Design, synthesis and activity evaluation study of novel substituted N-sulfonyl homoserine lactone derivatives as bacterial quorum sensing inhibitors

Article abstract of DOI:10.1007/s00044-017-2027-2

A novel series of N-sulfonyl homoserine lactone derivatives 7a–7m has been designed, synthesized, and evaluated for quorum sensing inhibitory activities through the violacein inhibition in Chromobacterium violaceum CV026. Compound 7e displayed the high level of inhibitory activity among all the compounds synthesized. Studies of structure-activity relationship indicated that compounds with thiophene group in side chain showed better activity than those substituted by furan, pyrrole, pyridyl, and phenethyl group. Thiophene substituted compounds which connected electron withdrawing group exhibited better inhibitory activity relate to those connected electron donating group. Further analysis indicated that compound bearing an electron withdrawing substituent at the position 2 of their thiophene ring exhibited superior activity against violacein production to those bearing the substituent at the position 3 and 4. Compound 7e in particular, with IC₅₀ value of 6.19 μM, were identified as promising lead compounds for further development.

Full text of DOI:10.1007/s00044-017-2027-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2027-2
ORIGINAL RESEARCH
Design, synthesis and activity evaluation study of novel substituted
N-sulfonyl homoserine lactone derivatives as bacterial quorum
sensing inhibitors
Qi Sun1 Mingming Zhao2 Jingwei Liang1 Junhai Xiao3 Fanhao Meng1
●
●
●
●
Received: 14 May 2017 / Accepted: 5 August 2017
© Springer Science+Business Media, LLC 2017
Abstract A novel series of N-sulfonyl homoserine lactone
derivatives 7a–7m has been designed, synthesized, and
evaluated for quorum sensing inhibitory activities through
the violacein inhibition in Chromobacterium violaceum
CV026. Compound 7e displayed the high level of inhibitory
activity among all the compounds synthesized. Studies of
structure-activity relationship indicated that compounds
with thiophene group in side chain showed better activity
than those substituted by furan, pyrrole, pyridyl, and phe-
nethyl group. Thiophene substituted compounds which
connected electron withdrawing group exhibited better
inhibitory activity relate to those connected electron
donating group. Further analysis indicated that compound
bearing an electron withdrawing substituent at the position
2 of their thiophene ring exhibited superior activity against
violacein production to those bearing the substituent at the
position 3 and 4. Compound 7e in particular, with IC50
value of 6.19 µM, were identified as promising lead com-
pounds for further development.
●
Keywords Bacterial quorum sensing inhibitor
●
●
●
Acylhomoserine lactone Design Synthesis Activity
evaluation
Introduction
In the growth process of bacteria, they can produce some
chemical signal molecules called auto-inducers and release
them into the surrounding environment. When the con-
centration of auto-inducers reaches to a certain threshold, a
variety of specific receptors (genes) associated with tran-
scription signals will be regulated to adapt the environ-
mental changes, which is known as the bacterial quorum
sensing (Swift et al. 2001; Yang et al. 2010). Several
important phenotypes are regulated by quorum sensing,
including biofilm formation and bioluminescent (Kim et al.
2009; Steven et al. 2001). Currently, quorum sensing has
been observed in more than 50 kinds of bacteria, including
Pseudomonas aeruginosa and Escherichia coli (Bassler
2002; Choudhary and Schmidt–Dannert 2010). Quorum
sensing inhibitors make pathogens lose pathogenicity by
preventing the expression of harmful genes, which do not
interfere with the normal physiological processes of bac-
teria, allowing for the discovery of new direction for the
development of novel antibacterial agents (Stewart and
William 2001; Galloway et al. 2011; Ng and Bassler 2009;
Reverchon et al. 2002).
* Junhai Xiao
13611090335@139.com
* Fanhao Meng
fhmeng@cmu.edu.cn
Qi Sun
qsun@cmu.edu.cn
Mingming Zhao
zhaomingmingbest@163.com
Jingwei Liang
liangjw89@sina.com
1
School of Pharmacy, China Medical University, 110122 Liaoning,
China
2
School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 110016 Liaoning, China
3
Laboratory of Computer-Aided Drug Design and Discovery,
Beijing Institute of Pharmacology and Toxicology, 100850
Beijing, China

Med Chem Res
Fig. 1 Structures of C6-HSL, CL and C10-HSL
Fig. 2 Design of the target compounds
lactone analogs bearing phenyl group in the tail chain
possessed superior quorum sensing inhibitory activity than
bearing aliphatic group (Castang et al. 2004). Based on
above, in order to increase the inhibitory activity, we
designed to replace the amide group with 4-
aminobenesulfonyl moiety between the nucleus and the
acyl side chain, and the compound should have aryl group
in tail chain.
To research the inhibitory activities of novel AHLs
quorum sensing inhibitors against violacein production, we
have made several changes to the structures of the existing
inhibitors CL and C10-HSL, while reserving the key
homoserine lactone nucleus which is the active essential
group of quorum sensing inhibitory activity (McClean et al.
1997). A 4-aminobenzenesulfonyl moiety was introduced
between nucleus and acyl side chain, which could improve
hydrophobicity of the compounds and exhibit broad biolo-
gical activity (Castang et al. 2004). In order to explore the
inhibitory activity effects of the five-aromatic, six-aromatic
heterocyclic group and phenethyl group in the tail chain
following the introduction of the 4-aminobenzenesulfonyl
moiety, a series of novel N-sulfonyl homoserine lactone
derivatives 7a–7m (Fig. 2) was designed, synthesized and
valuated as potential quorum sensing inhibitors on the basis
of their ability to inhibit the production of violacein, and a
preliminary analysis of the structure-activity relationship
(SAR) study of these target compounds was conducted.
