A new class of phenylhydrazinylidene derivatives as inhibitors of Staphylococcus aureus biofilm formation

Article abstract of DOI:10.1007/s00044-016-1535-9

In the struggle against the emergence of the antibiotic resistance, new molecules targeting biofilm formation could be useful as adjuvant of conventional antibiotics. This study focused on a new class of 2-phenylhydrazinylidene derivatives as antivirulence agents. The compound 12e showed interesting activities against biofilm formation of all tested Staphylococcus aureus strains with IC₅₀ ranging from 1.7 to 43 μM; compounds 12f and 13a resulted strong inhibitors of S. aureus ATCC 6538 and ATCC 29213 biofilm formation with IC₅₀ of 0.9 and 0.8 μM, respectively. A preliminary study on the mechanism of action was carried on evaluating the inhibition of sortase A transpeptidase. Compound 12e resulted not to be toxic at 1 mg/ml by using an in vivo model (the wax moth larva model, Galleria mellonella).

Full text of DOI:10.1007/s00044-016-1535-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1535-9
ORIGINAL RESEARCH
A new class of phenylhydrazinylidene derivatives as inhibitors
of Staphylococcus aureus biofilm formation
1
,2
1
1
1
•
Stella Cascioferro
Maria Grazia Cusimano
•
Benedetta Maggio
1
•
Demetrio Raffa
1
•
Maria Valeria Raimondi
1
•
Domenico Schillaci
1
•
Barbara Manachini
•
3
Ainars Leonchiks
•
Giuseppe Daidone
Received: 17 September 2015 / Accepted: 11 February 2016
Ó Springer Science+Business Media New York 2016
Abstract In the struggle against the emergence of the
antibiotic resistance, new molecules targeting biofilm for-
mation could be useful as adjuvant of conventional
antibiotics. This study focused on a new class of
Introduction
The emergence of multidrug-resistant Gram-positive and
Gram-negative bacteria continues to limit the clinical
efficacy of most of the currently marketed antibiotics
(Cascioferro et al., 2015b). The World Health Organiza-
tion, in a recent report on antimicrobial resistance in
common bacterial pathogens, highlights that a post-an-
tibiotic era may start the twenty-first century (WHO: http://
www.who.int/drugresistance/documents/surveillancereport/
en/). Furthermore, conventional antibiotics target plank-
tonic (free living) bacterial cells, but may be ineffective
against biofilm-associated infections due to the high-level
multifactorial resistance provided by biofilm mode of
growth (Yang et al., 2012). The biofilm is a multistratified
bacterial community that grows on a biological or artificial
surface, where single cells are embedded in a self-made
polymeric matrix. Biofilm-associated infections play a
direct role in the failure of implanted medical devices like
orthopedic prosthesis. The earlier removal of the device or/
and additional surgical interventions remain the most
effective means to treat biofilm-associated infections, with a
consequent high increase in healthcare costs due to longer
hospitalization periods and recurrent medical interventions
2
-phenylhydrazinylidene derivatives as antivirulence
agents. The compound 12e showed interesting activities
against biofilm formation of all tested Staphylococcus
aureus strains with IC50 ranging from 1.7 to 43 lM;
compounds 12f and 13a resulted strong inhibitors of S.
aureus ATCC 6538 and ATCC 29213 biofilm formation
with IC50 of 0.9 and 0.8 lM, respectively. A preliminary
study on the mechanism of action was carried on evaluat-
ing the inhibition of sortase A transpeptidase. Compound
12e resulted not to be toxic at 1 mg/ml by using an in vivo
model (the wax moth larva model, Galleria mellonella).
Keywords Phenylhydrazinylidene derivatives Á
Antibiofilm agents Á Sortase A Á Antivirulence agents Á
Bacterial adhesion
(Donlan and Costerton, 2002).
Here, we focus on Staphylococcus aureus, a Gram-
&
Stella Cascioferro
stellamaria.cascioferro@unipa.it
positive bacterium that may cause nosocomial infections
such as bacteremia, endocarditis, osteomyelitis, pneumo-
nia, and infections of the skin and soft tissues (Schaffer
et al., 2006; Diekema et al., 2001). Methicillin-resistant S.
aureus (MRSA) has been considered by the Antimicrobial
Availability Task Force of the Infectious Diseases Society
of America, as one of six high-priority dangerous ESKAPE
pathogens (Enterococcus, Staphylococcus, Klebsiella,
Acinetobacter, Pseudomonas, Enterobacter) (Chuang and
1
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche
e Farmaceutiche - Sezione di Chimica e Tecnologie
Farmaceutiche, Universit a` degli Studi di Palermo, Via
Archirafi 32, 90123 Palermo, Italy
2
3
IEMEST, Istituto Euromediterraneo di Scienza e Tecnologia,
Via Emerico Amari, 123, 90139 Palermo, Italy
Latvian Biomedical Research and Study Centre, Ratsupites 1,
Riga 1067, Latvia
123

Med Chem Res
Huang, 2013; Tenover and Goering, 2009; Talbot et al.,
Materials and methods
Chemistry
2
006).
Previously our research group synthesized a series of
-phenylhydrazinylidenealkanoic derivatives (Maggio
2
et al., 2016) that showed inhibitory activity against sortase
A (SrtA), a membrane transpeptidase responsible for the
anchoring of surface proteins to the cell wall envelope of
Gram-positive bacteria (Cascioferro et al., 2014b, 2015a).
These compounds also resulted to be able to reduce
staphylococcal biofilm formation, and the most active
derivatives, 1a,b, are reported in Fig. 1, whereas inhibition
data for sortase A and biofilm formation are showed in
Table 1.
