Inhibitory effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H- pyrazole-1-carbothioamide on Haemophilus spp. planktonic or biofilm-forming cells

Article abstract of DOI:10.1007/s00044-013-0700-7

During this study, we have investigated in vitro activity of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide derivatives with N-ethyl, N-(4-metoxyphenyl) and N-cyclohexyl substituents against Gram-negative Haemophilus influen

Full text of DOI:10.1007/s00044-013-0700-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1057–1066
DOI 10.1007/s00044-013-0700-7
ORIGINAL RESEARCH
Inhibitory effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-
1H-pyrazole-1-carbothioamide on Haemophilus spp. planktonic
or biofilm-forming cells
•
Urszula Kosikowska Anna Malm
•
•
Monika Pitucha Barbara Rajtar
•
Malgorzata Polz-Dacewicz
Received: 29 January 2013 / Accepted: 8 August 2013 / Published online: 23 August 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract During this study, we have investigated in vitro
activity of N-substituted-3-amino-5-oxo-4-phenyl-2,5-
dihydro-1H-pyrazole-1-carbothioamide derivatives with N-
ethyl, N-(4-metoxyphenyl) and N-cyclohexyl substituents
against Gram-negative Haemophilus influenzae and H. para-
influenzae bacteria. A spectrophotometric assay was used
in order to determine the bacterial growth and biofilm for-
mation using a microtiter plate to estimate minimal inhibi-
tory concentration (MIC) and minimal biofilm inhibitory
concentration (MBIC). Among the tested N-substituted
pyrazole derivatives, only N-ethyl-3-amino-5-oxo-4-phe-
nyl-2,5-dihydro-1H-pyrazole-1-carbothioamide showed a
significant in vitro activity against both planktonic cells of
H. parainfluenzae (MIC = 0.49–31.25 lg ml-1) and H. in-
fluenzae (MIC = 0.24–31.25 lg ml-1) as well as biofilm-
forming cells of H. parainfluenzae (MBIC = 0.24–31.25
lg ml-1) and H. influenzae (MBIC = 0.49 to C31.25
lg ml-1). The pyrazole compound exerted higher inhibitory
effect both on the growth of planktonic cells and biofilm
formation by penicillinase-positive and penicillinase-nega-
tive isolates of H. parainfluenzae than the activity of com-
monly used antibiotics such as ampicillin. No cytotoxicity of
the tested compound in vitro at concentrations used was
found. The tested pyrazole N-ethyl derivative could be
considered as a compound for the design of agents active
against both pathogenic H. influenzae and opportunistic H.
parainfluenzae, showing also anti-biofilm activity. This
appears important because biofilms are determinants of
bacterial persistence in long-term and recurrent infections
recalcitrant to standard therapy.
Keywords Haemophilus spp. Á Pyrazole derivatives Á
Antibacterial activity Á Anti-biofilm activity Á Cytotoxicity
Introduction
Biofilms are sessile aggregates of bacterial cells that are
created on either biotic surfaces (e.g., human tissues) or
abiotic surfaces (e.g., biomaterials, catheters) and act like a
single living organism that can exhibit differences in the
expression of surface molecules, antimicrobial resistance,
virulence factors, and pathogenicity (Costerton et al., 1999,
2003; Burmølle et al., 2010; Hall-Stoodley et al., 2012;
Bjarnsholt, 2013). In medicine, biofilms have been widely
associated with several chronic and recurrent diseases,
chronic wound infections, and foreign body infections
associated with implantable medical devices and indwell-
ing catheters, antibiotic-resistant and nearly impossible or
difficult to eradicate without aggressive and long-term
interventional strategies infections (Donlan, 2001; Steward
and Costeron, 2001; Gilbert et al., 2002; Stoodley et al.,
2004; Lasa et al., 2005; Sanclement et al., 2005; Macfar-
lane and Dillon, 2007; Vlastarakos et al., 2007; Macedo
and Abraham, 2009; Wolcott and Ehrlich, 2008; Coenye
and Nelis, 2010; Drago et al., 2012; Bjarnsholt, 2013).