Chromobacterium violaceum is a gram-negative bacteria
that synthesizes the violet pigment violacein as the result of
quorum sensing using N-acyl homoserine lactone (C6-HSL,
Fig. 1) as its auto-inducer (Choo et al. 2006). C. violaceum
CV026 (CV026) has been widely used as a bacterial model
to screen new quorum sensing inhibitors, since the phe-
nomenon of its quorum sensing system is easily observed
and facilitate quantitative analysis (Durán and Menck
2001).
A great number of N-acyl homoserine lactone (AHL)
derivatives have been developed on the basis of their inhi-
bition of violacein production. Chloro Lactone (CL) and
C10-HSL (Fig. 1) are the effective quorum sensing inhibi-
tors reported to date (Reverchon et al. 2002; Chen et al.
2011; Fuqua et al. 2001). AHLs can freely access cell
membrane and combine with receptor protein because they
possess a hydrophilic homoserine lactone ring together with
a hydrophobic aliphatic amide side chain. CviR is a cano-
nical transcription factor protein from C. violaceum. The
mechanism of inhibiting violacein production was that C10-
HSL allowed DNA binding but reduced or eliminated
transcriptional activation, suggesting that the CviR-C10-
HSL complex could not productively interact with RNA
polymerase. But CL prevented CviR from binding DNA
(Swem et al. 2009).
Among a number of AHLs quorum sensing inhibitors,
relatively few studies have focused on the amide moiety
between homoserine lactone nucleus and acyl side chain. To
research the function of the amide groups, ureidos, sulfo-
namides and heterocyclic rings were replaced (Frezza et al.
2006; Sabbah et al. 2012; Castang et al. 2004). Using
bioisosterism, the amide group was replaced by ureido
group. The resulting compounds exhibited good quorum
sensing inhibitory activity in Vibrio fischeri (Frezza et al.
2006). Furthermore, heterocyclic rings like triazoles and
tetrazoles replaced the amide group because they showed
some similarity with amide bonds (Sabbah et al. 2012).
Replacing amide group with sulfonamide, Castang et al.
synthesized a series of N-sulfonyl homoserine lactone
analogs which displayed quorum sensing inhibitory activity
in V. fischeri because of the widespread biological activity
of sulfonamides. Furthermore, N-sulfonyl homoserine
Results and discussion
Chemical synthesis
All the agents were commercially available and were
directly used without further purification unless mentioned
otherwise. The syntheses of the intermediates and target
compounds were completed according to the steps
demonstrated in Scheme 1. Commercially available L-
methionine 1 was reacted with bromoacetic acid at reflux to
give homoserine lactone hydrobromide 2, which was sub-
sequently reacted with 4-acetylamino-benzenesulfonyl
chloride to give compound 3. Using 95% ethanol as the

Med Chem Res
Scheme 1 Synthesis of target
compounds 7a–m. Reaction
conditions and reagents: a
bromoacetic acid, acetic acid,
H2O, 2-propanol, reflux, then
concentrated HCl, 55 °C, 30
min; b 4-acetylamino-
benzenesulfonyl chloride, TEA,
EtOH, 0 °C to rt., overnight; c 6
mol/L HCl, EtOH, reflux, 4 h; d
oxalyl chloride, CH2Cl2, 0 °C to
rt., overnight; e CH2Cl2, 0 °C to
rt., overnight
solvent, compound 3 which treated with refluxing in 6 mol/
L HCl for 2 h, produced the hydrolyzate compound 4. With
dichloromethane as the solvent, N,N-Dimethylformamide
(DMF) as a catalyst, a series of five or six membered aro-
matic carboxylic acid 5 produced the corresponding acid
chloride intermediate 6 under the action of oxalyl chloride.
Ultimately, according to amide Schotten–Baumann reac-
tion, compound 4 was reacted with intermediate 6 to give
the desired target compounds 7a–7m. The structures of all
the target compounds were characterized by methods of MS
(b)
(a) compound 7e
Fig. 3 Growth inhibition spot tests of compound 7e
1
and H-NMR.
target compound 7a–7m have no effect on the normal
growth of C. violaceum CV026 but inhibit bacterial QS
system. The violacein was produced by inducer C6-HSL.
The quorum sensing inhibitory activity of target com-
pounds 7a–7m were evaluated through the inhibition of the
violacein production in CV026, with compound C10-HSL
being used as a positive control. The experimental results
showed the white colonies in purple background of plates
were observed in all target compounds, which indicated
compounds 7a–7m have varying range of quorum sensing
inhibitory activity. The IC50 values for each synthesized
compound have been summarized in Table 1.
As shown in Table 1, compounds 7a–7b and 7d–7f
exhibited considerable levels of quorum sensing inhibitory
activity against the production of violacein, with IC50
values ranging from 6.19–130.74 μM. Among these newly
synthesized compounds 7e exhibited the best inhibitory
activity with IC50 value of 6.19 μM, which showed the
potential as lead compound for further development of
Evaluation of biological activity figures
The biological activity of target compounds were evaluated
through the violacein inhibition in C. violaceum CV026.