All commercial chemicals were purchased from Aldrich
(Sigma-Aldrich, St. Louis, MO, USA). Reaction progress
was monitored by TLC on silica gel plates (Merck 60, F254,
0.2 mm) and visualization on TLC was achieved by UV
light. Organic solutions were dried over Na SO . Evapora-
2
4
tion refers to the removal of solvent on a rotary evaporator
under reduced pressure. All melting points were determined
on a B u¨ chi 530 capillary melting point apparatus and are
uncorrected. IR spectra were recorded with a PerkinElmer
This work is concentrated on the synthesis of 11 novel
Spectrum RXI FTIR system spectrophotometer as solid in
1
KBr disk. H NMR spectra were recorded in CDCl at
2
-phenylhydrazinylidene derivatives, 12a–f and 13a–e,
structurally related to compounds 1a,b that bear the
-methyl-1,2-oxazole-3-yl or the 1-ethyl-3-methyl-pyra-
3
300.13 MHz, using a Bruker AC series 300-MHz spec-
trometer (tetramethylsilane as the internal standard):
Chemical shifts are expressed in d values (ppm). Micro-
analyses data (C, H, N) were obtained by an Elemental Vario
EL III apparatus and are within ± 0.4 % of the theoretical
values. Yields refer to products after crystallization. The
name of the compounds was obtained using the ACD/I-Lab
Web service (ACD/IUPAC Name Free 8.05).
5
zol-5-yl moiety at the place of the methyl group. These
compounds, having no effect on S. aureus growth but
interfering with biofilm formation, have a potential for the
development as antivirulence agents (Cascioferro et al.,
2
014a; Cascioferro, 2014) alternative or complementary to
conventional antibiotics in the treatment of infections
caused by this pathogen under biofilm form. The inhibi-
tion of the biofilm formation of the S. aureus reference
strains ATCC 25923, ATCC 29213, and ATCC 6538 was
investigated. We focused on inhibition of sortase A as a
possible mechanism of action. Finally, the toxicity of the
most active compound 12e was also evaluated in vivo by
the wax moth larva model (Galleria melonella, Lepi-
doptera: Pyralidae). Insects are considered a good model
to study the toxicity of different substances. In addition
this animal model is very useful considering that it is a
preliminary and alternative toxicity model that generates
in vivo data quickly and inexpensively (Desbois and
Coote, 2011).
General procedure for preparation of 3-oxopropanoates
(
11a,b)
To the appropriate acid derivative 7, (Jackson et al., 2012)
0
10 (0.021 mol) in 20 ml of THF, 3.6 g of N,N -car-
bonyldiimidazol (CDI) was slowly added under stirring.
After addition was complete, the solution was first mag-
netically stirred for 30 min at room temperature and then
heated to 60 °C for 1 h. The reaction mixture was cooled to
room temperature and treated with 2.3 g of MgCl and
2
4
3
.5 g of ethyl potassium malonate. After stirring (r.t.) for
h 15 ml of water followed by 5 ml of HCl 6 N was
added. The mixture was then concentrated under reduced
pressure, and the obtained white solid was collected by
filtration, washed with water, and dried. Spectroscopical
and physical data of compound 11a are in accordance with
those reported in the literature (Takagi et al., 2008).
The ethyl 3-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-3-oxo-
propanoate 11b was used crude for the next reaction.
O
O
O
O
H C
OH
H C
OH
3
3
N
N
HN
HN
General procedure for preparation of the ethyl-3-oxo-2-(2-
phenylhydrazinylidene)propanoates (12a–f)
Cl
A cold (0–10 °C) solution of the appropriate diazonium salts
3a–d (2 mmol) was adjusted to pH 5 by saturated sodium
acetate solution, and then a mixture of the proper 3-oxo-
propanoate 11a,b in 25 ml of ethanol with 0.24 g of sodium
acetate in 0.4 ml of water was slowly added. After addition
was complete, the solution was magnetically stirred for
Cl
NO2
a
b
Fig. 1 2-Phenylhydrazinylidenealkanoic derivatives with inhibitory
activity against Sortase A
1
23

Med Chem Res
Table 1 Ability of 1a,b to reduce the biofilm formation of S. aureus 6538, S. aureus 25923, and S. aureus 29213 bacterial strains and to inhibit
SrtA, expressed as IC50 (lM)
Compound
IC50 (lM)
S. aureus
2
S. aureus
29213
S. aureus
6538
SrtA
5923
1
1
a
91
1.6
21
39.3
18.2
50
57
b
43.4
3
0 min at 0–10 °C and for 90 min at room temperature. Then
ml of water was added, and the product was collected by
Anal. Calcd. for C15
H
13
F
2
N
3
O
4
: C, 53.42; H, 3.88; N,
3
12.46. Found C, 53.58; H, 3.94; N, 12.51.
filtration, washed with water, and dried.