Haemophilus spp. rods, generally known as Gram-neg-
ative microbiota of the upper respiratory tract, are able to
U. Kosikowska (&) Á A. Malm
Department of Pharmaceutical Microbiology, Medical
University of Lublin, Chodzki Str. 1, 20-093 Lublin, Poland
e-mail: u.kosikowska@umlub.pl
M. Pitucha
Department of Organic Chemistry, Medical University,
Chodzki Str. 4a, 20-093 Lublin, Poland
B. Rajtar Á M. Polz-Dacewicz
Department of Virology, Medical University, Chodzki Str. 1,
20-093 Lublin, Poland
123

1058
Med Chem Res (2014) 23:1057–1066
live as planktonic cells or colonize natural and artificial
surfaces as biofilm-forming cells (Hill et al., 2000; Chin
et al., 2005; Musk and Hergenrother, 2006; Galli et al.,
2007; Kilian, 2007; Moxon et al., 2008; Kosikowska and
as opportunistic, e.g., S. epidermidis, Bacillus spp. or Micro-
coccus luteus with MIC = 3.91–31.25 lg ml-1 (Pitucha
et al., 2010). The inhibitory effect against Gram-negative
bacteria––belonging to Enterobacteriaceae family (Esche-
richia coli, Klebsiella pneumoniae, and Proteus mirabilis)
or nonfermentative rods (Pseudomonas aeruginosa) was
weaker (MIC = 250–1,000 lg ml-1). In this paper, we have
investigated in vitro activity of pyrazole derivatives, among
which N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-
pyrazole-1-carbothioamide showed highest activity against
planktonic and biofilm-forming cells of H. influenzae and H.
parainfluenzae.
¨
Malm, 2009; Murphy et al., 2007; Drago et al., 2012; Unal
et al., 2012). Both pathogenic Haemophilus influenzae and
opportunistic H. parainfluenzae can cause acute, chronic,
invasive or non-invasive infections. These microorganisms
may form a biofilm which is a virulence determinant which
contributes to recurrent or chronic infections. H. influenzae
is the most pathogenic bacteria colonizing the mucous
membranes of the respiratory tract of young children or
sporadically elderly people. H. influenzae, mainly serotype
b (Hib), is frequently associated with different diseases,
e.g., otitis media, chronic bronchitis, and pneumonia
(Agrawal and Murphy, 2011). Other illnesses caused by
this species include osteomyelitis, arthritis, sepsis, phleg-
mon cellulitis, or abscesses. Non-typeable H. influenzae
(NTHi) is one of the main causes of airway infection in
chronic obstructive pulmonary disease, of recurrent otitis
media in infants and children, sinusitis in children and
adults, pneumonia in adults, lower respiratory tract infec-
tion in adults, and recurrent respiratory tract infections in
patients with chronic bronchitis (Murphy, 2003; Erwin and
Smith, 2007).
Results and discussion
According to our preliminary results, N-(4-metoxyphenyl)-
3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-car-
bothioamide compound had very low inhibitory effect on
the growth of planktonic cells of the reference strains of
Haemophilus spp. (MIC = 500–1,000 lg ml-1), similar
to N-cyclohexyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-
pyrazole-1-carbothioamide which inhibited the growth of
these bacteria with somewhat lower MIC = 125–500
lg ml-1. Among the tested pyrazole derivatives, N-ethyl-
3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbo-
thioamide derivative showed a significant in vitro potency
against the growth of planktonic cells of the tested Hae-
Haemophilus parainfluenzae is an opportunistic pathogen,
which may cause several endogenous diseases occasionally
and under predisposing conditions (e.g., chronic diseases or
immune disorders) such as respiratory tract infections, endo-
carditis, biliary tract infection, septic arthritis, thoracic empy-
ema, meningitis, secondary bacteremia, urethritis, and hepatic
abscesses (Chow et al., 1974; Cooney et al., 1981; Warman
et al.; 1981; Raoult et al., 1987; Darras-Joly et al., 1997; Das
et al., 1997; Bottone and Zhang,1995; Pillai et al., 2000;
Frankard et al., 2004; Cardines et al., 2009).
mophilus spp. strains with MIC \62.5 lg ml-1
.