Since quorum sensing inhibition is focused on the inhibition
of bacterical quorum sensing signal molecules and not on
the interference of the normal physiological activity of
bacteria, we designed to exclude the effect of growth inhi-
bition activity on quorum sensing before the evaluation of
inhibitory activity. The spot test method was performed to
test growth inhibition activity of compound 7a–7m against
C. violaceum CV026. The results showed the white colonies
in purple background of flatbeds (plates) and no transparent
growth inhibition zone even in the highest concentration of
compound 7e (Fig. 3a), and the same phenomenon had been
observed on other compound. As compared with them, Fig.
3b showed the transparent growth inhibition zone with a
sharp and regular edge. The above results indicated that

Med Chem Res
Table 1 The substituents and IC50 values of 7a–7m for inhibiting
violacein production
Table 1 continued
Compound
R
IC50(μM)
Compound
R
IC50(μM)
7l
NAa
7a
130.74 1.33
7m
NAa
7b
7c
102.69 2.76
NAa
C10-HSL
0.66 0.07
a
NA means no significant inhibition
novel quorum sensing inhibitors. Considering the SAR
studies derived from these synthesized compounds sug-
gested that the introduction of 4-aminobenzenesulfonyl
moiety between the homoserine lactone nucleus and acyl
side chain of CL and C6-HSL contributed to the enhance-
ment of inhibitory activity against violacein production. The
introduction of thiophene group at the R position provided
better inhibitory activity relate to the furan, pyrrole, pyridyl,
and phenethyl group, as exemplified by the comparison of
compound 7a and 7g, 7j, 7k, 7l. Thiophene substituted
compounds which connected electron withdrawing group
exhibited better inhibitory activity relate to those connected
electron donating group, as the example of the comparison
of 7e and 7d. Further analysis indicated that compound
bearing an electron withdrawing substituent at the position
2 of their thiophene ring exhibited superior activity against
violacein production to those bearing the substituent at the
position 3 and 4. Compound 7e containing position 2 sub-
stituted thiophene group at the R position exhibited the
highest level of inhibitory activity against violacein pro-
duction of all of the analogs.
7d
7e
7f
49.22 1.42
6.19 0.34
13.51 0.54
7g
7h
NAa
NAa
7i
7j
NAa
NAa
Molecular modeling
A molecular docking study was conducted with the CviR
structure (PDB entry 3QP1), which C6-HSL and compound
7e were docked into the structure using molecular operating
environment (MOE) 2014.09. C6-HSL could bind to the
CviR from bacteria C. violaceum to regulate the production
of violacein as AI. As shown in Fig. 4, compound 7e
occupied the space to that of the natural ligand C6-HSL.
Both of them bound to CviR through hydrogen bonding
interactions with the Asp 97 and Trp 84 residues.
7k
NAa
C6-HSL and compound 7e have been shown as pink and
green stick models, respectively. The key amino acid resi-
dues which provided interactions with C6-HSL and com-
pound 7e have been drawed as their respective chemical

Med Chem Res
Cambridgeshire, UK). The mass spectra were determined
using an API-3000 mass spectrometer. The mass spectra
were determined using an Agilent 5875 (EI) spectrometer
1
(Palo Alto, CA, USA). The H-NMR (400 MHz) and 13C-
NMR (100 MHz) spectra were recorded with a Bruker ARX
400 spectrometer (Karlsruhe, Germany), using TMS as a
reference material and DMSO as solvent. Thin—layer
chromatography silica gel GF254 (Qingdao Marine Che-
mical Products) was used to monitor the extent of reaction.
All solvents and reagents were purchased from commercial
sources and used without further purification.
Fig. 4 Molecular model of CviR with C6-HSL and compound 7e
bound to the active site
Synthesis of compound 2–4
(S)-3-aminodihydrofuran-2(3H)-one hydrobromide (2)
Compound 2 was synthesized with a well-established lit-
erature procedure (Nielsen and Givskov 2003). It was
obtained as a white solid;Yield 55%; mp 220–224 °C;
25
D
1
½αŠ = −24.2° (c = 0.1, H2O); H-NMR (400 Hz, DMSO-
d6) δ: 8.76 (s, 3H), 4.46 (t, 1H, J = 8.0 Hz,J = 7.6 Hz),
4.35–4.31 (m,2H), 2.56–2.51 (m,1H), 2.30–2.27 (m,1H);
EI-MS m/z: 102.1 [M + H]+.
(S)-N-(4-(N-(2-oxotetrahydrofuran-3-yl)sulfamoyl)phenyl)
acetamide (3)
L-homoserine lactone hydrobromide 2 (10.9 g, 60 mmol)
and triethylamine (25.5 mL, 120 mmol) were dissolved in
ethanol (120 mL). 4-acetylaminobenzenesulfonyl chloride
(16.8 g, 72 mmol) was added at 0 °C in a portion-wise
manner. The resulting mixture was stirred overnight at
ambient temperature. The next day, the reaction solution
was concentrated to 1/3 volume and poured into ice-cold
water (200 mL) with vigorous stirring for 1 h. Then the
white solid precipitate was collected by filtration. The filter
cake was washed with ice water and dried under vacuum
before being recrystallized from ethaol to give white solid 3.
Fig. 5 Comparison of C6-HSL molecular docking conformation and
eutectic conformation
structures. Cyan dotted lines represented the hydrogen
bonding interactions between the compounds and the amino
acid residues (Trp 84 and Asp 97).
