Synthesis and anti-biofilm activities of dihydro-pyrrol-2-one derivatives on Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.bmcl.2014.12.010

Biofilm formation is an important reason for bacterial resistance to antimicrobials. Many compounds with dihydro-pyrrol-2-one (DPO) have antibacterial effects. It is prospective to base on DPO skeleton to design new compounds for biofilm inhibition. DPO was designed by a novel method of tandem cyclization between ethyl glyoxalate and amines, the series of DPO derivatives were synthesized by change of the amines. Their activities were evaluated by the inhibition of biofilm in Pseudomonas aeruginosa. The interaction of DPO derivatives with mannitol dehydrogenase (MDH) or extracellular DNA (eDNA) in the biofilm was simulated by molecular docking to reveal possible mechanism. 19 new DPO derivatives were synthesized and identified, 15 of them had antibacterial activities, but only 5 of them had more than 50% inhibition on biofilm of P. aeruginosa at 50 μg/mL. The MDH activity and eDNA content in biofilm decreased significantly after treatment of the DPO derivatives in concentration dependence. The simulation reveals that strong interaction exists between the five DPO derivatives and MDH or eDNA, which are involved in anti-biofilm mechanism. The synthetic method of DPO derivatives is practical to provide effective anti-biofilm agents for P. aeruginosa, and they take effect through inhibition on MDH and eDNA of biofilm.

Full text of DOI:10.1016/j.bmcl.2014.12.010

Bioorganic & Medicinal Chemistry Letters 25 (2015) 597–601
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-biofilm activities of dihydro-pyrrol-2-one
derivatives on Pseudomonas aeruginosa
⇑
Yong Ye , Fei Fang, Yue Li
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Biofilm formation is an important reason for bacterial resistance to antimicrobials. Many compounds
with dihydro-pyrrol-2-one (DPO) have antibacterial effects. It is prospective to base on DPO skeleton
to design new compounds for biofilm inhibition. DPO was designed by a novel method of tandem cycli-
zation between ethyl glyoxalate and amines, the series of DPO derivatives were synthesized by change of
the amines. Their activities were evaluated by the inhibition of biofilm in Pseudomonas aeruginosa. The
interaction of DPO derivatives with mannitol dehydrogenase (MDH) or extracellular DNA (eDNA) in
the biofilm was simulated by molecular docking to reveal possible mechanism. 19 new DPO derivatives
were synthesized and identified, 15 of them had antibacterial activities, but only 5 of them had more than
Received 2 October 2014
Revised 17 November 2014
Accepted 5 December 2014
Available online 13 December 2014
Keywords:
DPO derivatives
Tandem reaction
Bacterial biofilm
Mannitol dehydrogenase
Extracellular DNA
50% inhibition on biofilm of P. aeruginosa at 50 lg/mL. The MDH activity and eDNA content in biofilm
decreased significantly after treatment of the DPO derivatives in concentration dependence. The simula-
tion reveals that strong interaction exists between the five DPO derivatives and MDH or eDNA, which are
involved in anti-biofilm mechanism. The synthetic method of DPO derivatives is practical to provide
effective anti-biofilm agents for P. aeruginosa, and they take effect through inhibition on MDH and eDNA
of biofilm.
Ó 2014 Elsevier Ltd. All rights reserved.
Dihydro-pyrrol-2-one (DPO), a key building block for construct-
ing a variety of bioactive ingredients or intermediates, widely
exists in pheromone, alkaloids, steroids, heme, chlorophyll and
other compounds, and it has very strong antibacterial and other
pharmacological activities.1 The inherent ring-conjugate system
and chirality of DPO contribute to its high region-selectivity and
stereo-selectivity in chemical reaction,2 which are conducive to
the functionalization of specific sites in the molecules.3 Therefore,
DPO synthesis plays an important role in rational drug design and
drug screening.
Many methods exist today for the synthesis of DPO derivatives,
including the multistep method,4 the two component method,5–7
the three component method8,9 and the multi-component
method.10,11 The first method involves a cumbersome and ineffi-
cient process of separation and purification, the second and the
third methods need the complex substrates, which are difficult to
synthesize, and the last one requires many kinds of raw materials.
We designed a new tandem reaction with two simple components
(aldehydes and amines) to synthesize DPO derivatives.
tance, leading to the deficiency of therapeutic effects.12 Biofilm for-
mation is an important reason for bacterial resistance and diseases
recurrence.13 It is meaningful to find out effective anti-biofilm
agents for therapy of P. aeruginosa induced infections. DPO has
small molecular structure, may play a role in anti-biofilm with
strong permeability. It is prospective to design and screen out
anti-biofilm agents with DPO skeleton based on new synthetic
methods.