Ethyl 3-(5-methyl-1,2-oxazol-3-yl)-2-[2-(4-nitrophenyl)hy-
drazinylidene]-3-oxopropanoate (12d) Orange powder
Ethyl 2-[2-(3,4-dichlorophenyl)hydrazinylidene]-3-(5-methyl-
1
,2-oxazol-3-yl)-3-oxopropanoate (12a) Orange powder
(EtOH); yield: 99 %, m.p.: 100–102 °C; IR (KBr) mmax
1
(
(
EtOH); yield: 20 %; m.p.: 125 °C; IR (KBr) mmax 3136
3160 (NH), 1671 (CO) cm-1; H NMR (CDCl
3
, 300 MHz):
), 4.3841 (q, 2H,
), 6.49 (s, 1H oxazole H-4), 7.42 (d, 2H, C ), 8.27
-
NH), 1678 (CO) cm ; H NMR (CDCl , 300 MHz):
1
1
d = 1.38 (t, 3H, CH
CH
(d, 2H, C
(CDCl , 300 MHz): d = 180.99 (CO, C-3), 170.43 (C,
), 2.56 (s, 3H, CH
3
3 3
d = 1.36 (t, 3H, CH ), 2.52 (s, 3H, CH ), 4.40 (q, 2H, CH ),
2
H
6 4
3
3
2
1
3
6
.42 (s, 1H oxazole H-4), 7.13–7.45 (3H, a set of signals,
H ), 12.96 (s, 1H, exchangeable, NH); C NMR
6 4
1
C H ), 12.88 (s, 1H, exchangeable, NH).; C NMR
3
6
3
3
(
CDCl , 300 MHz): d = 180.97 (CO, C-3), 169.98 (C, C,
isoxazole C-5); 162.82 (C, CO-OCH
0
2
CH
3
), 162.02 (C,
0
3
isoxazole C-5), 163.33 (C, CO-OCH CH ), 162.22 (C,
2
isoxazole C-3), 146.63 (C, C-1 ), 144.07 (CH, C-4 ), 129.03
3
0
0
0
0
0
isoxazole C-3), 141.18 (C, C-1 ), 133.60 (C, C-2), 131.17
(C, C-2), 125.76 (C, C-3 , 5 ), 115.64 (CH, C-2 , 6 ), 102.10
(CH, isoxazole C-4), 62.26 (CH , O-CH CH ), 13.96 (CH
O-CH CH ), 12.33 (CH , isoxazole C-5); Anal. Calcd. for
: C, 52.03; H, 4.07; N, 16.18. Found C, 52.15;
H, 4.14; N, 16.33.
0
0
0
(
(
CH, C-3 ), 128.31 (C, C-5 ), 127.12 (CH, C-4 ), 117.70
0
2
2
3
,
3
0
CH, C-6 ), 115.28 (CH, C-2 ), 102.26 (CH, isoxazole C-4),
1.85 (CH , O-CH CH ), 14.00 (CH , O-CH CH ), 12.30
2
3
3
6
C H N O
15 14 4 6
2
2
3
3
2
3
(
CH , isoxazole C-5); Anal. Calcd. for C H Cl N O : C,
3 15 13 2 3 4
4
8.67; H, 3.54; N, 11.35. Found C, 48.71; H, 3.68; N, 11.43.
Ethyl 2-[2-(3,4-dichlorophenyl)hydrazinylidene]-3-(1-ethyl-3-
methyl-1H-pyrazol-5-yl)-3-oxopropanoate (12e) Pale yel-
low powder (EtOH); yield: 98 %, m.p.: 198–199 °C; IR
Ethyl 3-(5-methyl-1,2-oxazol-3-yl)-2-[2-(2-nitrophenyl)hy-
drazinylidene]-3-oxopropanoate (12b) Bright yellow
powder (EtOH); yield: 98 %; m.p.: 102–104 °C. IR (KBr)
-
1 1
(KBr) mmax 3188 (NH), 1721 (CO) cm ; H NMR (CDCl
300 MHz): d = 1.36 (t, 3H, CH ), 1.46 (t, 3H, CH ), 2.31 (s,
3H, CH ), 4.37 (q, 2H, CH ), 4.53 (q, 2H, CH ), 6.49 (s, 1H
pyrazole H-4), 7.04–7.42 (3H, a set of signals, C ), 12.61
, 300 MHz):
CH ); 146.72
,
3
-
1 1
mmax 3144 (NH), 1687 (CO), 1666 (CO) cm ; H NMR
3
3
(
CDCl , 300 MHz): d = 1.38 (t, 3H, CH ), 2.54 (s, 3H,
3
2
2
3
3
CH ), 4.44 (q, 2H, CH ), 6.49 (s, 1H oxazole H-4),
3
H
6 3
2
1
3
7
.15–7.68 (4H, a set of signals, C H ), 14.30 (s, 1H,
6
(s, 1H, exchangeable, NH); C NMR (CDCl
3
4
1
3
exchangeable, NH);
C NMR (CDCl , 300 MHz):
3
d = 179.04 (CO, C-3), 163.47 (C, CO-OCH
2
3
0
d = 181.24 (CO, C-3), 170.39 (C, isoxazole C-5), 162.04
C, CO-OCH CH ), 161.49 (C, isoxazole C-3), 138.55 (C,
(C, pyrazole C-5), 141.44 (C, C-1 ), 138.14 (C, C-2), 133.92
0 0
(
(C, pyrazole C-3), 131.18 (CH, C-5 ), 128.38 (C, C-3 ),
2
3
0
0
0
0
0
C-1 ), 136.10 (CH, C-6 ), 135.16 (C, C-2), 130.75 (C,
0
127.72 (C, C-4 ), 116.94 (CH, C-2 ), 114.56 (CH, C-6 ),
113.51 (CH, pyrazole C-4), 61.86 (CH , O-CH CH ), 47.18
(CH , N-CH CH ), 15.88 (CH , N-CH CH ), 14.02 (CH
O-CH CH ), 13.34 (CH , pyrazole C-5); Anal. Calcd. for
C H18Cl N O : C, 51.40; H, 4.57; N, 14.10. Found C,
17 2 4 3
0
0
C-2 ), 125.84 (CH, C-3 ), 123.33 (CH, C-4 ), 118.00 (CH,
2
2
3
0
C-5 ), 102.14 (CH, isoxazole C-4), 62.26 (CH , O-CH
2
2
2
3
3
2
3
,
3
2
CH ), 14.05 (CH , O-CH CH ), 12.33 (CH , isoxazole
3
2
3
3
3
2
3
3
C-5); Anal. Calcd. for C H N O : C, 52.03; H, 4.07; N,
15 14 4 6
1
6.18. Found C, 52.12; H, 4.09; N, 16.25.
Ethyl 2-[2-(2,6-difluorophenyl)hydrazinylidene]-3-(5-methyl-
,2-oxazol-3-yl)-3-oxopropanoate (12c) Orange powder
51.53; H, 4.61; N, 14.25.