In order to validate the docking protocol,a self-docking
of C6-HSL into the binding pocket was firstly performed.
As shown in Fig. 5, C6-HSL docking pose stacked well with
the crystallographic ligand (RMSD = 0.9429), and the H-
bonds predicted by MOE were similar to those found in the
crystal structure.
1
(12 g, 67%); mp 174–176 °C; H-NMR (400 Hz, DMSO-
d6) δ: 10.33 (s, 1H), 8.16 (d, 1H, J = 8.0 Hz), 7.75 (s, 4H),
4.34–4.30 (m, 1H), 4.21 (m, 1H), 4.08 (m, 1H), 2.08–2.06
(m, 4H), 1.80(m, 1H); EI-MS m/z: 299.34 [M + H]+.
(S)-4-amino-N-(2-oxotetrahydrofuran-3-yl)
benzenesulfonamide (4)
Materials and methods
General procedures
6 mol/L HCl (20 mL) was added with stirring to a solution
of intermediate 3(10 g, 33 mmol) in ethanol (40 mL). After
heating at relux for 4 h, the reaction mixture was then
cooled and the solvent was evaporated to dryness in vacuo
to give the crude product, which was dissolved in water
(100 mL). The pH of the solution was adjusted to 8–9 with
1 mol/L NH4OH. After stirring for 1 h, the resulting white
The melting points of intermediates and target compounds
were determined with a YRT-4 melting point detector (P.I.
F. Tianjin University,Tianjin, China) and have been repor-
ted as uncorrected values. Optical rotations were measured
with a Polar 3005 polarimeter (Optical Activity Ltd.,

Med Chem Res
solid was filtrated under vacuum. The white solid was
collected by filtration. Then the filter cake was washed with
ice-cold water and dried under vacuum before being
recrystallied from ethaol to give product 4 (5.2 g, 57%); mp
(400 Hz, DMSO-d6) δ: 10.64 (s, 1H), 8.21 (d, 1H,
J = 8.0 Hz), 7.94 (m, 3H), 7.82 (d, 2H, J = 8.0 Hz), 7.32 (s,
1H), 4.37 (m, 1H), 4.22 (m, 1H), 4.10 (m, 1H), 2.14 (m,
1H), 1.83 (m, 1H); 13C-NMR (DMSO-d6) δ: 174.6(C-1′),
159.3(C = O), 142.2(C-4), 138.5(C-4”), 135.9(C-1), 134.7
(C-1”), 130.0(C-2”), 128.5(C-3”), 127.7(C-2,C-6), 119.9
(C-3,C-5), 65.2(C-4′), 51.4(C-2′), 29.5(C-3′); EI-MS m/z:
401.1 [M + H]+.
1
163–165 °C; H-NMR (400 Hz, DMSO-d6) δ: 7.72 (d, 1H,
J = 9.2 Hz), 7.45 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.8
Hz), 5.95 (m, 2H), 4.21 (m, 2H), 4.09 (m, 1H), 2.06(m, 1H),
1.78 (m, 1H); EI-MS m/z: 256.4 [M + H]+.
Synthesis of different aromatic chain substituted
chloride 6
(S)-4-methyl-N-(4-(N-(2-oxotetrahydrofuran-3-yl)
sulfamoyl)phenyl)thiophene-2-carboxamide (7c)
A five or six membered aromatic carboxylic acid 5 was
dissolved in CH2Cl2 with dropping three drop DMF. 1.5
times the amount of oxalylchloride was cautiously added
into the reaction solution at 0 °C. After stirring at ambient
temperature for 3 h, the reaction mixture was concentrated
under reduced pressure to give the corresponding acid
chloride intermediate 6. The remaining acid chloride were
synthesized according to the method described above with a
yield of 83–92%.
Compound 7c was obtained as a pale yellow solid (0.38 g,
50% yield) from compound 4 according to the same pro-
cedure used for the synthesis of 7a. mp 187–188 °C; H-
1
NMR (400 Hz, DMSO-d6) δ: 10.50 (s, 1H), 8.23 (d, 1H,
J = 8.0 Hz), 7.94 (m, 3H), 7.81 (d, 2H, J = 8.0 Hz), 7.51 (d,
1H, J = 8.0 Hz), 4.37 (m, 1H), 4.23 (m, 1H), 4.11 (m, 1H),
2.14 (m, 1H), 1.83 (m, 1H); 13C-NMR (DMSO-d6) δ: 174.6
(C-1′), 160.3(C = O), 142.6(C-4), 138.9(C-1”), 138.2(C-4”),
135.6(C-1), 131.7(C-2”), 128.1(C-3”), 127.6(C-2,C-6),
119.7(C-3,C-5), 65.2(C-4′), 51.4(C-2′), 29.5(C-3′), 15.5(C-
5”); EI-MS m/z: 381.1[M + H]+.
General procedure for the preparation of compound 7
(S)-N-(4-(N-(2-oxotetrahydrofuran-3-yl)sulfamoyl)phenyl)
thiophene-2-carboxamide(7a)
(S)-3-methyl-N-(4-(N-(2-oxotetrahydrofuran-3-yl)
sulfamoyl)phenyl)thiophene-2-carboxamide (7d)
Thiophene-2-carbonyl chloride (0.36 g, 2.44 mmol) was
cautiously added into a solution of compound 4 (0.52 g,
2.03 mmol) and triethylamine (0.41 g, 4.06 mmol) in dry
CH2Cl2 (15 mL) at 0 °C in a portion-wise manner, and the
reaction system was stirred at ambient temperature over-
night. The next day, the reaction mixture was filtrated under
reduced pressure before the filtrate was washed with water.