The synthetic route of DPO derivatives was designed as shown
in Figure 1. They were synthesized by ethyl glyoxalate and amines
with anhydrous sodium sulfate as water absorbent, Pd (TFA)2 as
catalyst and toluene as solvent. Each substrate was repeated five
times to ensure the synthesis yield.
Conditions for the palladium-catalyzed cascade cyclization of
ethyl glyoxalate with p-anisidine were optimized in our previous
research,14 DPO structure was formed in tandem reaction. Differ-
ent DPO derivatives were synthesized by using different amines.
19 kinds of DPO derivatives were obtained when 20 amines
reacted with ethyl glyoxalate. The structures and yields of DPO
derivatives are listed in Table 1.
The synthesis of DPO was previously achieved by the multi-step
reaction like other five member nitrogen heterocyclic rings.10,11
but the entire synthesis process is extremely cumbersome and
complicated because the by-product must be purified in each step.
Pseudomonas aeruginosa is the major pathogenic bacterium for
pulmonary nosocomial infection, and it easily acquire drug resis-
⇑
Corresponding author. Tel.: +86 20 87110234.
E-mail address: yeyong@scut.edu.cn (Y. Ye).
http://dx.doi.org/10.1016/j.bmcl.2014.12.010
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

598
Y. Ye et al. / Bioorg. Med. Chem. Lett. 25 (2015) 597–601
coupling or cyclization.15,16 The reaction mechanism is illustrated
O
O
C
R
in Figure 2. Arylamines have good adaptability with either
electron-withdrawing groups or electron-donating groups such
as methyl, alkyl, alkoxy, halogen, ester, acetyl aniline and ketone,
and can react with ethyl glyoxalate to reach high yield. However,
arylamines with meta-substituted group have slight decrease in
yield than with para-substituted group, it may be attributed to
steric hindrance on amino nitrogen atom. p-Nitro aniline
cannot generate the desired product, it may be due to the strong
electron withdrawing effect of p-nitro inhibiting the formation of
imine.17
N
Pd(TFA)2 (5mol%)
RHN
EtO2C
H
C
O
OEt
R
NH2
CO2Et
toluene, 70
ഒ
Figure 1. The synthetic route of dihydro-pyrrol-2-one.
We designed a new synthetic strategy in tandem catalytic system
to achieve the diversity and complexity of products by adjusting
the variety and portion of the substrates. Tandem reaction makes
raw materials to form complex structure without isolation of the
intermediates. It greatly reduces expenditure of solvent, energy
and time, and is a promising method in the field of organic
synthesis.
Based on low-cost raw materials, ethyl glyoxalate and amines
are chosen to build DPO structure. The mechanism is that ethyl
glyoxalate interacts with equivalent amine to form acyl imines
by dehydration. At the same time, palladium combines with ethyl
glyoxalate to be a chelate by oxidative addition. Finally, acyl imines
and the chelate continuously react to get DPO through cross
DPO derivatives exhibit different biological activities if there is
change of substituent in
substituted compounds because its enamine structure can be
further functionalized.18 For example,
,b-unsaturated lactam
c-lactam ring, especially the 3-amino
a
structure of DPO has Michael addition reaction with stable carbon
anions, nitrogen nucleophiles and Gilman reagents, which are
owing to the presence of a double bond in the ring prone to epox-
idation and hydroxylation.19 In addition, DPO dihydropyrrolo ring
can be oxidized to be pyrrole, and reduced to be pyrrolidone, which
Table 1
Yields of DPO derivatives in ethyl glyoxalate reaction with amines
Sample no.
1
Amine
Yield (%)
Sample no.
11
Amine
Yield (%)
Br
NH2
NH2
NH2
O
67.5 3.4
73.7 2.6
F
F
NH2
2
3
61.0 2.4
36.3 2.0
12
13
71.5 3.1
36.3 3.6
NH2
NH2
NH2
NH2
NH2
4
5
6
70.5 3.6
63.2 3.3
57.5 4.6
14
15
16
40.8 2.5
57.7 4.5
69.7 3.7
NH2
EtO2C
EtO
NH2
NH2
EtO2C
O
NH2
NH2
O
7
8
38.2 2.9
34.8 2.3
17
18
26.0 3.8
N
H
NH2
NH2
O2N
0.0 0.0
NH2
NH2
Cl
9
78.2 2.8
64.2 3.5
19
20
55.3 4.9
34.8 3.9
NH2
NH2
10
Cl
Yield was expressed as mean standard deviation (n = 5).