Ethyl 2-[2-(2,6-difluorophenyl)hydrazinylidene]-3-(1-ethyl-
3-methyl-1H-pyrazol-5-yl)-3-oxopropanoate (12f) Yellow
powder (EtOH); yield: 41.2 %; m.p.: 102–103 °C; IR (KBr)
1
(
EtOH); Yield: 48.6 %; m.p.: 104–105 °C; IR (KBr) mmax
1
245 (NH), 1671 (CO), 1656 (CO) cm-1; H NMR (CDCl ,
-1
1
3
3
mmax 3245 (NH), 1671 (CO) cm ; H NMR (CDCl
300 MHz): d = 1.28 (t, 3H, CH ), 2.52 (s, 3H, CH ), 4.38 (q,
2H, CH ), 6.66 (s, 1H oxazole H-4), 7.53–8.31 (4H, a set of
signals, C ), 12.26 (s, 1H, exchangeable, NH); Anal.
3
,
3
00 MHz): d = 1.43 (t, 3H, CH ), 2.51 (s, 3H, CH ), 4.45
3
3
3
3
(
q, 2H, CH ), 6.54 (s, 1H oxazole H-4), 6.66–7.26 (3H, a
2
2
set of signals, C H ), 13.69 (s, 1H, exchangeable, NH);
6
H
6 4
3
123

Med Chem Res
-
(OH), 3232 (NH), 1711 (CO) cm ; H NMR (CDCl ,
1
1
Calcd. for C H F N O : C, 56.04; H, 4.98; N, 15.38.
3
1
7
18
2
4
3
Found C, 56.28; H, 5.03; N, 15.53.
300 MHz): d = 2.50 (s, 3H, CH ); 6.63 (s, 1H oxazole
3
H-4); 7.51–8.23 (4H, a set of signals, C H ); 11.79 (s, 1H,
6
4
1
3
General procedure for preparation of the 3-oxo-2-(2-
exchangeable, OH); 12.47 (s, 1H, exchangeable, NH);
C
phenylhydrazinylidene)propanoic acids (13a–e)
NMR (CDCl , 300 MHz): d = 186.61 (CO, C-3), 171.03
3
(
C, isoxazole C-5); 164.80 (C, COOH), 160.85 (C, isoxa-
0 0
To a solution of 1.91 mmol of the appropriate esther
derivative 12a–f in 7 ml of ethanol, 1.68 ml of NaOH 5 N
was added, and the reaction mixture was magnetically
stirred at room temperature for 20 h. The sodium salt was
then filtered under reduced pressure, washed with ethanol,
and solubilized in water. This solution was treated with 5 N
aqueous HCl affording the crude compound 13a–e. The
product was filtered and purified by crystallization.
zole C-3), 145.76 (C, C-1 ), 145.49 (C, C-4 ), 125.83 (CH,
0
0
0
0
C-3 , 5 ), 124.59 (C, C-2), 117.63 (C, C-2 , 6 ), 103.00 (CH,
isoxazole C-4), 12.33 (CH , isoxazole C-5); Anal. Calcd.
3
for C H N O : C, 49.06; H, 3.17; N, 17.61. Found C,
13 10 4 6
49.25; H, 3.31; N, 17.84.
2
-[2-(3,4-Dichlorophenyl)hydrazinylidene]-3-(1-ethyl-3-
methyl-1H-pyrazol-5-yl)-3-oxopropanoic acid (13e) Yel-
low orange (EtOH); yield: 78 %; m.p.: 131–133 °C; IR
-
1 1
2
-[2-(3,4-Dichlorophenyl)hydrazinylidene]-3-(5-methyl-1,2-
(KBr) mmax 3654 (OH), 3401 (NH), 1662 (CO) cm ; H
oxazol-3-yl)-3-oxopropanoic acid (13a) Brownish orange
powder (EtOH); yield: 48 %, m.p.: 201–202 °C IR (KBr)
NMR (CDCl , 300 MHz): d = 1.45 (t, 3H, CH ); 2.36 (s,
3H, CH ); 6.74 (s, 1H pyrazole H-4); 7.19–7.55 (3H, a set
3
3
3
-
1
;
mmax 3435 (OH), 3218 (NH), 1660 (CO), 1674 (CO) cm
of signals, C H ); 13.80–14.20 (s, 1H, exchangeable, OH,
6
3
1
13
s, 1H, exchangeable, NH); C NMR (CDCl , 300 MHz):
H NMR (CDCl , 300 MHz): d = 2.56 (s, 3H, CH ), 6.48
3
3
3
(
s, 1H oxazole H-4), 7.26–7.67 (3H, a set of signals, C H ),
6
d = 184.06 (CO, C-3), 165.71 (C, COOH); 147.22 (CH,
3
3
1
0
1
3.31 (s, 1H, OH), 14.23 (s, 1H, exchangeable, NH);
C
pyrazole C-4), 140.34 (C, C-1 ), 135.94 (C, C-2), 134.41
0 0
NMR (CDCl , 300 MHz): d = 186.45 (CO, C-3), 170.81
(C, pyrazole C-5), 131.60 (CH, C-5 ), 130.46 (C, C-3 ),
0
3
0
0
(
C, isoxazole C-5); 165.29 (C, COOH), 160.39 (C, isoxa-
0
124.03 (C, C-4 ), 118.60 (CH, C-2 ), 116.19 (CH, C-6 ),
114.93 (CH, pyrazole C-3), 47.63 (CH , N-CH CH ),
zole C-3), 139.84 (C, C-1 ), 134.32 (C, C-2), 131.54 (C,
0
2
2
3
0
0
C-3 ), 131.01 (CH, C-5 ), 123.89 (C, C-4 ), 119.24 (CH,
0
15.97 (CH , N-CH CH ), 13.28 (CH , pyrazole C-5); Anal.
