Synthesis and biological evaluation of novel L-homoserine lactone analogs as quorum sensing inhibitors of pseudomonas aeruginosa

Article abstract of DOI:10.1248/cpb.c19-00359

In this study, we synthesized four series of novel L-homoserine lactone analogs and evaluated their in vitro quorum sensing (QS) inhibitory activity against two biomonitor strains, Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PAO1. Studies of the structure–activity relationships of the set of L-homoserine lactone analogs indicated that phenylurea-containing N-dithiocarbamated homoserine lactones are more potent than (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (C30), a positive control for biofilm formation. In particular, compared with C30, QS inhibitor 11f significantly reduced the production of virulence factors (pyocyanin, elastase and rhamnolipid), swarming motility, the formation of biofilm and the mRNA level of QS-related genes regulated by the QS system of PAO1. These results reveal 11f as a potential lead compound for developing novel antibacterial quorum sensing inhibitors.

Full text of DOI:10.1248/cpb.c19-00359

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Chem. Pharm. Bull. 67, 1088–1098 (2019)
Vol. 67, No. 10
Regular Article
Synthesis and Biological Evaluation of Novel L-Homoserine Lactone
Analogs as Quorum Sensing Inhibitors of Pseudomonas aeruginosa
Haoyue Liu,a,b,c Qianhong Gong,d Chunying Luo,a,b,c Yongxi Liang,a,b,c Xiaoyan Kong,a,b,c
,a,b,c
Chunli Wu,*
Pengxia Feng,a,b,c Qing Wang,a,b,c Hui Zhang,a,b,c and M.A. Wirekoa,b,c
b
a School of Pharmaceutical Sciences, Zhengzhou University; Zhengzhou 450001, P. R. China: Key Laboratory of
Technology of Drug Preparation (Zhengzhou University), Ministry of Education of China; Zhengzhou 450001, P. R.
c
China: Key Laboratory of Henan Province for Drug Quality and Evaluation; Zhengzhou 450001, P. R. China: and
d Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean
University of China; Qingdao 266003, P. R. China.
Received May 3, 2019; accepted July 3, 2019
In this study, we synthesized four series of novel L-homoserine lactone analogs and evaluated their in
vitro quorum sensing (QS) inhibitory activity against two biomonitor strains, Chromobacterium violaceum
CV026 and Pseudomonas aeruginosa PAO1. Studies of the structure–activity relationships of the set of L-
homoserine lactone analogs indicated that phenylurea-containing N-dithiocarbamated homoserine lactones
are more potent than (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (C30), a positive control for bio-
film formation. In particular, compared with C30, QS inhibitor 11f significantly reduced the production of
virulence factors (pyocyanin, elastase and rhamnolipid), swarming motility, the formation of biofilm and the
mRNA level of QS-related genes regulated by the QS system of PAO1. These results reveal 11f as a potential
lead compound for developing novel antibacterial quorum sensing inhibitors.
Key words quorum sensing; L-homoserine lactone; Pseudomonas aeruginosa; dithiocarbamate; phenylurea;
benzothiazolyl
the interest of scientific researchers. For example, cefazolin
Introduction
N-Acyl homoserine lactones (AHLs) are well-known quo- exhibits a potent inhibitory effect against Pneumococcus and
rum sensing molecules of Gram-negative bacteria.1,2) For Haemophilus influenza.20) Moreover, the aromatic thioether
example, Pseudomonas aeruginosa regulates its physiologi- group has long been used as a linker to connect different
cal activity by releasing N-(3-oxododecanoyl)-L-homoserine biologically active moieties in the design of novel com-
lactone3,4) (OdDHL, Fig. 1). Previous studies have shown that pounds.21–23) The phenylurea moiety has biological activities,
AHLs regulate the production of biofilm and virulence factors including antibacterial, anticancer and insecticide24) activities,
that include elastase and pyocyanin.5–7) Thus, many bacteria and phenylurea derivatives have been found to be fungicidal
tend to express virulence factors when the concentration of against Rhizoctonia solani and Pellicularia spp.25)
AHLs they have generated is high enough to overcome inher-
Brominated furanones (BFs) are a leading class of anti-viru-
lence compounds and have been proven to prevent the normal
ent host defenses.8,9)
Expression of virulence genes is also connected with las, operation of the QS system, which competes with AHLs for a
rhl and Pseudomonas quinolone signal (PQS) systems which receptor protein.26) We chose the frequently used (Z)-4-bromo-
are important quorum sensing (QS) systems.10–13) LasI, for 5-(bromo-methylene)-2(5H)-furanone (C30, Fig. 1) as a posi-
example, directs the synthesis of OdDHL in the las QS tive control for inhibition of the las system for our evaluation
system. Once OdDHL reaches a critical threshold concentra- of the anti-biofilm formation activity of our analogs.27)
tion, it binds to transcriptional regulatory protein lasR.14) The
Inspired by previous research about the antibacterial evalu-
QS network of P. aeruginosa is very complicated, with each ation of AHL analogs containing thioester linkages,28) we
signaling mechanism related to and affecting another. The las herein report the design and synthesis of novel L-homoserine
system is thought to be at the top of the regulatory network, lactone analogs by introducing a thioether group (Fig. 1 7a–d,
which can positively regulate expression of the rhlI/R gene, 8a–c) or a phenylurea group (Fig. 1 11a–i, 12a–c) at the side
the pqsABCDE gene cluster and the pqsR gene.3)
chain terminus, and the antibacterial activities of these ana-
Bacteria gain resistance to antibiotics due to the ease with logs against the P. aeruginosa QS system. Structure–activity
which their biofilm and virulence factors induce mutations relationships (SARs) and molecular docking studies were con-
that allow them to evade the toxic effects of antibiotics.15,16) ducted to rationalize the mechanism of the action of these
Quorum sensing inhibitors (QSIs) are thus considered a new QSIs.
direction for antibacterial drug discovery because they only
inhibit the virulence factors of bacteria and do not interfere
with the normal physiological activities of pathogens, so a re-
sistance response is not triggered.17–19)
Results and Discussion
Chemistry The L-homoserine lactone hydrochloride 4
was efficiently synthesized from methionine (1) following
Aromatic thioethers are a class of important sulfur-contain- previously reported methods29) (Chart 1). The reported optical
ing derivatives with biological activities, and have attracted rotation of compound 4 ([α]2D0=−27.5° (c 0.2, water)) was used
*To whom correspondence should be addressed. e-mail: kedi2009@126.com
© 2019 The Pharmaceutical Society of Japan

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to confirm its stereochemistry.30) With crucial intermediate 4 activity of the las QS system, and were more potent than the
in hand, four series of novel L-homoserine lactone (HL) ana- positive control (C30). However, analogs 7a, 7d and 8a had no
logs were synthesized by introducing different moieties onto effect on QS. 7b, 8b and 8c showed excellent inhibitory activ-
the amine.