Then the yellow solid was precipitated from the organic
phase and was filtrated under vacuum to provide the desired
Compound 7d was obtained as a white solid (0.40 g, 53%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 176–178 °C; 1H-NMR
(400 Hz, DMSO-d6) δ: 10.36 (s, 1H), 8.23 (d, 1H, J = 8.0
Hz), 7.88 (d, 2H, J = 8.0 Hz), 7.79 (d, 2H, J = 8.0 Hz), 7.72
(m, 1H), 7.06 (d, 1H, J = 8.0 Hz), 4.36 (m, 1H), 4.23 (m,
1H), 4.11 (m, 1H), 2.14 (m, 1H), 1.83 (m, 1H); 13C-NMR
(DMSO-d6) δ: 174.6(C-1′), 161.8(C = O), 142.7(C-4),
141.6(C-1”), 135.6(C-1), 131.6(C-4”), 130.9(C-2”), 128.5
(C-3”), 127.6(C-2,C-6), 119.9(C-3,C-5), 65.2(C-4′), 51.4(C-
2′), 29.5(C-3′), 15.5(C-5”); EI-MS m/z: 381.0[M + H]+.
1
product 7a (0.44 g, 60%); mp 114–119 °C; H-NMR (400
Hz, DMSO-d6) δ: 10.57 (s, 1H), 8.25 (1H, d, J = 8.0 Hz),
8.22 (d, 1H, J = 8.0 Hz), 8.07 (d, 1H, J = 8.0 Hz), 7.93 (m,
3H), 7.82 (d, 2H, J = 8.0 Hz), 7.26 (d, 1H, J = 8.0 Hz), 4.37
(m, 1H), 4.23 (m, 1H), 4.11 (m, 1 H), 2.14 (m, 1H), 1.84
(m, 1H); 13C-NMR (DMSO-d6) δ: 174.6(C-1′), 160.3
(C = O), 142.5(C-4), 139.4(C-1”), 135.7(C-1), 132.7(C-4”),
129.9(C-2”), 128.3(C-3”), 127.6(C-2,C-6) 119.9(C-3,
C-5), 65.2(C-4′), 51.4(C-2′), 29.5(C-3′); EI-MS m/z: 366.9
[M + H]+.
(S)-3-chloro-N-(4-(N-(2-oxotetrahydrofuran-3-yl)
sulfamoyl)phenyl)thiophene-2-carboxamide (7e)
Compound 7e was obtained as a white solid (0.42 g, 52%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 219–221 °C; 1H-NMR
(400 Hz, DMSO-d6) δ: 10.63 (s, 1H), 8.23 (d, 1H, J = 8.0
Hz), 7.96 (m, 1H),7.87 (d, 2H, J = 8.0 Hz), 7.82 (d, 2H,
J = 8.0 Hz), 7.24(d, 1H, J = 8.0 Hz), 4.37 (m, 1H), 4.23 (m,
1H), 4.11 (m, 1H), 2.13 (m, 1H), 1.83 (m, 1H); 13C-NMR
(DMSO-d6) δ: 174.6(C-1′), 160.1(C = O), 142.7(C-4),
136.1(C-1), 134.7(C-1”), 132.6(C-2”), 130.5(C-4”), 127.6
(C-2,C-6),119.7(C-3,C-5), 110.7(C-3”), 65.2(C-4′), 51.4(C-
2′), 29.5(C-3′); EI-MS m/z: 401.3[M + H]+.
(S)-5-chloro-N-(4-(N-(2-oxotetrahydrofuran-3-yl)
sulfamoyl)phenyl)thiophene-2-carboxamide (7b)
Compound 7b was obtained as a yellow solid (0.45 g, 55%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 243–249 °C; 1H-NMR

Med Chem Res
(S)-3-bromo-N-(4-(N-(2-oxotetrahydrofuran-3-yl)
sulfamoyl)phenyl)thiophene-2-carboxamide (7 f)
Hz), 7.45 (d, 1H, J = 8.0 Hz), 6.87 (d, 1H, J = 8.0 Hz), 4.36
(m, 1H), 4.23 (m, 1H), 4.10 (m, 1H), 2.13 (m, 1H), 1.83 (m,
1H); 13C-NMR (DMSO-d6) δ: 174.6(C-1′), 155.4(C = O),
148.8(C-4”), 142.1(C-4), 135.9(C-1), 127.6(C-2,C-6), 126.1
(C-1”), 119.9(C-3,C-5), 118.1(C-2”), 114.5(C-3”), 65.2(C-
4′), 51.4(C-2′), 29.5(C-3′); EI-MS m/z: 453.1[M + Na]+.