3 2 3 3
0
C-2 ), 116.69 (CH, C-6 ), 103.04(C, isoxazole C-4), 12.26
Calcd. for C H Cl N O : C, 48.80; H, 3.82; N, 15.18.
15 14 2 4 3
(
CH , isoxazole C-5); Anal. Calcd. for C H Cl N O : C,
2 3 4
Found C, 48.95; H, 3.94; N, 15.33.
3
13
9
4
1
5.64; H, 2.65; N, 12.28. Found C, 45.75; H, 2.94; N,
2.53.
Biology
3-(5-Methyl-1,2-oxazol-3-yl)-2-[2-(2-nitrophenyl)hydrazinyli-
dene]-3-oxopropanoic acid (13b) Orange powder (EtOH).
Yield: 50 %, m.p. [205 °C; IR (KBr) mmax 3435 (OH),
Microbial strains
-
1
1
3
144 (NH), 1666 (CO), 1687 (CO) cm
;
H NMR
The staphylococcal reference strains used were: S. aureus
ATCC 29213, S. aureus ATCC 25923 and S. aureus ATCC
6538.
(
DMSO, 300 MHz): d = 2.50 (s, 3H, CH ); 7.11–8.18
3
(
4H, a set of signals, C H , s, 1H oxazole H-4); 14.55 (s,
6
4
1
H, exchangeable, NH); 4.93 (s, 1H, exchangeable, OH);
Anal. Calcd. for C H N O : C, 49.06; H, 3.17; N, 17.61.
13 10 4 6
Minimum inhibitory concentrations (MIC)
Found C, 49.15; H, 3.24; N, 17.73.
MICs were determined by a micromethod described pre-
2
2
-[2-(2,6-Difluorophenyl)hydrazinylidene]-3-(5-methyl-1,
viously (Schillaci et al., 2008; Raimondi et al., 2012).
-oxazol-3-yl)-3-oxopropanoic acid (13c) Orange pow-
der (EtOH); yield: 51 %, m.p.: 190–192 °C; mp
01–202 °C; IR (KBr) mmax 3419 (OH), 3158 (NH), 1633
Evaluation of biofilm formation
2
-
1 1
(
CO) cm ; H NMR (CDCl , 300 MHz): d = 2.56 (s, 3H,
3
All the bacterial reference strains were tested for their
ability to form biofilms. Briefly, bacteria were grown in
tryptic soy broth (TSB, Sigma) containing 2 % glucose
overnight at 37 °C in a shaking bath and then diluted 1:200
CH ), 6.48 (s, 1H oxazole H-4), 7.26–7.67 (3H, a set of
3
signals, C H ), 13.31 (s, 1H, OH), 14.23 (s, 1H,
6
3
exchangeable, NH); Anal. Calcd. for C H F N O : C,
13 9 2 3 4
5
1
0.49; H, 2.93; N, 13.59. Found C, 50.58; H, 2.97; N,
3.61.
to a suspension with optical density (OD) of about 0.040 at
6
5
70 nm corresponding to *10 CFU/ml. Polystyrene
3
-(5-Methyl-1,2-oxazol-3-yl)-2-[2-(4-nitrophenyl)hydrazinyli-
24-well tissue culture plates were filled with 2 ml of
diluted suspension and incubated for 24 h at 37 °C. Then,
the wells were washed three times with 1 ml of sterile
dene]-3-oxopropanoic acid (13d) Yellow powder (ethyl
acetate) Yield: 20 %, m.p.: 210 °C; IR (KBr) mmax 3418
1
23

Med Chem Res
phosphate-buffered saline (PBS) and stained with 1 ml of
crystal violet 0.1 % v/v for 1 min. The excess stain was
removed by placing the plates under running tap water.
Plates were dried overnight in inverted position at
NaCl and 1 mM DTT, was used for the final purification.
The fractions containing the protein were collected and
concentrated to 10 mg/ml. The purified protein was ana-
lyzed using matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectroscopy and SDS-
PAGE Coomassie blue staining.
3
7 °C. Crystal violet-stained adherent bacteria in each well
were redissolved to homogeneity in 1 ml of ethanol, and
the OD was read at 600 nm. Each assay was performed in
triplicate and repeated at least twice (Schillaci et al., 2010).
FRET-based screening of compounds 12a–f and 13a–e
Biofilm prevention assay
All the compounds were prepared as 10 mM stock solu-
tions in dimethyl sulfoxide (DMSO) and used for the IC50
determination. The compounds were screened at a single
dose of 100 lM (1 % DMSO) in black 384-well plates
(Greiner Bio-One). Two known sortase inhibitors, phenyl
vinyl sulfone (Frankel et al., 2004) and 1-(3,4-dichlor-
ophenyl)-3-(dimethylamino)propan-1-one (Maresso et al.,
2007), were used as the positive controls. The inhibitory
activity of all of the compounds was determined by
quantifying the increase in fluorescence intensity upon
cleavage of the FRET-peptide dabcyl-QALPETGEE-
edans, which was used as the sortase substrate. A previ-
ously published method (Ton-That et al., 1999) was used
with slight modifications. Briefly, the reactions were per-
formed in a volume of 100 ll containing 50 mM Tris–HCl,
Procedure described above was used to evaluate the
activity of all the synthesized compounds in preventing
biofilm formation. Polystyrene 24-well tissue culture
plates were filled with 1 ml of diluted bacterial suspension
(
OD of about 0.040 at 570 nm), obtained and diluted as
previously seen, and sub-MIC concentrations, ranging
from 300 to 0.3 lM of each compound were directly
added to the bacterial suspension at time zero and incu-
bated at 37 °C for 24 h. After that time the wells were
washed and stained with crystal violet as seen in biofilm
forming assay. By comparing the average optical density
of the growth in control wells with that in the sample
wells, the following formula was used to calculate the
percentages of inhibition for each concentration of the
sample:
5 mM CaCl , 150 mM NaCl, pH 7.5, 10 lM S. aureus
2
SrtA, 20 lM fluorescent peptide substrate dabcyl-QAL-
PETGEE-edans, and the prescribed concentrations of the
test compounds or positive controls.