As shown in Table 1, detailed studies with N-ethyl-3-
amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothio-
amide revealed that this compound possessed good activity
against planktonic cells of the reference strains of H. para-
influenzae ATCC 7901 (MIC = 0.49 lg ml-1), H. para-
influenzae ATCC 51505 (MIC = 7.81 lg ml-1), and H.
influenzae ATCC 10211 (MIC = 0.49 lg ml-1). This
compound was also active against planktonic cells of 20
clinical isolates of H. parainfluenzae (MIC = 1.95–31.25
lg ml-1) and of 11 clinical isolates of H. influenzae
(MIC = 0.24–31.25 lg ml-1). Moreover, the activity of
the tested compound against H. parainfluenzae and H. in-
fluenzae biofilm-forming cells was also determined––it
inhibited biofilm formation by reference strains of
H. parainfluenzae ATCC 7901 (minimal biofilm inhibitory
concentration [MBIC] = 1.95 lg ml-1) and H. parainflu-
enzae ATCC 51505 (MBIC = 15.63 lg ml-1) or by 20
clinical isolates of H. parainfluenzae (MBIC = 0.24–31.25
lg ml-1). The tested compound showed the inhibitory
effect against biofilm-forming cells of H. influenzae ATCC
10211 (MBIC = 15.63 lg ml-1) or seven H. influenzae
clinical isolates (MBIC = 0.49–31.25 lg ml-1). In case of
four clinical isolates of H. influenzae, MBIC were found to
Nitrogen heterocycles, including pyrazoles, are important
groupofnaturalorsyntheticderivativeswithabroadspectrum
of biological and pharmaceutical activities, e.g., antibacterial,
antifungal, antiviral, anti-inflammatory, antipyretic, antican-
cer, and anticonvulsant (Comber et al., 1991; Mahajan et al.,
1991; Chauhan et al., 1993; Sugiura et al. 1977; Bekhit and
¨
Abdel-Aziem, 2004; Gokhan-Kelekc¸i et al., 2007; Lin et al.,
2007; Kumar et al., 2012). Much attention has been paid to
pyrazole derivatives due to their wide range of antibacterial
activities as potential and selective inhibitors against DNA
gyrase capable of causing bacterial cells’ death (Reece and
Maxwell, 1991; Maxwell, 1997; Tanitame et al., 2004; Liu
et al., 2008; Shiroya et al., 2011). N-ethyl-3-amino-5-oxo-4-
phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide, synthe-
sized according to Pitucha et al. (2010) appears to be a
promising precursor of agents with good activity mainly
against Gram-positive bacteria––both pathogenic, including
Staphylococcus aureus (MIC = 7.81–62.5 lg ml-1) as well
be [31.25 lg ml-1
.
123

Med Chem Res (2014) 23:1057–1066
1059
Table 1 The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-
1H-pyrazole-1-carbothioamide on the growth of Haemophilus spp.
planktonic (MIC) or biofilm-forming (MBIC) cells
Species
Growth
Biofilm formation
MIC
No. of MBIC No. of
(lg ml-1) strains (lg ml-1) strains
Haemophilus ATCC
parainfluenzae 7901
0.49
7.81
1
1
1.95
1
1
ATCC
51505
15.63
Clinical
isolates
(n = 20)
0.24
0.98
1.95
3.91
7.81
15.63
31.25
0.49
0
0
1
1
3
7
8
1
0.24
1
1
3
3
0
6
6
1
0.98
1.95
3.91
7.81
15.63
31.25
15.63
Fig. 1 The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-
1H-pyrazole-1-carbothioamide compound on biofilms formation by
Haemophilus spp. on the basis of MBIC/MIC ratio
Haemophilus
influenzae
ATCC
10211
Clinical
isolates
(n = 11)
0.24
0.49
1
1
3
1
1
0
2
2
0
0.24
0.49
0
1
1
2
1
1
0
1
4
tested compound. The most widely used as a measurement of
compound’s toxicity is the half maximal effective concen-
tration (EC50), as the concentration of the compound where
50 % of its maximal effect is observed; in case of the tested
compound EC50 = 278.8 lg ml-1. This means that this
compound was not toxic to eukaryotic cells at concentrations
exerting inhibitory effect against Haemophilus spp.,
including anti-biofilm activity.
