biofilm
formation inhibitory activity. Considering its prominent effect in reducing pyocyanin
production and the desired MIC value, compound 29 showed the most promising
potency in biofilm inhibition. To explore the possible mechanism of compound 29

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2
9
inhibiting the LasR, a brief molecular docking study was conducted (Figure 7).
Superimposition of AHLs and compound 29 reveled similar interactions and the
aromatic substituent laid exactly where the alkyl chain is, which conformed to our
hypothesis.
[Figure 7]
According to the QS Inhibition assays, compound 29 can inhibit the lasB
expression in a dose-dependent manner. In addition to this, rhlA-gfp and pqsA-gfp
bioreporters in the same stain which correlate rhamnolipid and PQS molecules
respectively were also studied and the results were proven to be affirmative. The
cytotoxicity and ADME stability assay also give promising result in the future
development of compound 29.
5
. Conclusion
In conclusion, we have used natural and chemically synthesized halogenated
furanones as beginning to prepare analogs bearing alkyl chain, vinyl bromide key or
aromatic ring. Optimization efforts established the furanone core structure and
hydrophobic side chain. Combination of these findings ultimately led to the discovery
of compound 29, a potent new approach to combat the drug resistant bacteria strains.
On the basis of our findings, compound 29 was advanced into a promising lead in
combating drug resistant bacteria. As potent QSI candidates, compound 29 which
have no effect in suppressing the regular life cycle of the bacteria, may bring us a new
anti-bacterial mechanism that trigger no drug resistance system while the
pathogenicity and virulence factors decline.

ACKNOWLEDGMENTS
This study is supported by the research grant from the National Natural Science Fund
of China (No. 81573294, 81773593, 81872759), the Excellent Young Teachers
Program of Guangdong Provincial colleges and universities (YQ 2015061), the Major
Project for the Innovation of Industry and Research of Guangzhou City (No.
2
01508020128), the Pearl River S&T Nova Program of Guangzhou (201610010100),

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and the Fundamental Research Funds for the Central University (No. 21617478). All
authors would also like to thank Dr. Liang Yang from the Singapore Centre for
Environmental Life Sciences Engineering, Nanyang Technological University for
gifting the PAO1-lasB-gfp reporter strain, the rhlA-gfp and pqsA-gfp plasmids.

ABBREVIATIONS
QS, quorum sensing; MIC, minimum inhibitory concentration; MDR, multiple drug
resistant; QSIs, quorum sensing inhibitors; AHLs, acyl homoserine lactones; 3OC12,
N-3-oxododecanoyl homoserine lactone; SEM, scanning electron microscopy.

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Figure 1. Natural products to 5-hydroxyl-3,4-halogenated-5H-furan-2-ones.

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Figure 2. Scanning electron micrographs (5000×) of PAOA on the surface of catheter
for seven days in the absence (blank) or presence of compound 29.