Y. Ye et al. / Bioorg. Med. Chem. Lett. 25 (2015) 597–601
599
O
O
R
O
Compound 1
HN
(a)
Compound 4
Compound 5
Compound 10
Compound 11
Erythromycin
OEt
EtO
EtO
0.5
0.4
0.3
0.2
0.1
0.0
H
O
O
N
R
Pd
O
R
HN
O
Pd(II)
OEt
O
O
OEt
O
R
NH2
H
N
H
R
OEt
EtO
O
O
HN
OEt
R
R
HN
O
H
N
O
-Pd
OEt
OH
R
OEt
EtO
Pd(II)
O
EtO
O
O
0
10
20
30
40
50
Figure 2. Reaction mechanism of ethyl glyoxalate and amines.
Concentration (μg/mL)
has various pharmacological activities, such as anti-inflammation,
anti-tumor, anti-HIV and preventing senile dementia.20
(b)
Our research disclosed the effects of DPO derivatives on bacte-
rial growth and biofilm formation. Bacterial biofilm were measured
0.3
according to the reference.21 50% inhibitory concentrations (IC50
)
of the compounds on bacterial growth and biofilm formation were
determined by dilution until the inhibition rate reached 50%.
Of 19 DPO derivatives synthesized by our method, fifteen had a
0.2
0.1
0.0
certain inhibitory effect on P. aeruginosa at 50 lg/mL concentra-
tion, eight of them had more than 50% growth inhibition rate,
and the compound nos. 1 and 10 had no significant difference from
erythromycin on the inhibition of bacterial growth. The results are
shown in Table 2. Five compounds (1, 4, 5, 10 and 11) had more
than 50% inhibition on biofilm of P. aeruginosa, and were more
effective than erythromycin, which is one of popular drugs cur-
rently for inhibition of P. aeruginosa and was used as the control
in the experiment, but had less effect due to bacterial resistance.
Other anti-biofilm agents such as carbenicilli and roxithromyci
Compound 1
Compound 4
Compound 5
Compound 10
Compound 11
Erythromycin
0
10
20
30
40
50
Concentration (μg/mL)
have IC50 from 64 to 256
pound no. 10 has the lowest IC50 on both bacterial growth
(27.8 g/mL) and biofilm formation (28.8 g/mL) among DPO
l
g/mL on growth of P. aeruginosa.22 Com-
Figure 3. Activity of mannitol dehydrogenase (a) and content of extracellular DNA
(b) in bacterial biofilm affected by the DPO derivatives (x Æ s, n = 5). They were
measured, respectively, by a decrease in the absorbance of reactive mixture at
340 nm and biofilm lysate at 260 nm compared to untreated controls. ⁄⁄, p <0.01,
compared with erythromycin.
l
l
derivatives. It is different from other pyrrole–imidazole alkaloids
such as fluconazol, ketoconazole and clotrimazole, those only show
effects on fungi with 64 lg/mL of IC50 but no inhibitory effect on
P. aeruginosa.23 It suggests that compound no. 10 [diethyl 1-(3-chlo-
rophenyl)-4-((3-chlorophenyl)amino)-5-oxo-2,5-dihydro-1H-pyrrole-
2,3-dicarboxylate] is a good candidate of antibiofilm agents.
The expression of specific genes is involved in biofilm formation
and responsible for bacterial drug resistance.24 The main difference
between biofilm bacteria and planktonic bacteria is that biofilm
bacteria are tightly packed and wrapped in its own secreted extra-
cellular polysaccharide matrix called extracellular polymeric sub-
stances (EPS). The main components of EPS is alginate.25
Mannitol dehydrogenase (MDH) is a key enzyme in alginate syn-
thesis process of P. aeruginosa.26 In addition, large amount of extra-
cellular DNA (eDNA) is found in the biofilm,27 it not only affects the
formation of biofilms, but also increases the resistance of biofilms
by chelating cation.28 It is proved that eDNA enzymes can clear
early and immature biofilm in vitro,29 and biofilm formation can
be regulated by MDH and eDNA.30
Table 2
Inhibition of DPO derivatives on bacterial growth and biofilm formation of Pseudo-
monas aeruginosa
Compound
no.