OD570 growth control À OD sample
570
Inhibition ð% Þ ¼
OD growth control
570
The peptide substrate without the recombinant SrtA was
incubated in the same manner and used as a negative
control. The reactions were conducted for 24 h at 37 °C,
and the fluorescence emitted with an excitation wavelength
of 350 nm, and an emission wavelength of 495 nm after
substrate cleavage was recorded.
 100
Each assay was performed in triplicate and assays were
repeated at least twice.
Enzyme activity assay
Endpoint determination of product formation was used
as a criterion for the primary screening. This determination
was made by measuring the total product fluorescence 24 h
after the initiation of the reaction. The relative inhibition
activity was determined as %I = 100 % - (Fsample/Fcon-
Expression of recombinant sortase A
Recombinant and catalytically active sortase A with the
N-terminal deletion of residues 1–59 and possessing
C-terminal hexahistidine sequence (SrtADN59-6His) was
used for the enzyme activity assay. SrtADN59-6His was
prepared according to a slightly modified previously pub-
lished method (Zhulenkovs et al., 2014b). Briefly, after the
IPTG induction of the E. coli BL21 (DE3)-transformed
strain, the cell pellet was collected by centrifugation and
resuspended in lysis buffer, and the recombinant protein
was purified by affinity chromatography on a Ni–NTA
column (Qiagen). The enzyme was eluted with an imida-
zole gradient, and the fractions containing the protein were
further purified by the gel filtration. A Superdex 75 (GE
Healthcare) column, which was equilibrated with 10 mM
sodium phosphate (pH 7.0) buffer containing 100 mM
9 100 %), where Fsample is the fluorescence intensity of
trol
the well containing the corresponding test compound and
Fcontrol is the fluorescence of the positive control reaction
without inhibition.
For the IC50 determination, 10 lM S. aureus sortase A
was preincubated in the reaction buffer with increasing
concentrations of the inhibitory compounds (x–y lM) for
1 h at 37 °C prior to the addition of the dabcyl-QAL-
PETGEE-edans substrate. The total fluorescence was
recorded at 1-min intervals for 1 h, and the progress curves
were constructed. The initial velocities of the biphasic
reactions were obtained through nonlinear regression, as
previously described (Zhulenkovs et al., 2014a; Huang
et al., 2003). The IC50 values were determined by fitting
123

Med Chem Res
the obtained data to a default four-parameter variable-slope
sigmoidal function in SigmaPlot 12.5 using a nonlinear
least-squares algorithm.
propan-2-one, achieving the ethyl 2,4-dioxopentanoate 5
(Kamal et al., 2014). Refluxing of the latter compound with
hydroxylamine hydrochloride led to the formation of the ethyl
5
-methyl-1,2-oxazole-3-carboxylate 6 (Baraldi et al., 1982)
which, in turn, was hydrolyzed to the corresponding acid 7
Jackson et al., 2012) that, finally, by reaction with ethyl
Insects
(
Larvae of greater wax moth G. mellonella were reared on a
natural diet honeybee nest debris at 30 °C in the dark. Last
instar larvae of an average mass of in 521 mg (± 110 mg)
were selected for this study.
potassium malonate lead to the obtaining of11a (Takagi et al.,
2008).
The starting product ethyl 3-(1-ethyl-3-methyl-1H-
pyrazol-5-yl)-3-oxopropanoate 11b was obtained by
reacting the ethyl 2,4-dioxopentanoate 5 with hydrazine
reaching initially the ethyl 3-methyl-1H-pyrazole-5-car-
boxylate 8. The latter was ethylated obtaining 9 which was
hydrolyzed to the corresponding acid 10. Finally, the
derivative 11b was obtained by reacting 10 with ethyl
potassium malonate.
Evaluation of toxicity
To evaluate the toxicity of the compound 12e, a single
treatment dose of 1 mg/kg was given to each larva. In
the experiments, there were two group controls: one
untreated and the other was injected with PBS containing
At this point, the diazonium salts 3a–d afforded from
the anilines 2a–d were reacted with the ethyl propanoates
11a,b to give the ester derivatives 12a–f which, in turn,
were transformed in the corresponding acids 13a–e
(Scheme 1).
3
% DMSO, and the same solvents were used to dissolve
the tested compound. A sample group was treated with
the compound 12e. Three replicates of four larvae for
each group were disposed. Injection of solvents or
compound solution was given into alternate prolegs
moving up the body toward the head such that each
proleg was injected only once. Larvae were stored in
Petri dishes in the dark at 37 °C for 72 h in dark con-
ditions. Larvae were inspected every 24 h, and the sur-
vival rate and the weight was observed. Moreover, to
detect potential deleterious effect of the compound 12e,
the variation in larval weight and the time pass from the
inoculum at the later larval stage to the pupation were
also checked.