0.98
0.98
1.95
1.95
3.91
3.91
7.81
7.81
15.63
31.25
[31.25
15.63
31.25
[31.25
Although the control of bacterial infections has been
effective since the discovery of antimicrobial drugs, wide-
spread drug resistance among bacteria has led to a search for
new antibacterial agents. However, the finding of biofilm
phenotype bacteria, showing usually intrinsic insensitivity to
available drugs at standard dosing effective against plank-
tonic cells, has created a necessity to pay more attention to
targeted anti-biofilm agents. In this work, we have found that
the N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyr-
azole-1-carbothioamide possessed good in vitro activity
either against free-floating (planktonic) or biofilm-forming
cells of Haemophilus spp. Haemophili rods, e.g., pathogenic
H. influenzae or opportunistic H. parainfluenzae are found to
be a part of proper microflora of the upper respiratory tract
(Kilian, 2007; Murphy et al., 2007). Under favorable con-
ditions, these bacterial species may be the etiologic agents of
various and unspecified infections, including those associ-
ated with biofilm formation (Trollfors et al., 1985; Black
et al., 1988; Mitchell and Hill, 2000; Chin et al., 2005; Musk
and Hergenrother, 2006; Rele et al., 2006; Galli et al., 2007;
Moxon et al., 2008; Cardines et al., 2009; Drago et al., 2012;
Bjarnsholt, 2013). It has been estimated that the biofilms
protect microbes from the immune system, antimicrobials,
predation or stresses, and are crucial for the development of
recurrent and opportunistic diseases (Costerton et al., 1999,
To determine the power of the tested compound as an
anti-biofilm agent, the MBIC/MIC ratio was assessed. The
most frequently MBIC/MIC ratio ranged from 0.5 to
2 lg ml-1, indicating comparable activity of the com-
pound either against planktonic or biofilm-forming cells of
H. parainfluenzae and H. influenzae (Fig. 1). Only in some
cases, MBIC/MIC ratio was lower for H. parainfluenzae
and was higher for H. influenzae, indicating strain- and
species-dependent activity of the tested compound.
Figure 2 shows the activity of N-ethyl-3-amino-5-oxo-4-
phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide on the
growth or biofilm formation by penicillinase-negative
(S85Pen-) and penicillinase-positive (S86Pen?) H. para-
influenzae. In the case of penicillinase-positive isolate, the
activity of the compound was significantly higher both on
the growth and on the biofilm formation.