ity against CV026 in Figure S1. According to the biological
Acyl compounds 5 and 6 were formed by reacting 4 with data for series I and II against CV026, it can be seen that the
chloroacetyl chloride and 3-chloropropionyl chloride, respec- extra carbon atom of 8c increased its inhibitory activity com-
tively, under Schotten–Baumann conditions at room tempera- pared with that of its analog 7c.
ture.31,32) Compound series I and II were prepared by reacting
Surprisingly, almost all compounds of series III, containing
different mercapto compounds with 5 and 6, respectively, both phenylurea groups and dithiocarbamate groups, exhibited
in the presence of K2CO333) (Chart 2). After the reaction of remarkable QS inhibitory (QSI) activity against CV026 (Fig.
compound 4 with CS2, compound series III were synthesized S1) and PAO1 (Fig. 2). However, only the analogs with the
by one-pot reaction with compounds 10a–i34) (Chart 3). Com- electron-donating groups on the terminal phenyl group (11b–f,
pound series IV were synthesized by coupling compounds 11h and 11i) showed powerful inhibitory activity against the
10a–c with compound 435) (Chart 3). The structures and yields las system. These results indicated that the position and elec-
of these four synthetic series are shown in Table 1 and Table 2. tronic character of the substituents on the terminal phenyl ho-
Biological Evaluation A QSI screening model (las system moserine lactone analogs series III are critical for this series’
model: QSIS-lasI36)) was used to evaluate the inhibitory activ- QSI activity. Specifically, 2-methoxy (11b) and 2-methyl (11h)
ity of newly synthesized compounds against P. aeruginosa. analogs were more potent than 4-methoxy (11d) and 3-methyl
This screening model is based on the sucrose lethal gene sacB (11i) analogs. Biological evaluation of compounds of series IV
being activated after self-expressed or externally added signal revealed that they had weak QSI activity. This clearly shows
molecules bind to the LasR protein expressed by the P. aeru- that the dithiocarbamate group of series III is critical to its
ginosa las system. The growth of bacteria can be inhibited in QSI activity.
LB medium containing sucrose. Thus, if QSIs are added into
To explore the mechanism of action of active compounds,
LB medium, the expression of the sacB gene will be inhibited, we tested the effects of analogs 7b, 8b, 8c, 11b, 11f and 11i
meaning the bacteria can grow normally. This screening princi- on the production of virulence factors such as the inhibition
ple is consistent with the series of high throughput QSI screen- of pyocyanin production, reduction of elastase activity and
ing models constructed by Rasmussen’s laboratory.37) The in- reduction of rhamnolipid production. Meanwhile, we exam-
hibitory effects of all compounds of the four series against the ined the effects of these active compounds on the formation of
P. aeruginosa PAO1 las system are shown in Fig. 2.
biofilm and swarming motility. As shown, analogs 7b, 8b, 8c,
It can be seen from Fig. 2 that analogs 7b, 8b, 7c and 8c 11b, 11f, 11i and the positive control C30 had no significant
containing acyl homoserine lactones with benzothiazolyl or effects on the growth of PAO1 at 15µM (Fig. 3A). Thus, we
4-chlorophenyl substituents showed significant inhibitory decided to examine their impact on virulence factors at this
concentration and found that analogs 7b, 8b, 8c, 11b, 11f and
11i could inhibit the production of pyocyanin. Specifically,
these analogs decreased the production of pyocyanin in PAO1
by 14.2, 15.1, 27.5, 33.0, 34.5 and 30.3%, respectively, at sub-
minimum inhibitory concentrations (sub-MIC), with the in-
hibitory activity of analogs 8c, 11b, 11f and 11i being greater
than that of C30 (31.6%) (Fig. 3B). 11b, 11f and 11i decreased
the production of elastase by 14.3, 13.7 and 18.4%, respective-
ly, almost as much as the reduction by C30 (16.5%) (Fig. 3C).
7b, 8b, 8c, 11b, 11f, 11i and C30 decreased the production of
rhamnolipid in PAO1 by 20.5, 22.2, 17.6, 20.8, 28.1, 16.7 and
25.8%, respectively. Overall, 11f was the most potent against
virulence factor production compared with C30 (Fig. 3D).
Interestingly, all active compounds also inhibited biofilm
formation (Fig. 3E), with this decreased by 28.6, 38.0, 34.1,
35.3, 36.2, 40.3 and 28.6% by 7b, 8b, 8c, 11b, 11f, 11i and
C30, respectively. Moreover, analogs 7b, 11f and 11i showed
stronger inhibitory effects on the swarming motility of Pseu-
domonas than C30 (Fig. 3F), with 11f being the most active.
These results verified that analogs 7b, 8b, 8c, 11b, 11f and 11i
Fig. 1. Structures of OdDHL, C30 and the Active Core Group of the
Three Series of Target Compounds
Reagents and conditions: (a) MeOH/H2O, CH3I, r.t., 20h; (b) NaHCO3(aq), r.t.→ reflux 12h; (c) 6M HCl, 30% H2O2, reflux, 12h.
Chart 1. Synthesis of L-Homoserine Lactone 4

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Reagents and conditions: (a) chloroacetyl chloride, NaHCO3, CH2Cl2 H2O (2:1), r.t., 6h; (b) NaHCO3, 3-chloropropionyl chloride, CH2Cl2:H2O (2:1), r.t., 7h; (c) K2CO3,
acetone, 50°C, 5h.
Chart 2. Synthesis of Compounds of Series I and II
Reagents and conditions: (a) 2-chloroacetamide, oxalamide, CH2Cl2, 0°C for 1h then 90°C, reflux, 5h; (b) CS2, acetone, Na3PO4·12H2O, r.t., 6h; (c) acetonitrile,
NaHCO3, 75°C, reflux, 8h.
Chart 3. Synthesis of Compounds of Series III and IV
Table 1. The Structures and Yields of Compounds of Series I and II
Table 2. The Structures and Yields of Compounds of Series III and IV
Compd.
R
Yield
Compd.
R
Yield
11a
11b
11c
11d
11e
11f
3-Cl
44%
51%
61%
40%
45%
47%
11g
11h
11i
4-i-Pr
2-CH3
3-CH3
3-Cl
49%
42%
55%
45%
37%
56%
2-OCH3
4-CH3
4-OCH3
2-OC2H5
2,3-Ar
12a
12b
12c
2-OCH3
4-CH3
and the regulation of QS proteins (rhlR, lasR, pqsR).3) The
results showed that the analogs inhibited the expression of
lasI, lasR, pqsA, pqsR, rhlI and rhlR. For example, the expres-
sion of lasI was downregulated by 50.5, 32.9, 47.9, 38.9, 55.1,
inhibited the bacterial QS system by selectively attenuating its 59.2 and 26.9% by analogs 7b, 8b, 8c, 11b, 11f, 11i and C30,
production of virulence factors, especially those that promoted respectively (Fig. 4A). 8b was almost equipotent with C30 for
PAO1 biofilm formation.
downregulation of the expressions of lasA, and 11f (71.0%)
To further verify the mechanism of action of analogs 7b, and 11i (64.1%) were superior to C30 (58.5%) (Fig. 4B). 7b, 8c
8b, 8c, 11b, 11f and 11i, the mRNA expression of QS-related and 11f were most effective at inhibiting expression of pqsA,
genes rhlI, lasI, pqsA, rhlR, lasR and pqsR was analyzed by with 11f downregulating pqsA expression by 67.7%. (Fig. 4C).
real-time fluorescence quantitative PCR. These genes are re- Analogs 8c, 11b, 11f, 11i and C30 downregulated the expres-
lated to the synthesis of QS signal molecules (rhlI, lasI, pqsA) sions of pqsR by 53.5, 47.9, 72.5, 57.5 and 45.0%, respectively

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Fig. 2. Inhibitory Effects of All Compounds of Four Series against the P. aeruginosa PAO1 las QS System
The extent of the red turbidity circle represents the degree of inhibition of QS activity by the compound i.e., the larger the circle of turbidity, the greater the inhibitory
activity.