The structures of the new compounds were determined by
analytical and spectroscopic measurements. The IR spectra
of compounds 12a–f showed adsorptions in the range
-
1
1666–1685 cm as distinct bands for the carbonyl groups.
1
The H NMR spectra of the above compounds confirmed the
phenylhydrazinylidene structure as the showed the NH
signal in the range 12.26–14.31 d. The presence of electron
withdrawing groups in the ortho positions of the phenyl ring
produced a low field shift than the unsubstituted derivatives.
The acid derivatives 13 produced in the IR spectra carbonyl
-
1
bands in the 1660–1711 cm region together with multiple
-
bands in the 2500–3250 cm for the OH and NH groups.
1
Statistical analysis
1
The H NMR spectra showed signal in the 13.2–15.2 d
For survival and weight variation of G. mellonella larvae
data analysis, collated datasets were compared using
analysis of variance (ANOVA) with homogeneity of vari-
ances performed using Cochran’s test prior to ANOVA
analysis. The statistical significances of the parameter
analyzed were calculated by one-way ANOVA and
Tukey’s pairwise comparisons. Statistical significance was
assessed by all applied tests (P \ 0.05) using Statistica 6.0
which disappeared by addition of deuterium oxide,
attributable to NH and OH groups. In the case of derivative
13b COOH signal was observed at 4.93 d superimposed
with H O signal.
2
Biological activity
Compounds 12a–f and 13a–e were primarily evaluated for
their antistaphylococcal activity against three reference
strains of S. aureus, namely ATCC 25923, ATCC 29213,
ATCC 6538. The minimum inhibitory concentration (MIC)
of the tested compounds was evaluated, and the results
showed the compounds have no effect on the microbial
viability (MIC [ 200 lM).
(
StatSoft, Tulsa, OK, USA).
Results and discussion
Chemistry
Esters 12a–f and acids 13a–e were also tested for their
inhibitory activity on SrtA, utilizing the FRET technics
(Table 2) (Sekar and Periasamy, 2003). Among the tested
compounds, the ester derivative ethyl 2-[2-(2,6-difluo-
rophenyl)hydrazinylidene]-3-(1-ethyl-3-methyl-1H-pyrazol-
The phenylhydrazinylidene derivatives 12a–f and 13a–e were
obtained as reported in scheme 1. In particular, the interme-
diate ethyl 3-(5-methyl-1,2-oxazol-3-yl)-3-oxopropanoate
1
1a was obtained by reacting diethyl ethanedioate 4 with
1
23

Med Chem Res
R1
R2
O
NH2
R5
H C
3
H
3
C
O
O
O
O
c
b
a
N
OEt
EtO
N
H C
OEt
3
R3
O
O
COOEt
R4
COOH
b'
7
5
4
2a-d
6
d
e
H C
3
H C
3
H C
3
R1
c
b''
O
O
R2
+
2
-
N
d
N
Cl
N
N
COOEt
N
H
COOH
COOEt
N
N
+
X
O
CH3
C H
C H
2 5
R3
R5
2
5
11a-b
R4
10
9
8
f
3a-d
O
O
X
O
X
O
O
R1
NH N
R5
OH
R1
g
NH N
R5
R2
CH3
R2
R3
R4
R3
R4
13a-e
12a-f
2
2
2
2
3
3
3
3
1
1
1
a R
b R
1
=H, R
2
=H, R
, R = H, R
=H, R =H, R
=H, R = NO
=H, R =Cl, R
, R = H, R =H, R
=H, R =H, R =H, R
=H, R = NO , R =H, R
3
=Cl, R
=H, R
=H, R
, R =H, R
=Cl, R =H
=H, R =H
=F
4
=Cl, R
=H, R
=F
5
=H
12b X=5-methyl-1,2-oxazole-3-yl, R
12c X=5-methyl-1,2-oxazole-3-yl, R
12d X=5-methyl-1,2-oxazole-3-yl, R
12e X=1-ethyl-3-methyl-1H-pyrazole-5-yl, R
12f X=1-ethyl-3-methyl-1H-pyrazole-5-yl, R
13a X=5-methyl-1,2-oxazole-3-yl, R =R =R
13b X=5-methyl-1,2-oxazole-3-yl, R = NO , R
=R =F, R
= R = R
=R
1
= NO
2
, R
2
= R
3
= R
=R =H,
=H, R
4
= R
5
= H,
1
= NO
=F, R
=H, R
=H, R
= NO
=F, R
=H, R
2
2
3
4
5
=H
1
=R
5
=F, R
= R
=R
=R
=H, R
2
=R
3
4
c R
d R
1
2
3
4
5
1
= R
2
4
=, R
5
3
= NO
=R =Cl,
=R =H,
2
,
1
2
3
2
4
5
=H
1
2
=R
5
=H, R
3
4
a R
b R
1
2
3
4
5
1
5
=F, R
3
2
=R
=Cl
= R
=H
=H, R
=R =H, R
3
4
1
2
2
3
4
5
1
2
5
=R
4
c R
1
2
3
4
5
1
2
2
= R
=R
3
= R
=R
4
5
= H
d R
1
2
3
2
4
5
=H
13c X=5-methyl-1,2-oxazole-3-yl, R
13d X=5-methyl-1,2-oxazole-3-yl, R
1
5
2
3
4
1a X=5-methyl-1,2-oxazole-3-yl
1b X=1-ethyl-3-methyl-1H-pyrazole-5-yl
2a X=5-methyl-1,2-oxazole-3-yl, R =R =R
1
2
4
=, R
5
3
= NO
2
,
13e X=1-ethyl-3-methyl-1H-pyrazole-5-yl, R
1
2
5
3
=R =Cl
4
1
2
5
=H, R
3
=R =Cl,
4
Scheme 1 Reagents and conditions: (a) EtONa, acetone, stirring
–10 °C, 4 h; (b) EtOH, hydroxylamine hydrochloride, reflux, 3 h;
10 h, HCl; (d) THF, CDI, MgCl
rt, 4 h, (e) HCl 5 N, KNO
acetate, stirring, 0–10 °C, 2 h; (g) EtOH, NaOH 5 N, stirring, rt, 20 h
2
, ethyl potassium malonate, stirring,
0
2
, stirring, 0–5 °C; (f) EtOH, sodium
0
00
(
b ) Acetic acid, hydrazine monohydrochloride, reflux, 2 h; (b )
Diethyl sulfate, 150–160 °C, 2 h; (c) EtOH, NaOH (10 %), stirring,
5
-yl)-3-oxopropanoate 12f and the 2-[2-(2,6-difluorophenyl)
Even if the compounds 12a–f and 13a–e were less active
than derivatives 1a,b against SrtA, they resulted, in some
cases (Table 2), more potent as inhibitors of staphylococcal
biofilm formation, the compounds 13a and 12e showed the