Compound 7 f was obtained as a yellow solid (0.52 g, 58%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 151–154 °C; 1H-NMR
(400 Hz, DMSO-d6) δ: 10.71 (s, 1H), 8.24 (d, 1H, J = 8.0
Hz), 7.92 (m, 1H),7.87 (d, 2H, J = 8.0 Hz), 7.83 (d, 2H,
J = 8.0 Hz), 7.27 (d, 1H, J = 8.0 Hz), 4.37 (m, 1H), 4.23
(m, 1H), 4.11 (m, 1H), 2.13 (m, 1H), 1.83 (m, 1H);
13C-NMR (DMSO-d6) δ: 174.6(C-1′), 159.8(C = O), 142.1
(C-4), 136.2(C-1), 132.8(C-1”), 131.5(C-2”), 130.5(C-4”),
127.7(C-2,C-6), 119.7(C-3,C-5), 111.7(C-3”), 65.2(C-4′),
51.4(C-2′), 29.5(C-3′); EI-MS m/z: 444.9[M + H]+.
(S)- N-(4-(N-(2-oxotetrahydrofuran-3-yl)sulfamoyl)phenyl)
-1H-pyrrole-2-carboxamide (7j)
Compound 7j was obtained as a pale yellow solid (0.34 g,
50% yield) from compound 4 according to the same pro-
1
cedure used for the synthesis of 7a. mp 170–174 °C; H-
NMR (400 Hz, DMSO-d6) δ: 11.79 (d, 1H, J = 8.0 Hz),
10.10 (s, 1H),8.17 (d, 1H, J = 8.0 Hz), 7.95 (d, 2H, J = 8.0
Hz), 7.80 (d, 2H, J = 8.0 Hz), 7.12 (d, 1H, J = 8.0 Hz), 7.02
(d, 1H, J = 8.0 Hz), 6.20 (d, 1H, J = 8.0 Hz), 4.36 (m, 1H),
4.23 (m, 1H), 4.11 (m, 1H), 2.13 (m, 1H), 1.83 (m, 1H);
13C-NMR (DMSO-d6) δ: 174.6(C-1′), 161.3(C = O), 142.3
(C-4), 135.6(C-1), 127.6(C-2,C-6), 123.7(C-1”), 120.3(C-
4”), 119.7(C-3,C-5), 110.5(C-3”), 65.2(C-4′), 51.4(C-2′),
29.5(C-3′); EI-MS m/z: 365.0[M + H]+.
(S)-N-(4-(N-(2-oxotetrahydrofuran-3-yl)sulfamoyl)phenyl)
furan-2-carboxamide (7 g)
Compound 7 g was obtained as a yellow solid (0.46 g, 65%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 195–196 °C;1H-NMR (400
Hz, DMSO-d6) δ: 10.49 (s, 1H), 8.18 (d, 1H, J = 8.0 Hz),
7.96 (m, 3H),7.82 (d, 2H, J = 8.0 Hz), 7.40 (d, 1H, J = 8.0
Hz), 6.72(d, 1H, J = 8.0 Hz), 4.36 (m, 1H), 4.23 (m, 1H),
4.11 (m, 1H), 2.17 (m, 1H), 1.86 (m, 1H); 13C-NMR
(DMSO-d6) δ: 174.6(C-1′), 156.5(C = O), 147.0(C-1”),
146.3(C-4”), 142.4(C-4), 135.7(C-1), 127.6(C-2,C-6), 119.9
(C-3,C-5), 115.7(C-2”), 112.4(C-3”), 65.2(C-4′), 51.4(C-2′),
29.5(C-3′); EI-MS m/z: 351.1[M + H]+.
(S)-N-(4-(N-(2-oxotetrahydrofuran-3-yl)sulfamoyl)phenyl)
isonicotinamide (7k)
Compound 7k was obtained as a white solid (0.32 g, 45%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 174–185 °C; 1H-NMR
(400 Hz, DMSO-d6) δ: 10.86 (s, 1H), 8.81 (m, 2H), 8.23 (d,
1H, J = 8.0 Hz), 7.97 (d, 2H, J = 8.0 Hz), 7.86 (m, 4H),
4.36 (m, 1H), 4.23 (m, 1H), 4.11 (m, 1H), 2.14 (m, 1H),
1.83 (m, 1H); 13C-NMR (DMSO-d6) δ: 174.6(C-1′), 164.7
(C = O), 150.4(C-3”,C-4”), 142.3(C-4), 151.6(C-1”), 136.2
(C-1), 127.6(C-2,C-6), 121.7(C-2”,C-5”), 120.12(C-3,
C-5), 65.2(C-4′), 51.4(C-2′), 29.5(C-3′); EI-MS m/z: 362.1
[M + H]+.
(S)-N-(4-(N-(2-oxotetrahydrofuran-3-yl)sulfamoyl)phenyl)
furan-3-carboxamide (7 h)
Compound 7 h was obtained as a pale yellow solid (0.40 g,
58% yield) from compound 4 according to the same pro-
cedure used for the synthesis of 7a. mp 202–209 °C; H-
1
NMR (400 Hz, DMSO-d6) δ: 10.28 (s, 1H), 8.44 (s, 1H),
8.21 (d, 1H, J = 8.0 Hz), 7.92 (m, 5 H), 7.02 (d, 1H, J = 8.0
Hz), 4.36 (m, 1H), 4.23 (m, 1H), 4.10 (m, 1H), 2.13 (m,
1H), 1.82 (m, 1H); 13C-NMR (DMSO-d6) δ: 174.6(C-1′),
160.9(C = O), 146.4(C-4”), 144.5(C-3”), 142.7(C-4), 135.5
(C-1), 127.6(C-2,C-6), 122.7(C-1”), 119.7(C-3,C-5), 109.3
(C-2”), 65.2(C-4′), 51.4(C-2′), 29.5(C-3′); EI-MS m/z: 351.1
[M + H]+.