(Fig. 4D), but surprisingly only 11f achieved equipotency with docking results correlated well with the biological evaluation
C30 for inhibition of rhlI expression (Fig. 4E). Furthermore, results.
11f and 11i were only slightly more inhibitory of rhlR expres-
sion (58.8 and 55.5%, respectively) than C30 (48.4%) (Fig. 4F).
In summary, analog 11f was the most potent inhibitor of
Conclusion
In conclusion, we have designed and synthesized four series
the expression of most QS-regulated genes in P. aeruginosa of novel L-homoserine lactone analogs and evaluated their
PAOI. However, 11f showed more powerful inhibitory ac- inhibitory activity against QS in P. aeruginosa. The results
tivity of lasR, pqsR and rhlR expression than it did of lasI, indicated that 7b and 7c of series I and 8b and 8c of series
pqsA and rhlI expression. These results confirmed that active II, representing L-homoserine lactone analogs with benzothia-
compounds inhibited virulence expression by controlling the zolyl and 4-chlorophenyl substituents, respectively, showed
expression of related genes (Fig. 4).
greater inhibitory activity against CV026 and PAO1 than 7a,
7d and 8a, with 4,5-dihydro-thiazol or 1,3,4-thiadiazole sub-
stituents. In addition, most compounds of series III with a
Analysis of Molecular Docking Results
To verify the results of this bioassay evaluation, we used sidechain comprising a phenylurea and dithiocarbamate group
AutoDock molecular simulation software (Molecular model- exhibited remarkable inhibition of QS in CV026 and PAO1.
ing was performed using the autodock 4 software from the Compounds of series IV, with a sidechain comprising only a
Scripps Research Institute) to simulate the binding of the ac- phenylurea group, had weak QSI activity. These results indi-
tive compounds to the receptor protein LasR.38) OdDHL and cated that the dithiocarbamate group was critical to the QSI
analogs 7b and 8b interacted with Thr-75, Tyr-93 and Ser-129 activity of series III.
groups which are located inside the receptor pocket of LasR,
The results of mechanism of action studies verified that
via three hydrogen bonds (i.e., 7b and 8b each formed one hy- analogs 7b, 8b, 8c, 11b, 11f and 11i inhibited the P. aerugi-
drogen bond with each of the three residues) (Figs. 5A–C). 8c nosa QS system by selectively attenuating the expression of
interacted with the amino acid residues Tyr-93, Thr-75, Asp-73 virulence factors, especially biofilm formation by PAO1. The
and Ser-129 to form four hydrogen bonds (Fig. 5D); 11b activity of compound 11f was superior to that of all other ana-
formed two hydrogen bonds by interacting with Asp-73 and logs, and the results of molecular docking indicated that 11f
Ser-129 (Fig. 5E); 11f linked with Ser-129 and Arg-61 to form competed with OdDHL to bind with LasR. This 11f–LasR
three hydrogen bonds (Fig. 5F); 11i interacted with Tyr-56, interaction inhibited the expression of the las system genes,
Tyr-47 and Ser-129 via three hydrogen bonds (Fig. 5G).
PQS system genes and rhl system-related genes, thereby in-
The fact that 11f was the most potent compound and also hibiting the production of virulence factors and the formation
the only analog that interacted with Arg-61 suggested that of PAO1 biofilm.
Arg-61 may be more important for QS than other amino
Our research has enriched the chemical space around L-
acid residues in the active site of LasR. Thus, the molecular homoserine lactones, and afforded a new lead compound (11f)

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Fig. 3. (A) Growth Curve Analysis of Paeruginosa PAO1 Treated with Active Compounds at Sub-MICs (15µM)
(B) Effects of active compounds on pyocyanin production. (C) Effects of active compounds on elastase activity. (D) Effects of active compounds on rhamnolipid produc-
tion. (E) Effects of active compounds on biofilm formation. (F) Effects of active compounds on swarming motility. C30 is used as a positive control, and DMSO is used
as a negative control.
for the development of QS inhibitors as antibacterial agents 2.42–2.27 (m, 1H, -O-CH2-CH2-CH).
with less likelihood of triggering the development of resis-
General Procedure for the Synthesis of Compounds
tance. We expect that further investigation and expansion of 5 and 6 Compounds 5 and 6 were efficiently synthesized
the SAR of 11f will afford more efficient and highly selective from L-homoserine lactone hydrochloride 4 following our
QSIs for antibacterial drug development.
previously reported methods.39) The acyl group of 5 and 6 was
introduced by condensation with chloroacetyl chloride and
3-chloropropionyl chloride, respectively.
Experimental
General Remarks All chemical reagents and solvents
(S)-2-Chloro-N-(2-carbonyl Tetrahydrofuran-3-yl)acetamide 5
White solid: (TLC acetone:petroleum ether 2:1), (yield
were purchased from commercial sources and used with-
1
out further purification. H-NMR (400MHz) and 13C-NMR 68.2%), mp 121–122°C; 1H-NMR (400MHz, DMSO-d6)
spectra (100MHz) were recorded on a Bruker (DPX-400) δ: 8.76 (d, J=8.0Hz, 1H, -CO-NH), 4.63 (dd, J=20.0Hz,
spectrometer. High resolution (HR)-MS were recorded on a J=8.0Hz, 1H, -O-CH2), 4.35 (t, J=8.0Hz, 1H, -CH-NH),
Waters Micromass Q-Tof Micromass spectrometer by electro- 4.21 (dd, J=20.0Hz, J=8.0Hz, 1H, -O-CH2), 4.14 (s, 2H,
spray ionization (ESI). Melting points were determined on X-5 -CH2-Cl), 2.44–2.36 (m, 1H, -O-CH2-CH2-CH), 2.24–2.15 (m,
Microdigital melting point (mp) apparatus and are reported 1H, -O-CH2-CH2-CH-).
uncorrected.