The in vitro cytotoxicity of the tested N-ethyl-3-amino-5-
oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide com-
pound was presented as percentage viability of vero cells
used as an experimental model. According to the results
shown in Table 2, after 48 h of incubation, no cytotoxic
effect was observed up to 200 lg ml-1 concentration of the
123

1060
Med Chem Res (2014) 23:1057–1066
Fig. 2 The effect of N-ethyl-3-
amino-5-oxo-4-phenyl-2,5-
dihydro-1H-pyrazole-1-
carbothioamide compound and
ampicillin on the penicillinase-
negative (filled diamond
S85Pen-) and penicillinase-
positive (filled square
S86Pen?) Haemophilus
parainfluenzae planktonic or
biofilm-forming cells (broth
without bacteria:
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
S85Pen(–) S86Pen +)
S85Pen(–)
S6Pen(+)
OD570 = 0.09–0.11)
0
5
10 15 20 25 30 35
Concentration [µg ml-1]
0
5
10 15 20 25 30 35
Concentration [µg ml-1]
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
S85Pen(–)
S6Pen(+)
S85Pen(–)
S6Pen(+)
0
5
10 15 20 25 30 35
Concentration [µg ml-1]
0
5
10 15 20 25 30 35
Concentration [µg ml-1]
e.g., pyrazole or thioamide derivatives, may act as quorum-
sensing inhibitors (Hentzer and Givskov, 2003; Schillaci
et al. 2008; Brackman et al., 2009; Kociolek, 2009; Oancea,
2010). Quorum-sensing phenomenon, which is one of the
ways to control biofilms, is a chemical form of bacterial
communication via signaling molecules essential for bacte-
rial communities to regulate the group and to synchronize
the behavior (Hastings and Greenberg, 1999; Van Houdt
et al., 2004; Raffa et al., 2005; Waters and Bassler, 2005;
Musk and Hergenrother, 2006; Bjarnsholt and Givskov,
2007; Amer et al., 2008; Labandeira-Rey et al., 2009; Deep
et al., 2011). In agreement with the data provided by the
literature, pyrazole compounds may act as inhibitors that
target this cell–cell signaling mechanism (Tanitame et al.,
2004; Musk and Hergenrother, 2006; Tse-Dinh, 2007;
Schillaci et al., 2008; Brackman et al., 2009; Oancea, 2010).
The number of literature data dealing with regulatory
mechanisms controlling the haemophili biofilm formation
and a possible effect of different chemical compounds on
this process is strongly limited. In our opinion, comparable
activity of the tested compound having the ethyl substituent
against planktonic or biofilm-forming cells of haemophili
rods may be due to the dual activity of pyrazole––main
inhibitory effect against DNA gyrase and additional activity
Table 2 The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-
1H-pyrazole-1-carbothioamide on vero cells line viability
Compound concentration (lg ml-1
)
Cell viability (in %) SD
x
500
37.95 7.7
82.15 5.7
89 6.6
200
100
50
93.55 4.2
97.4 1.7
98.05 1.8
98.75 2.0
100 0.0
100 0.0
25
12.5
6.25
3.15
0 (control)
2003; Donlan, 2002; Prakash et al., 2003; Jain et al., 2007;
Wolcott and Ehrlich, 2008).
The pyrazole derivatives are potent and selective inhibi-
tors against DNA gyrase (Reece and Maxwell, 1991; Tani-
tame et al., 2004; Tse-Dinh, 2007; Farag et al. 2008; Liu
et al., 2008; Shiroya et al., 2011). Considering a possible
mechanism of anti-biofilm activity of N-ethyl-3-amino-5-
oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide, it
should be noted that several classes of chemical compounds,
123

Med Chem Res (2014) 23:1057–1066
1061
associated with the disorder of quorum-sensing phenomenon
and biofilm formation. We did not find existing studies
dealing with effect of the pyrazole compounds on formation
or eradication of biofilms created by H. influenzae and H.
parainfluenzae.
nyl-2,5-dihydro-1H-pyrazole-1-carbothioamide, and N-cyc-
lohexyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-
carbothioamide were tested against H. influenzae and H.
parainfluenzae in form of planktonic or biofilm-forming cells.
Our study shows that the pyrazoles can be inhibitors acting on
planktonic or biofilm-forming cells of Haemophilus spp.
Additionally, these results allow to expect that this compound
will be the starting substance in the search of antimicrobials
with low toxicity, showing inhibitory effect against Gram-
negative haemophili rods and including anti-biofilm activity.
Further investigations should clarify the mechanism of pyr-
azoles against biofilm formed by haemophili rods.
It should be mentioned that Lux-S family of quorum-
sensing regulatory systems involved in production of au-
toinducer 2 (AI-2), occurring in many bacterial species
and functioning as interspecies signaling system, have
been identified in H. influenzae or H. ducrei (Bassler,
1999; Vendeville et al., 2005; Armbruster et al., 2009;
¨
Swords, 2012). Unal et al. (2012) have showed that in
nontypeable H. influenzae, the two-component signaling
system QseB/C was involved in biofilm formation. Daines
et al. (2005) and Armbruster et al. (2009) have observed
the role of LuxS and AI-2 luxS-dependent factors which
control biofilm formation in non-typable H. influenzae,
but they considered as controversial its importance as
virulence factor in pathogenesis of the biofilm-associated
infections.