(S)-N-(4-(N-(2-oxotetrahydrofuran-3-yl)sulfamoyl)phenyl)-
2-(o-tolyl)acetamide (7l)
Compound 7 l was obtained as a white solid (0.36 g, 46%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 191–193 °C; 1H-NMR
(400 Hz, DMSO-d6) δ: 10.56 (s, 1H), 8.17 (d, 1H, J = 8.0
Hz), 7.78 (m, 4H), 7.16 (m, 4H), 4.35 (m, 1H), 4.21 (m,
1H), 4.08 (m, 1H), 3.73 (s, 2H), 2.10 (m, 1H), 1.81 (m, 1H);
13C-NMR (DMSO-d6) δ: 174.6(C-1′), 169.8(C = O), 142.9
(C-4), 136.8(C-1), 135.2(C-2”), 134.3(C-3”), 130.1(C-4”),
130.0(C-7”), 127.8(C-2,C-6), 126.9(C-5”), 125.9(C-6”),
118.8(C-3,C-5), 65.2(C-4′), 51.4(C-2′), 41.0(C-1”), 29.5(C-
3′), 19.5(C-8”); EI-MS m/z: 389.2[M + H]+.
(S)-5-bromo-N-(4-(N-(2-oxotetrahydrofuran-3-yl)
sulfamoyl)phenyl)furan-2-carboxamide (7i)
Compound 7i was obtained as a pale yellow solid (0.53 g,
61% yield) from compound 4 according to the same pro-
cedure used for the synthesis of 7a. mp 190–196 °C; H-
NMR (400 Hz, DMSO-d6) δ: 10.56 (s, 1H), 8.24 (d, 1H, J
= 8.0 Hz), 7.94 (d, 2H, J = 8.0 Hz), 7.81 (d, 2H, J = 8.0
1

Med Chem Res
(S)-2-(2-chlorophenyl)-N-(4-(N-(2-oxotetrahydrofuran-3-
yl)sulfamoyl)phenyl)acetamide (7m)
inhibitory concentration) and were taken to be average of
three determinations. All the IC50 values were calculated
with the Origin 8.0 software.
Compound 7 m was obtained as a white solid (0.43 g, 52%
yield) from compound 4 according to the same procedure
used for the synthesis of 7a. mp 223–228 °C; 1H-NMR
(400 Hz, DMSO-d6) δ: 10.64 (s, 1H), 8.18 (d, 1H, J = 8.0
Hz), 7.78 (m, 4H), 7.44 (m, 4H), 4.33 (m, 1H), 4.21 (m,
1H), 4.08 (m, 1H), 3.89 (s, 2H), 2.11 (m, 1H), 1.81 (m, 1H);
13C-NMR (DMSO-d6) δ: 174.6(C-1′), 168.7(C = O), 142.8
(C-4), 135.2(C-1), 133.8(C-3”), 133.6(C-2”), 132.4(C-7”),
129.1(C-4”), 128.8(C-5”), 127.8(C-2,C-6), 127.2(C-6”),
118.7(C-3,C-5), 65.2(C-4′), 51.4(C-2′), 40.9(C-1”), 29.5(C-
3′); EI-MS m/z: 409.2[M + H]+.
Conclusions
A novel series of N-sulfonyl homoserine lactone derivatives
7a–7m has been designed, synthesized and evaluated for
quorum sensing inhibitory activities against violacein pro-
duction. Preliminary SAR studies indicated that compounds
with thiophene groups at the R position showed better
inhibitory activity than those substituted by furan, pyrrole,
pyridyl and phenethyl groups at the same position. Thio-
phene substituted compounds which connected electron
withdrawing substituent displayed better levels of inhibitory
activity relate to those connected electron donating sub-
stituent. Of the compounds synthesized, compound 7e
exhibited promising levels of inhibitory activity towards
violacein production, with IC50 values of 6.19 μM and are
currently being identified as promising lead compound for
further structural transformation studies.
Evaluation of the biological activity
Through the primary screening of the synthesized com-
pounds, the inhibitory activities against violacein produc-
tion were determined according to a spot test method with
compound C10-HSL being used as a positive control. Spe-
cifically, CV026 (400 μL) was cultured in molten semi-solid
medium. The phosphate buffered solution (PBS) solution of
C6-HSL (15 μL, 0.125 mM) was then added and the mixture
was uniformly mixed with the semi-solid LB agar (5 mL).
The resulting mixture was subsequently dumped on the
surface of the solid LB agar. Upon solidification of the
mixture plate, the test compounds were spotted on the
plates. The plates were then placed in an oven and being
cultured for 16–18 h at 30℃. The inhibitory activities of the
compounds towards violacein production were determined
by the presence of white colonies in purple background of
the plates. If the transparent growth inhibition zone were
observed, it would suggest that the compounds had the
growth-inhibitory effect.
For the further screening of the compounds which
inhibited violacein production, CV026 were cultured to log
phase in LB medium and then placed in 12-well plates with
the LB medium. The PBS solution of C6-HSL (15 μL,
0.125 mM) and different concentrations of the compounds
which showed inhibitory activities in the primary screening
were added to the wells. After an incubation period of
16–18 h, a portion (1 mL) of cultures was removed from
each mixture to Eppendorf tube at 12,470 × g for 10 min to
precipitate the insoluble violacein and the culture super-
natant was discarded. Following DMSO (500 μL) was
added to each Eppendorf tube to completely dissolve the
violacein, the resulting mixtures were centrifuged at
12,470 × g for 10 min to remove cells. The upper layer of
the above violacein solution (200 μL) was then removed
and placed into a 96-well microplate, and its absorbance
value at 585 nm was measured(Olivero et al. 2011). The
final results were expressed as IC50 (half maximum
Acknowledgements This work was supported by Chinese national
science foundation with Grant No. 81274182, 81573687.