(S)-2-Chloro-N-(2-carbonyl Tetrahydrofuran-3-yl)propylamide
Synthesis of L-Homoserine Lactone Hydrochloride (4)
L-Homoserine lactone hydrochloride (4) was efficiently syn-
6
White solid (TLC: acetone:petroleum ether 2:1), (yield
thesized from methionine following our previously reported 64.5%), mp127–129°C; 1H-NMR (400MHz, DMSO-d6)
methods.28)
δ: 8.50 (d, J=7.8Hz, 1H, -CO-NH), 4.52 (dt, J=10.8Hz,
L-Homoserine lactone hydrochloride 4, white solid, (3 8.9Hz, 1H, -O-CH2), 4.28 (td, J=8.8, 1.8Hz, 1H, -CH-NH),
steps, yield 59%), mp 218–220°C, [α]2D0−27.5°, (c 0.2, water)30); 4.15 (ddd, J=10.4, 8.8, 6.5Hz, 1H, -O-CH2), 3.73–3.69 (m,
1H-NMR (400MHz, dimethyl sulfoxide (DMSO)-d6) δ: 9.04 (s, 2H, -CH2-Cl), 2.56 (td, J=6.2, 1.7Hz, 2H, -CH2-CH2-Cl),
3H, NH2·HCl), 4.45 (t, J=8.8Hz, 1H, -O-CH2), 4.37–4.21(m, 2.39–2.31 (m, 1H, -O-CH2-CH2-CH), 2.12–2.01 (m, 1H, -O-
2H, -O-CH2, -CH-CO), 2.62–2.51 (m, 1H, -O-CH2-CH2-CH), CH2-CH2-CH).

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Fig. 4. Inhibition of QS-regulated Gene ((A)–(F)) Expression in P. aeruginosa PAO1 by Active Compounds
(A) lasI. (B) lasR. (C) pqsA. (D) pqsR. (E) rhlI. (F) rhlR.
General Procedure for the Synthesis of Compounds
7a–d A solution of HS-R2 (0.92mmol) and K2CO3 (120mg) yl)acetamide 7b
in acetone was added to compound 5 (0.84mmol). After 5h White solid (TLC: petroleum ether:EtOAc 1:3), (yield
(S)-2-(Benzo[d]thiazol-2-ylthio)-N-(2-oxotetrahydrofuran-3-
1
stirring at 50°C, the solvent was removed under vacuum, and 44%), mp 133–135°C; H-NMR (400MHz, DMSO-d6) δ: 8.88
the resulting residue was extracted with ethyl acetate and (d, J=7.8Hz, 1H, -CO-NH-), 8.02 (d, J=8.0Hz, 1H, Ar-H-),
water. The organic layer was dried over MgSO4 and evapo- 7.84 (d, J=8.1Hz, 1H, Ar-H-), 7.49–7.45 (m, 1H, Ar-H-), 7.37
rated under vacuum, and the resulting residue was purified by (m, 1H, Ar-H-), 4.63 (dt, J=10.7, 8.8Hz, 1H, -O-CH2-), 4.35
column chromatography, affording white solid 7a–d.
(S)-2-((4,5-Dihydrothiazol-2-yl)thio)-N-(2-oxotetrahydrofuran-3- -CH2-S-), 2.46–2.39 (m, 1H, -O-CH2-CH2-CH-), 2.17 (m, 1H,
yl) Cetamide 7a
-O-CH2-CH2-CH-). 13C-NMR (100MHz, DMSO-d6) δ: 174.9,
(td, J=8.8, 1.5Hz, 1H, -CH-NH-), 4.25–4.17 (m, 3H, -O-CH2-,
White solid (TLC: petroleum ether:EtOAc 1:6), (yield 166.6, 165.9, 152.5, 134.8, 126.4, 124.5, 121.8, 121.1, 65.3, 48.4,
1
58%), mp114–116°C; H-NMR (400MHz, DMSO-d6) δ: 8.65 36.2, 28.1. HRESI-MS m/z calcd for [M+Na]+ C13H12N2O3S2:
(d, J=7.8Hz, 1H, -CO-NH-), 4.60–4.53 (m, 1H, -O-CH2-), 331.0182. Found: 331.0198.
4.34 (td, J=8.8, 1.7Hz, 1H, -CH-NH-), 4.20 (ddd, J=10.4,
8.8, 6.5Hz, 1H, -O-CH2-), 4.12 (t, J=8.0Hz, 2H, -CO-CH2- acetamide 7c
S-), 3.94–3.86 (m, 2H, -CH2-S-), 3.46 (t, J=8.0Hz, 2H, White solid (TLC: petroleum ether:EtOAc 1:3), (yield
(S)-2-((4-Chlorophenyl)thio)-N-(2-oxotetrahydrofuran-3-yl)-
1
-CH2-N-), 2.44–2.36 (m, 1H, -O-CH2-CH2-CH-), 2.18–2.08 53%), mp 139–141°C; H-NMR (400MHz, DMSO-d6) δ: 8.69
(m, 1H, -O-CH2-CH2-CH-). 13C-NMR (100MHz, DMSO-d6) (d, J=7.8Hz, 1H, -CO-NH-), 7.40–7.36 (m, 4H, Ar-H-), 4.57
δ: 174.9, 166.8, 162.6, 65.3, 63.8, 48.29, 35.55, 35.42, 28.11. (dt, J=10.9, 8.9Hz, 1H, -O-CH2-), 4.34 (td, J=8.8, 1.5Hz,
HRESI-MS m/z calcd for [M+H]+ C9H12N2O3S2: 261.0362. 1H, -CH-NH-), 4.20 (ddd, J=10.4, 8.8, 6.5Hz, 1H, -O-CH2-),
Found: 261.0368.
3.71 (s, 2H, -CH2-S-), 2.40–2.33 (m, 1H, -O-CH2-CH2-CH-),

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Fig. 5. The Results of the Docking Pose of OdDHL (A)38) and Compounds 7b (B), 8b (C), 8c (D), 11b (E), 11f (F) and 11i (G) with the Receptor
Protein LasR
Hydrogen bonds are shown as green balls.
2.11 (m, 1H, -O-CH2-CH2-CH-). 13C-NMR (100MHz, DMSO- 2.53–2.50 (m, 2H, -CH2-CH2-S-), 2.43–2.36 (m, 1H, -O-CH2-
d6) δ: 174.9, 167.9, 134.9, 130.7, 129.9, 128.8, 65.3, 48.2, 36.2, CH2-CH-), 2.23–2.12 (m, 1H, -O-CH2-CH2-CH-). 13C-NMR
28.0. HRESI-MS m/z calcd for [M+Na]+ C12H12ClNO3S: (100MHz, DMSO-d6) δ: 195.0, 175.2, 170.0, 65.3, 57.3, 48.0,
308.0124. Found: 308.0117.
(S)-2-((1,3,4-Thiadiazol-2-yl)thio)-N-(2-oxotetrahydrofuran-3- C10H14N2O3S2: 297.0338. Found: 297.0331.