Growth
inhibition
rate (%)
IC50
(
l
g/mL)
Biofilm
inhibition
rate (%)
IC50 (lg/mL)
1
2
3
4
5
6
8
9
10
11
13
15
16
19
20
80.8 5.9
34.2 4.0
61.4 4.8**
13.6 4.6
29.3 3.3
53.2 2.7**
53.8 3.9**
35.5 4.5
28.9 3.2
20.7 2.2
70.2 5.2**
65.4 3.1**
27.5 5.8
37.8 2.4
42.0 2.2
11.8 3.1
41.3 5.4
45.7 4.1
40.2 4.1
/
/
47.4 4.0
48.4 3.8
/
/
34.0 4.0**
55.5 6.6**
65.8 2.5**
63.1 3.6**
50.4 6.0**
32.5 5.5**
68.6 3.8**
83.0 4.8
/
48.4 5.1**
42.2 3.0**
46.6 5.6**
50.0 2.5**
/
41.6 3.2**
/
27.8 4.0
38.4 3.0
/
/
/
/
/
28.8 4.5
39.0 4.4
76.8 5.8**
20.3 6.4**
44.7 5.8**
15.4 6.6**
26.6 6.0**
40.9 6.0**
In order to discuss the mechanism of DPO derivatives inhibiting
bacterial biofilm, MDH or eDNA in biofilm were measured, respec-
tively, by a decrease in the absorbance of reactive mixture at
340 nm and biofilm lysate at 260 nm compared to untreated con-
/
/
/
/
/
/
trols according to the references.31,32
DA340 and DA260 increased
Erythromycin 67.6 5.9
33.2 5.6
significantly (p <0.01) after the treatments of the DPO derivatives
at concentration dependence as shown in Figure 3. It suggests that
they play a role in the inhibition of MDH and eDNA in the biofilms.
Data were presented as mean standard deviation (n = 5).
**
p <0.01, compared with erythromycin.

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Y. Ye et al. / Bioorg. Med. Chem. Lett. 25 (2015) 597–601
Table 3
Binding energy of DPO derivatives with mannitol dehydrogenase and eDNA
DPO derivatives
Mannitol dehydrogenase
CDOCKER energy (kcal/mol) CDOCKER interaction energy (kcal/mol)
eDNA
CDOCKER energy (kcal/mol)
CDOCKER interaction energy (kcal/mol)
1
4
5
10
11
À22.21 2.40
À23.84 1.96
À19.75 1.96
À28.18 0.94
À15.27 0.71
41.88 3.36
43.18 2.81
44.57 3.81
38.60 1.12
45.47 1.93
À40.67 0.64
À45.03 1.10
À37.43 1.37
À46.39 0.55
À37.62 1.12
20.09 1.04
18.82 1.37
21.94 1.29
18.99 1.54
20.87 0.68
Data were presented as mean standard deviation (n = 10 poses).
Figure 4. Diagram of DPO derivative binding with mannitol dehydrogenase (a) and eDNA (b).
Simulation of molecular docking provides a simple way to
robenzene in DPO derivatives have little differences, but cannot act
with MDH and eDNA. It suggests that the substituent groups, the
size of atoms and the charge all affect the three-dimensional struc-
ture of the final product, and then influence its binding force.
Mimic diagram of compound 10 binding to MDH and DNA was
shown in Figure 4, it illustrates that they can well bind with each
other by hydrogen bonds.
The docking simulation shows a good correlation between
interaction and anti-biofilm effect. It suggests that molecular dock-
ing can be used to predict the drug’s effect on biofilm inhibition.
The interaction between DPO derivatives and MDH and eDNA
can be used to speculate the biofilm inhibition.
research the interaction between DPO derivatives and MDH or
eDNA. Based on the lock-key principle and complementary struc-
tural hypothesis, molecular docking simulates mutual interaction
between ligand and receptor.33 If receptor and ligand can interact,
they must approach each other, and then combines in a particular
conformation of the binding site, finally reach stable complex by
adjusting conformation. It is helpful to drug design through adding
or removing some electron withdrawing or donating groups to
obtain the binding substance in the lowest energy according to
principle of energy complementation. Correct affinity prediction
is conducive to drug design and screening. The semi-flexible dock-
ing applied in this research is suitable for docking of macromole-
cule receptor and small molecule ligand. The main factors
affecting the binding stability of ligand and receptor are hydropho-
bic force and bonding force. Free energy value is an important
parameter for evaluation of docking affinity, binding activity and
stability of receptor and ligand, and it can be used to judge the
interaction of ligand and receptor. The simulation shows that only
five (1, 4, 5, 10 and 11) among the 19 DPO derivatives can bind to
MDH and eDNA. Each molecule successfully docks in 10 poses. The
binding energy is listed in Table 3. They all have positive CDOCKER
interactive energy and negative CDOCKER energy, suggesting that
they can spontaneously bind to MDH and eDNA, and interact with
them. Compound No.10 has the highest CDOCKER energy on both
MDH and eDNA suggesting that the strong molecular interaction
contributes to its anti-biofilm activity.
Acknowledgments
The authors gratefully thank for financial support from National
Natural Science Foundation of China (No. 81173646).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
12.010.
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