Compliance with ethical standards
Conflicts of interest The authors declare that they have no com-
peting interests.
References
Bassler BL (2002) Small talk:Cell-to-cell communication in bacteria.
Cell 109:421–424
Castang S, Chantegrel B, Deshayes C, Dolmazon R, Gouet P, Haser R,
Reverchon S, Nasser W, Hugouvieux-Cotte-Pattat N, Doutheau
A (2004) N-Sulfonyl homoserine lactones as antagonists of
bacterial quorum sensing. Bioorg Med Chem Lett 14:5145–5149
Chen G, Swem LR, Swem DL, O’Loughlin CT, Jeffrey PD, Bassler
BL, Hughson FM (2011) A strategy for antagonizing quorum
sensing. Mol Cell 42:199–209
Choo JH, Rukayadi Y, Hwang JK (2006) Inhibition of bacterial
quorum sensing by vanilla extract. Lett Appl Microbiol
42:637–641
Choudhary S, Schmidt-Dannert C (2010) Applications of quorum
sensing in biote -chnology. Appl Microbiol Biotechnol
86:1267–1279
Durán N, Menck CF (2001) Chromobacterium violaceum: a review of
pharmacological and industiral perspectives. Crit Rev Microbiol
27:201–222
Frezza M, Castang S, Estephane J, Soulere L, Deshayes C, Chantegrel
B, Nasser W, Queneau Y, Reverchon S, Doutheau A (2006)
Synthesis and biological evaluation of homoserine lactone
derived ureas as antagonists of bacterial quorum sensing. Bioorg
Med Chem 14:4781–4791

Med Chem Res
Fuqua C, Parsek MR, Greenberg EP (2001) Regulation of gene
expression by cell-to-cell communication Acyl-homoserine lac-
tone quorum sensing. Annu Rev Genet 35:439–468
Galloway WR, Hodgkinson JT, Bowden SD, Welch M, Spring DR
(2011) Quorum sensing in gram negative bacteria: small-
molecule modulation of AHL and AI-2 quorum sensing path-
ways. Chem Rev 111:28–67
Kim C, Kim J, Park HY, Park HJ, Kim CK, Yoon J, Lee JH (2009)
Development of inhibitors against TraR quorum-sensing system
in agrobacterium tumefaciens by molecular modeling of the
ligand-receptor interaction. Mol Cells 28:447–453
McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR, Camara M,
Daykin M, Lamb JH, Swift S, Bycroft BW, Stewart GS, Williams
P (1997) Quorum sensing and Chromobacterium violaceum:
exploitation of violacein production and inhibition for the detec-
tion of N-acylhomoserine lactones. Microbiology 143:3703–3711
Ng WL, Bassler BL (2009) Bacterial quorum-sensing network archi-
tectures. Annual Review of Genetic 43:197–222
Reverchon S, Chanteqrel B, Deshayes C, Doutheau A, Cotte-Pattat N
(2002) New synthetic analogues of N-Acyl homoserine lactones
as agonists or antagonists of transcriptional regulators involved in
bacterial quorum sensing. Bioorg Med Chem 12:1153–1157
Sabbah M, Fanny F, Grand L, Boukraa M, Efrit ML, Doutheau A,
Soulère L, Queneau Y (2012) Synthesis and biological evaluation
of new N-acyl-homoserine-lactone analogues, base on triazole
and tetrazole scaffolds, acting as LuxR-dependent quorum sen-
sing modulators. Bioorg Med Chem 20:4727–4736
Steven AM, Queneau Y, Soulère L, von Bodman S, Doutheau A
(2001) Mechanisms and synthetic modulators of AHL-dependent
gene regulation. Chem Rev 111:4–27
Stewart PS, William CJ (2001) Antibiotic resistance of bacteria in
biofilms. The Lancet 358:135–138
Swem LR, Swem DL, O’Loughlin CT, Gatmaitan R, Zhao B, Ulrich
SM, Basster BL (2009) A quorum-sensing antagonist targets both
membrane-bound and cytoplasmic receptors and controls bac-
terial pathogenicity. Mol Cell 35(2):143–453
Nielsen J, Givskov M (2003) Compounds and Methods for controlling
bacterial virulence field of the invention. WO03/106445 A1, 24
December 2003.
Olivero JT, Pájaro NP, Stashenko E (2011) Antiquorum sensing
activity of essential oils isolated from different species of the
genus piper. Vitea revista de la facultad de quimica farmaceutica
18:2145–2166
Swift S, Downie JA, Whitehead NA, Barnard AML, Salmond GPC,
Williams P (2001) Quorum sensing as a population -density-
dependent determinant of bacterial physiology. Adv Microb
Physiol 45:199–270
Yang L, Liu Y, Stemberg C, Molin S (2010) Evaluation of Enoyl-Acyl
carrier protein reductase inhibitors as pseudomonas aeruginosa
quorum-quenching reagents. Molecules 15:780–792