45.1, 32.2, 28.0, 27.1. HRESI-MS m/z calcd for [M+Na]+
yl)acetamide 7d
(S)-3-(Benzo[d]thiazol-2-ylthio)-N-(2-oxotetrahydrofuran-3-
White solid (TLC: acetone:EtOAc 1:6), (yield 52%), yl)propanamide 8b
mp 96–97°C; 1H-NMR (400MHz, DMSO-d6) δ: 9.52 (s,
White solid (TLC: petroleum ether:EtOAc 1:4), (yield
1H, -CH-N-), 8.84 (d, J=7.8Hz, 1H, -CO-NH-), 4.64–4.57 50%), mp137–138°C; 1H-NMR (400MHz, DMSO-d6) δ:
(m, 1H, -O-CH2-), 4.35 (td, J=8.8, 1.7Hz, 1H, -CH-NH-), 8.53 (d, J=7.9Hz, 1H, -CO-NH-), 8.01 (d, J=8.0Hz, 1H,
4.24–4.18 (m, 1H, -O-CH2-), 4.15 (d, J=2.5Hz, 2H, -CH2-S- Ar-H-), 7.88 (d, J=8.0Hz, 1H, Ar-H-), 7.49–7.45 (m, 1H,
), 2.45–2.38 (m, 1H, -O-CH2-CH2-CH-), 2.20–2.09 (m, 1H, Ar-H-), 7.39–7.35 (m, 1H, Ar-H-), 4.59 (dt, J=10.7, 8.8Hz,
-O-CH2-CH2-CH-). 13C-NMR (100MHz, DMSO-d6) δ: 174.8, 1H, -O-CH2-), 4.35 (td, J=8.8, 1.6Hz, 1H, -CH-NH-), 4.22
166.5, 164.9, 154.2, 65.3, 48.4, 37.0, 28.1. HRESI-MS m/z (ddd, J=10.3, 8.8, 6.5Hz, 1H, -O-CH2-), 3.54 (t, J=6.9Hz,
calcd for [M+Na]+ C8H9N3O3S2: 281.9978. Found: 281.9977.
2H, -CH2-S-), 2.78–2.69 (m, 2H, -CH2-CH2-S-), 2.46–2.38
General Procedure for the Synthesis of Compounds 8a–c (m, 1H, -O-CH2-CH2-CH-), 2.14 (m, 1H, -O-CH2-CH2-CH-).
A solution of the mercapto compound (0.92mmol) and K2CO3 13C-NMR (100MHz, DMSO-d6) δ: 175.7, 170.5, 167.0, 153.2,
(120mg) in acetone was added to compound 6 (0.78mmol). 135.0, 126.8, 124.9, 122.3, 121.6, 65.8, 48.5, 35.0, 28.9, 28.8.
After stirring at 50°C for 5h, the solvent was removed under HRESI-MS m/z calcd for [M+Na]+ C14H14N2O3S2: 345.0338.
vacuum and the resulting was extracted with ethyl acetate and Found: 345.0346.
water. The organic layer was dried over MgSO4 and evapo-
rated under vacuum to give a residue, which was then purified propanamide 8c
by column chromatography, affording a white solid (8a–c). White solid (TLC: petroleum ether:EtOAc 1:3), (yield
(S)-3-((4,5-Dihydrothiazol-2-yl)thio)-N-(2-oxotetrahydrofuran-3- 58%), mp 88–89°C; H-NMR (400MHz, DMSO-d6) δ: 8.47 (d,
yl)propanamid 8a J=7.9Hz, 1H, -CO-NH-), 7.41–7.35 (m, 4H, Ar-H-), 4.57 (dt,
(S)-3-((4-Chlorophenyl)thio)-N-(2-oxotetrahydrofuran-3-yl)-
1
White solid (TLC: petroleum ether:EtOAc 1:10), (yield J=10.8, 8.8Hz, 1H, -O-CH2-), 4.35 (td, J=8.8, 1.7Hz, 1H,
1
55%), mp 128–130°C; H-NMR (400MHz, DMSO-d6) δ: 8.59 -CH-NH-), 4.21 (ddd, J=10.4, 8.7, 6.5Hz, 1H, -O-CH2-), 3.16
(d, J=7.9Hz, 1H, -CO-NH-), 4.55 (dt, J=10.8, 8.8Hz, 1H, (t, J=7.2Hz, 2H, -CH2-S-), 2.46 (t, J=7.3Hz, 2H, -CH2-CH2-
-O-CH2-), 4.35 (td, J=8.7, 1.5Hz, 1H, -CH-NH-), 4.24–4.19 S-), 2.42–2.36 (m, 1H, -O-CH2-CH2-CH-), 2.17–2.06 (m, 1H,
(m, 1H, -O-CH2-), 4.14 (t, J=7.9Hz, 2H, -CH2-N-), 3.88 (t, -O-CH2-CH2-CH-). 13C-NMR (100MHz, DMSO-d6) δ: 175.2,
J=6.9Hz, 2H, -CH2-CH2-N-), 3.33–3.29 (m, 2H, -CH2-S-), 170.1, 135.0, 130.4, 129.9, 129.0, 65.3, 47.9, 34.5, 28.3, 28.0.

Vol. 67, No. 10 (2019)
Chem. Pharm. Bull.
1095
HRESI-MS m/z calcd for [M+Na]+ C13H14ClNO3S: 322.0275. 3H, -O-CH2-, -CH2-S-), 3.74 (s, 3H, Ar-O-CH3), 2.59 (m, 1H,
Found: 322.0285.
-O-CH2-CH2-CH-), 2.25–2.20 (m, 1H, -O-CH2-CH2-CH-).
General Procedure for the Synthesis of Compounds 13C-NMR (100MHz, DMSO-d6) δ: 197.3, 173.5, 169.9, 155.6,
11a–i A solution of compound 4 (150mg, 1.09mmol) and 150.5, 130.4, 121.4, 114.1, 65.5, 55.2, 54.6, 39.0, 27.9. HRESI-
Na3PO4·12H2O (620mg, 1.63mmol) in acetone was added MS m/z calcd for [M+Na]+ C15H17N3O5S2: 406.0507. Found:
to CS2 (160 µL) and stirred for 2h at room temperature. The 406.0438.
resulting solution was added to compounds 10a–i (1.20mmol) 2-(3-(2-Ethoxyphenyl)ureido)-2-oxoethyl(S)-(2-oxotetrahy-
to react for 6h at room temperature. The solvent was removed drofuran-3-yl)carbamodithi-oate 11e
under vacuum, extracted with ethyl acetate and water. The
White solid (TLC: petroleum ether:EtOAc 1:1), (yield
organic layer was dried over MgSO4 and evaporated under 45%), mp 157–159°C; 1H-NMR (400MHz, DMSO-d6) δ:
vacuum to give the residue, which was then purified by col- 10.95 (s, 1H, -CO-NH-CO-), 10.73 (s, 1H, -CS-NH-), 10.49
umn chromatography, affording a white solid (11a–i).