best activity against S.aureus ATCC 29213 with an IC ,
hydrazinylidene]-3-(5-methyl-1,2-oxazol-3-yl)-3-oxopropa-
noic acid 13c showed the best inhibitory activity on the
enzyme with IC50 of 75 and 76.5 lM, respectively, better
than that one shown by a known inhibitor as phenyl vinyl
sulfone, PVS, (IC50 = 736 lM) (Frankel et al., 2004) and
very close to the IC50 of some previously reported analogues
5
0
respectively, of 0.8 and 1.7 lM. Interestingly, the com-
pounds 13e and 12f interfere with biofilm formation of all the
tested staphylococcal strains with IC ranging, respectively,
(
submitted data).
5
0
123

Med Chem Res
Table 2 Ability of 12a–f and 13a–e to reduce the biofilm formation of S. aureus 6538, S. aureus 25923 and S. aureus 29213 bacterial strains and
to inhibit SrtA, expressed as IC50 (lM)
Compounds
S. aureus
ATCC 25923
S. aureus
ATCC 29213
S. aureus
ATCC 6538
SrtA IC50
1
1
1
1
1
1
1
1
1
1
1
2a
2b
2c
2d
2e
2f
277
48
123
21
109
153
159
55
[200
[200
[200
[200
[200
75
41
10
83
177
1.7
13
10
43
42
0.9
53
3a
3b
3c
3d
3e
85
0.8
52
[200
195
64
506
254
88
68
29
76.5
231
20
26
[200
[200
736
16
11
PVS
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
1
-(3,4-Dichlorophenyl)-3-dimethylamino)propan-1-one
16.91
Derivative 12e, which showed the best antibiofilm
activity against two of the three tested strains of S. aureus,
was tested by administering a single dose of 1 mg/kg dis-
solved in a mixture of PBS/DMSO (3 %) at time 0 to
groups of 10 larvae and observing the effect after 24, 48,
and 72 h. The toxicity is evaluated on the basis of survival
percentage (see Fig. 2).
The comparison between survival percentage in the
group of larvae treated with compound 12e and the control
group (injected with the used solvents only) indicates a
lack of toxicity up to the examined dose and regimen under
examination.
Fig. 2 Effect of treatment with a single dose (1 mg/kg) of compound
Conclusions
1
2e on survival of Galleria mellonella larvae at 37 °C. Survival was
checked at 24, 48, and 72 h after injection. Results are given as mean
n = 3 replicates) with standard deviation. Data entries with different
(
In this study we found that the activity of the 2-phenyl-
hydrazinylidene derivatives is not directed against the
planktonic form of staphylococcal strains but against bio-
film formation.
letter are statistically different at post hoc Tukey’s t test for multiple
comparisons within each character (P \ 0.05)
from 11 to 20 and from 0.9 to 42 lM. It is very difficult to
correlate SrtA inhibition with the ability to interfere with
biofilm formation; for example, the compound 12f is a good
inhibitor of SrtA and of staphylococcal biofilm formation,
but the compound 13e, despite its property to prevent biofilm
formation, showed a not significant activity as SrtA inhibitor.
However, we must stress that it is difficult to compare the
data obtained with two completely different experimental
models; in fact in the evaluation of antibiofilm activity, we
use bacterial cells, but in the activity of inhibition of SrtA, a
biochemical model.
In conclusion, this study demonstrated that these novel
agents do not affect bacterial growth (MIC [ 200 lM)
but are biofilm inhibitors targeting a virulence mecha-
nism like adhesion and biofilm formation of S. aureus
strains.
With the aim to investigate the mechanism of action of
these molecules, we evaluated the activity as SrtA inhibi-
tors, and we found that there is no correlation between the
activity on the biofilm formation and the inhibition of the
transpeptidase even if it is very difficult to compare two so
different experimental models (cellular and biochemical).
Other possible mechanisms could be involved, e.g.,
The toxicity of the most active compound 12e was also
evaluated in in vivo model.
1
23

Med Chem Res
quorum sensing inhibition, so a further and more detailed
investigation is needed to clarify the mode of action.
In any case, the biological activity of such compounds is
that typical of antivirulence agents because they do not
target bacterial viability and they should impose a low
selection pressure rendering less probable the development
of resistance (Cascioferro et al., 2014a). In addition, the
absence of in vivo toxicity guarantees the active compound
to be pushed forward for further studies.
wall protein anchoring and virulence in Staphylococcus aureus.
J Am Chem Soc 126:3404–3405. doi:10.1021/ja0390294
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Compliance with ethical standards
Conflict of interest We declare that we have no conflict of interest.
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