2-(3-(3-Chlorophenyl)ureido)-2-oxoethyl(S)-(2-oxotetrahy- Ar-H), 7.05–7.00 (m, 2H, Ar-H), 6.94–6.90 (m, 1H, Ar-H),
drofuran-3-yl)carbamodithi-oate 11a
(d, J=7.9Hz, 1H, Ar-NH-CO-), 8.16 (d, J=8.1Hz, 1H,
5.50 (dt, J=10.6, 8.6Hz, 1H, -O-CH2-), 4.40 (t, J=8.4Hz,
White solid (TLC: petroleum ether:EtOAc 1:1), (yield 1H, -CH-NH-), 4.33–4.27 (m, 3H, -O-CH2-, -CH2-S-), 4.10
1
44%), mp 140–142°C; H-NMR (400MHz, DMSO-d6) δ: 11.04 (q, J=7.0Hz, 2H, -O-CH2-CH3), 2.58 (m, 1H, -O-CH2-
(s, 1H, -CO-NH-CO-), 10.51 (d, J=7.9Hz, 1H, -CS-NH-), CH2-CH-), 2.25–2.20 (m, 1H, -O-CH2-CH2-CH-), 1.37 (t,
10.40 (s, 1H, Ar-NH-CO-), 7.79 (t, J=1.9Hz, 1H, Ar-H), 7.41 J=6.9Hz, 3H, -O-CH2-CH3). 13C-NMR (100MHz, DMSO-d6)
(d, J=8.9Hz, 1H, Ar-H), 7.34 (t, J=8.0Hz, 1H, Ar-H), 7.15 δ: 197.3, 173.5, 169.8, 150.2, 147.2, 127.4, 123.5, 120.6, 119.0,
(dd, J=7.8, 0.8Hz, 1H, Ar-H), 5.50 (dt, J=10.9, 8.6Hz, 1H, 112.0, 65.5, 64.2, 54.6, 39.0, 27.9, 14.6. HRESI-MS m/z calcd
-O-CH2-), 4.40 (t, J=8.4Hz, 1H, -CH-NH-), 4.33–4.27 (m, for [M+Na]+ C16H19N3O5S2: 420.0658. Found: 420.0664.
3H, -O-CH2-, -CH2-S-), 2.59–2.55 (m, 1H, -O-CH2-CH2-CH-),
2-(3-(Naphthalen-1-yl)ureido)-2-oxoethyl(S)-(2-oxotetrahy-
2.22 (m, 1H, -O-CH2-CH2-CH-). 13C-NMR (100MHz, DMSO- drofuran-3-yl)carbamodith-ioate 11f
d6) δ: 197.2, 173.5, 170.0, 150.4, 136.5, 128.8, 127.4, 121.4,
White solid (TLC: petroleum ether:EtOAc 1:1), (yield
65.5, 54.7, 30.7, 27.9. HRESI-MS m/z calcd for [M+Na]+ 47%), mp 151–153°C; 1H-NMR (400MHz, DMSO-d6) δ:
C14H14ClN3O4S2: 410.0006. Found: 409.9986.
2-(3-(2-Methoxyphenyl)ureido)-2-oxoethyl(S)-(2-oxotetrahy- (d, J=7.8Hz, 1H, -CS-NH-), 8.12 (d, J=7.5Hz, 1H, Ar-H),
drofuran-3-yl)carbamodithioate 11b 7.99 (d, J=8.1Hz, 1H, Ar-H), 7.93 (d, J=8.5Hz, 1H, Ar-H),
11.22 (s, 1H, -CO-NH-CO-), 10.99 (s, 1H Ar-NH-CO-), 10.55
White solid (TLC: petroleum ether:EtOAc 1:1), (yield 7.75 (d, J=8.1Hz, 1H, Ar-H), 7.66 (t, J=7.3Hz, 1H, Ar-H),
51%), mp 130–131°C; 1H-NMR (400MHz, DMSO-d6) δ: 7.61–7.57 (m, 1H, Ar-H), 7.53 (t, J=7.9Hz, 1H, Ar-H), 5.53
10.99 (s, 1H, -CO-NH-CO-), 10.70 (s, 1H, -CS-NH-), 10.51 (d, (m 1H, -O-CH2-), 4.44–4.39 (m, 3H, -CH-NH-, -CH2-S-),
J=8.0Hz, 1H, Ar-NH-CO-), 8.16 (d, J=7.8Hz, 1H, Ar-H), 4.33–4.29 (m, 1H, -O-CH2-), 2.61 (m, 1H, -O-CH2-CH2-CH-),
7.05 (d, J=3.8Hz, 2H, Ar-H), 6.95–6.91 (m, 1H, Ar-H), 5.50 2.27–2.22 (m, 1H, -O-CH2-CH2-CH-). 13C-NMR (100MHz,
(dt, J=11.0, 8.6Hz, 1H, -O-CH2-), 4.41 (t, J=8.4Hz, 1H, DMSO-d6) δ: 197.3, 173.5, 170.8, 150.9, 133.6, 132.4, 128.7,
-CH-NH-), 4.34–4.27 (m, 3H, -O-CH2-, -CH2-S-), 3.86 (s, 3H, 126.7, 126.2, 125.8, 125.4, 124.4, 120.2, 117.9, 65.5, 54.7,
Ar-O-CH3), 2.61–2.57 (m, 1H, -O-CH2-CH2-CH-), 2.25–2.20 30.7, 27.9. HRESI-MS m/z calcd for [M+Na]+ C18H17N3O4S2:
(m, 1H, -O-CH2-CH2-CH-). 13C-NMR (100MHz, DMSO-d6) 426.0558. Found: 426.0462.
δ: 197.3, 173.5, 169.8, 150.2, 148.0, 126.9, 123.5, 120.6, 119.0,
110.8, 65.5, 55.9, 54.7, 39.0, 27.9. HRESI-MS m/z calcd for drofuran-3-yl)carbamodi-thioate 11g
[M+Na]+ C15H17N3O5S2: 406.0502. Found: 406.0504.
White solid (TLC: petroleum ether:EtOAc=1:1), (yield
2-Oxo-2-(3-(p-tolyl)ureido)ethyl(S)-(2-oxotetrahydrofuran-3- 49%), mp 142–144°C; H-NMR (400MHz, DMSO-d6) δ: 10.90
yl)carbamodithioate 11c (s, 1H, -CO-NH-CO-), 10.50 (d, J=7.8Hz, 1H, -CS-NH-),
2-(3-(4-Isopropylphenyl)ureido)-2-oxoethyl(S)-(2-oxotetrahy-
1
White solid (TLC: petroleum ether:EtOAc 1:1), (yield 10.23 (s, 1H, Ar-NH-CO-), 7.43 (d, J=8.5Hz, 2H, Ar-H), 7.20
1
61%), mp 143–145°C; H-NMR (400MHz, DMSO-d6) δ: 10.91 (d, J=8.5Hz, 2H, Ar-H), 5.52–5.47 (m, 1H, -O-CH2-), 4.41
(s, 1H, -CO-NH-CO-), 10.50 (d, J=7.9Hz, 1H, -CS-NH-), (t, J=8.5Hz, 1H, -CH-NH-), 4.34–4.28 (m, 3H, -O-CH2-,
10.22 (s, 1H, Ar-NH-CO-), 7.40 (d, J=8.3Hz, 2H, Ar-H), -CH2-S-), 2.85 (dd, J=13.8, 6.9Hz, 1H, Ar-CH-), 2.61–2.56
7.13 (d, J=8.3Hz, 2H, Ar-H), 5.50 (m, 1H, -O-CH2-), 4.40 (m, 1H, -O-CH2-CH2-CH-), 2.25–2.20 (m, 1H, -O-CH2-
(t, J=8.5Hz, 1H, -CH-NH-), 4.33–4.27 (m, 3H, -O-CH2-, CH2-CH-), 1.19 (d, J=6.9Hz, 6H, Ar-CH-(CH3)2). 13C-NMR
-CH2-S-), 2.58 (m, 1H, -O-CH2-CH2-CH-), 2.26–2.19 (m, 4H, (100MHz, DMSO-d6) δ: 197.3, 173.5, 169.9, 150.4, 143.9,
-O-CH2-CH2-CH-, Ar-CH3). 13C-NMR (100MHz, DMSO-d6) 135.2, 126.7, 119.8, 65.5, 54.6, 39.0, 32.8, 27.9, 23.9. HRESI-
δ: 197.3, 173.5, 170.0, 150.4, 134.9, 132.7, 129.3, 119.6, 65.5, MS m/z calcd for [M+H+CH3CN]+ C17H21N3O4S2: 437.1312.
54.6, 39.0, 27.9, 20.4. HRESI-MS m/z calcd for [M+Na]+ Found: 437.1876.
C15H17N3O4S2: 390.0553. Found: 390.0557.
2-(3-(4-Methoxyphenyl)ureido)-2-oxoethyl(S)-(2-oxotetrahy- yl)carbamodithioate 11h
drofuran-3-yl)carbamodit-hioate 11d White solid (TLC: petroleum ether:EtOAc 1:1), (yield
2-Oxo-2-(3-(o-tolyl)ureido)ethyl(S)-(2-oxotetrahydrofuran-3-
1
White solid (TLC: petroleum ether:EtOAc 1:1), (yield 42%), mp 96–97°C; H-NMR (400MHz, DMSO-d6) δ: 11.04
1
40%), mp 100–101°C; H-NMR (400MHz, DMSO-d6) δ: 10.89 (s, 1H, -CO-NH-CO-), 10.51 (d, J=7.9Hz, 1H, -CS-NH-),
(s, 1H, -CO-NH-CO-), 10.51 (d, J=7.3Hz, 1H, -CS-NH-), 10.28 (s, 1H, Ar-NH-CO-), 7.94 (d, J=8.0Hz, 1H, Ar-H), 7.21
10.15 (s, 1H, Ar-NH-CO-), 7.44 (d, J=9.0Hz, 2H, Ar-H), (m, 2H, Ar-H), 7.03 (t, J=7.4Hz, 1H, Ar-H), 5.50 (dt, J=10.8,
6.91 (d, J=9.0Hz, 2H, Ar-H), 5.51 (dt, J=9.8, 8.8Hz, 1H, 8.6Hz, 1H, -O-CH2-), 4.40 (t, J=8.4Hz, 1H, -CH-NH-),
-O-CH2-), 4.41 (t, J=8.4Hz, 1H, -CH-NH-), 4.34–4.29 (m, 4.33–4.27 (m, 3H, -O-CH2-, -CH2-S-), 2.62–2.55 (m, 1H,

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Vol. 67, No. 10 (2019)
-O-CH2-CH2-CH-), 2.28–2.17 (m, 4H, -O-CH2-CH2-CH-, Ar- 2H, Ar-H), 7.15 (d, J=8.3Hz, 2H, Ar-H), 4.33 (td, J=8.6,
CH3). 13C-NMR (100MHz, DMSO-d6) δ: 197.3, 173.5, 170.4, 2.1Hz, 1H, -O-CH2-), 4.19–4.13 (m, 1H, -O-CH2-), 3.68 (t,
150.5, 135.9, 130.3, 127.4, 126.4, 123.9, 120.7, 65.5, 54.7, 39.0, J=9.1Hz, 1H, -CH-NH-), 3.59–3.49 (s, 2H, -CH2-CO-), 2.94
27.9, 17.5. HRESI-MS m/z calcd for [M+Na]+ C15H17N3O4S2: (s, 1H, -CH-NH-), 2.45 (m, 1H, -O-CH2-CH2-CH-), 2.27 (s,
390.0553. Found: 390.0560.
2-Oxo-2-(3-(m-tolyl)ureido)ethyl(S)-(2-oxotetrahy- (100MHz, DMSO-d6) δ: 177.2, 174.2, 150.3, 135.0, 132.7,
drofuran-3-yl)carbamodithioate 11i 129.3, 119.6, 65.5, 55.1, 49.8, 29.5, 20.3. HRESI-MS m/z calcd
White solid (TLC: petroleum ether:EtOAc 1:1), (yield for [M+Na]+ C14H17ClN3O4: 314.1117. Found: 314.0547.
3H, Ar-CH3), 2.00 (m, 1H, -O-CH2-CH2-CH-). 13C-NMR
55%), mp 130–131°C; 1H-NMR (400MHz, DMSO-d6) δ:
Bacterial Strains and Media Bacterial strains are pro-
10.93 (s, 1H, -CO-NH-CO-), 10.51 (d, J=7.9Hz, 1H, -CS- vided by Ocean University of China.36) Unless otherwise
NH-), 10.27 (s, 1H, Ar-NH-CO-), 7.36 (s, 1H, Ar-H), 7.32 stated, strains were grown in LB medium.
(d, J=8.2Hz, 1H, Ar-H), 7.21 (t, J=7.8Hz, 1H, Ar-H), 6.92
(d, J=7.5Hz, 1H, Ar-H), 5.51 (dt, J=11.0, 8.6Hz, 1H, -O-
CH2-), 4.40 (dd, J=8.8, 7.9Hz, 1H, -CH-NH-), 4.33–4.30
QS Inhibitory Activity Assay
P. aeruginosa PAO1 las System Assay
Preparation of the QSIS-lasI plate was conducted by
(m, 3H, -O-CH2-, -CH2-S-), 2.59 (m, 1H, -O-CH2-CH2-CH-), the following method. 20mL of Luria–Bertani agar (LB
2.29–2.23 (m, 4H, -O-CH2-CH2-CH-, Ar-CH3). 13C-NMR agar) (20g/L) was melted and cooled to 50°C. Gentamicin
(100MHz, DMSO-d6) δ: 197.3, 173.5, 170.0, 150.4, 138.3, (80 µg/mL) sucrose (1mL), OdDHL (30nM) and TTC (2, 3,
137.4, 128.8, 124.4, 120.1, 116.7, 65.5, 54.6, 39.0, 27.9, 21.0. 5-triphenyltetrazolium chloride) (250µL) were added, the re-
HRESI-MS m/z calcd for [M+Na]+ C15H17N3O4S2: 390.0558. sulting QSIS-lasI solution mixed, and then cultured overnight
Found: 390.0489.
in LB medium. Agar plates were subsequently prepared by
General Procedure for the Synthesis of Compounds pouring 15mL of the mixture into Petri dishes. The medium
12a–c In a 25mL round bottom flask, the solution of com- was allowed to solidify, and then wells were made in the solid
pound 4 (150mg, 1.09mmol) in acetonitrile was added to gel. All samples including synthesized compounds (5µL,
corresponding compounds 13a–c (1.20mmol) and NaHCO3 1.27 µg/µL), the positive control C30 (5µL, 5mM) and the
(230mg, 2.74mmol). After stirring for 8h at 75°C, the solvent negative control DMSO (5µL) were added to wells, and the
was removed under vacuum, extracted with ethyl acetate and plate was then incubated overnight at 37°C.
water. The organic layer was dried over MgSO4 and evapo-
rated under vacuum to give a residue, which was then subject
to column chromatography, affording a white solid (12a–c).
Chromobacterium Violaceum CV026 Inhibition Assay
CV026 was cultured overnight. The cultures (100µL), ka-
namycin (15µL, 20mg/mL) and C6-HSL (15µL, 5mmol/L)
(S)-N-((3-Chlorophenyl)carbamoyl)-2-((2-oxotetrahy- were added to LB medium (15mL, 40°C), mixed and poured
drofuran-3-yl)amino)acetamide 12a
into Petri dishes. The medium was allowed to solidify, and
White solid (TLC: petroleum ether:EtOAc 1:2), (yield then the wells were made. All samples including synthesized
1
45%), mp 133–134°C; H-NMR (400MHz, DMSO-d6) δ: 10.48 compounds (5µL, 1.27µg/µL), the positive control group (C30;
(s, 2H, -CO-NH-CO-, Ar-NH-CO-), 7.78 (d, J=1.9Hz, 1H, 5µL, 5mM) and the negative control group (DMSO; 5µL)
Ar-H), 7.41–7.34 (m, 2H, Ar-H), 7.16 (d, J=7.5Hz, 1H, Ar-H), were added to wells, and the plate was then incubated at 30°C
4.33 (td, J=8.6, 2.2Hz, 1H, -O-CH2-), 4.20–4.13 (m, 1H, -O- for 24h. The experimental results are shown in Figure S1.
CH2-), 3.68 (t, J=9.2Hz, 1H, -CH-NH-), 3.55 (s, 2H, -CH2-
CO-), 2.94 (s, 1H, -CH-NH-), 2.45 (m, 1H, -O-CH2-CH2-CH-),
2.03–1.98 (m, 1H, -O-CH2-CH2-CH-). 13C-NMR (100MHz,
Virulence Factor Assays
Growth Curve Analysis
P. aeruginosa PAO1 was cultured overnight, diluted to
DMSO-d6) δ: 177.2, 174.2, 150.4, 139.1, 133.2, 130.6, 123.4, OD600=0.05, and then transferred into 96-well plates with
119.2, 118.2, 65.5, 55.1, 49.8, 29.5. HRESI-MS m/z calcd for active compounds (to give a final concentration of 15µM).
[M+Na]+ C13H14ClN3O4: 334.0571. Found: 334.0530.
Bacterial cultures were incubated for 24h at 37°C, and optical
(S)-N-((2-Methoxyphenyl)carbamoyl)-2-((2-oxotetrahy- density was measured at 600nm every 2h.
drofuran-3-yl)amino)acetamide 12b
Detection of Pyocyanin Production
White solid (TLC: petroleum ether:EtOAc 1:2), (yield
P. aeruginosa PAO1 was cultured overnight, diluted with
37%), mp 121–123°C; 1H-NMR (400MHz, DMSO-d6) δ: LB medium, and then transferred into 96-well plates with ac-
10.80 (s, 1H, -CO-NH-CO-), 10.42 (s, 1H, Ar-NH-CO-), 8.16
(d, J=8.2Hz, 1H, Ar-H), 7.07 (m, 2H, Ar-H), 6.96–6.93 (m,
1H, Ar-H), 4.32 (td, J=8.6, 2.3Hz, 1H, -O-CH2-), 4.20–4.15
(m, 1H, -O-CH2-), 3.88 (s, 3H, Ar-O-CH3), 3.68 (t, J=9.2Hz,
1H, -CH-NH-), 3.54 (s, 2H, -CH2-CO-), 2.96 (s, 1H, -CH-
NH-), 2.44 (m, 1H, -O-CH2-CH2-CH-), 2.01 (m, 1H, -O-CH2-
CH2-CH-). 13C-NMR (100MHz, DMSO-d6) δ: 177.2, 174.0,
150.2, 148.0, 127.0, 123.5, 120.6, 119.0, 110.9, 65.5, 55.9, 55.1,
49.7, 29.5. HRESI-MS m/z calcd for [M+Na]+ C14H17N3O5:
330.1066. Found: 330.1021.
tive compounds (to give a final concentration of 15µM). The
bacterial cultures were incubated at 37°C for 12h, and then
optical density was measured at 600nm. 1mL of bacterial cul-
ture was taken from 96-well plates and centrifuged at 4°C for
10min. After centrifugation, 1mL of chloroform was added to
the liquid supernatant and mixed thoroughly. After separation,
250µL hydrochloric acid (0.2mol/L) was added to the chlo-
roform layer. The aqueous phase was separated after 3min
was measured by enzyme-labeled instrument
and the OD520
(RT6000 from Rayto, America).
(S)-N-((4-Methylphenyl)carbamoyl)-2-((2-oxothiotetrahy-
drofuran-3-yl)amino)acetamide 12c
Elastase Activity Assay
PAO1 bacterial cultures (OD600 ≈1.0) was diluted, trans-
White solid (TLC: petroleum ether:EtOAc 1:2), (yield ferred to peptone tryptic soy broth (PTSB) medium. When
56%), mp 119–121°C; 1H-NMR (400MHz, DMSO-d6) δ: OD600 ≈0.2, the active compounds into PTSB medium con-
10.32 (s, 2H, -CO-NH-CO-, Ar-NH-CO-), 7.41 (d, J=8.4Hz, taining PAO1, and then cultured them in the shock conditions.

Vol. 67, No. 10 (2019)
Chem. Pharm. Bull.
1097
Bacterial cultures were centrifuged at 4°C for 10min, and interest.
then 500µL of Congo red buffer was added into 500µL of the
liquid supernatant. After reacting for 8h at 37°C, the mixture
Supplementary Materials The online version of this ar-
was centrifuged at 4°C for 10min, and sample of the superna- ticle contains supplementary materials.
tant analyzed by OD490, to reveal the elastase activity.
Determination of Rhamnolipid Production
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