Materials and methods
N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-
1H-pyrazole-1-carbothioamide derivatives
Three N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-
pyrazole-1-carbothioamide derivatives have been screened for
the antibacterial investigations. Obtained compounds were
characterized and described earlier (Pitucha et al., 2010).
N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-
pyrazole-1-carbothioamide derivatives were prepared
according to Scheme 1. The starting 1-cyanophenylacetic
acid hydrazide was prepared in the reaction of corre-
sponding ethyl 1-cyanophenylacetate with 80 % hydrazine
hydrate at room temperature. Next, this compound was
converted to the 1-(cyanophenylacetyl-4-subtituted)thio-
semicarbazide in the reaction of 1-cyanophenylacetic acid
hydrazide with ethyl or 4-methoxyphenyl isothiocyanate.
Cyclization of these compounds in alkaline or hydrochloric
acid medium led to appropriate N-substituted-3-amino-5-
oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide.
N-cyclohexyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-
pyrazole-1-carbothioamide was obtained in the reaction of
1-cyanophenylacetic acid hydrazide with cyclohexyl iso-
thiocyanate. The reaction was carried out in the diethyl
ether at room temperature without the separation of linear
product.
The change in the structure of the substituent has a
significant impact on the physicochemical properties of the
compound (Hulzebos et al., 2001; Martin et al., 2002). In
our study, we synthesized and tested derivatives differing
from each other by the type of substituents in the thioamide
group. The best results were obtained for the compound
having the ethyl substituent. From the microbiological
point of view, the key factor is the presence of ethyl group
which only slightly increases the mass and the volume of
the compound compared to derivatives with cyclohexyl or
4-metoxyphenyl substituents. Additionally, lower molecu-
lar weight of ethyl derivative can have a significant effect
on the antimicrobial properties of this compound. In our
opinion, replacement of ethyl group on cyclohexyl or
4-metoxyphenyl in the tested pyrazole derivatives causes a
significant decrease of their activity against Haemophilus
spp. Besides, ethyl substituent has a limited conformational
freedom which may affect selectivity (Graham, 2001). This
is very important information from the point of view of the
further modifications of these derivatives and their activity
against either biofilm-forming cells or against mature bio-
film of Haemophilus spp. In addition, further work is
needed to evaluate the role of pyrazole derivatives during
biofilm formation and their influence either on adhesive
capabilities of haemophili rods or on quorum-sensing
phenomenon.
Bacterial strains
The haemophili reference species from American Type
Culture Collection (ATCC)––H. influenzae ATCC 10211, H.
parainfluenzae ATCC 7901, and H. parainfluenzae ATCC
51505 were included. Besides, 20 clinical isolates of H.
parainfluenzae and 11 clinical isolates of H. influenzae from
the museum of Department of Pharmaceutical Microbiology
of Medical University of Lublin were used.
Conclusions
N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-
carbothioamide, N-(4-metoxyphenyl)-3-amino-5-oxo-4-phe-
123

1062
Med Chem Res (2014) 23:1057–1066
O
O
O
C
NH
NH R
S
C
OC2H5
C
NH
R-NCS
NH NH
2
2
HC
NH C
HC
HC
NH2
C H OH
2
5
CN
CN
CN
NaOH, HCl
C H NCS
6
11
H
N
H
N
H2N
H2N
C
NH
S
C
NH
C6H11
R
N
N
C
C
C
C
C
C
S
O
O
R = C H , 4-CH OC H
6 4
2
5
3
Scheme 1 Synthesis and structure of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide
Growth conditions
Growth assay
The Haemophilus chocolate agar (HAEM, bioMerieux)
medium with PolyVitex and hemoglobin or tripticasein soy
broth (TSB) ? Haemophilus test medium supplement
(HTMS)––TSB (Biocorp) medium supplemented with
HTMS (HTMS SRO158E, Oxoid) with growth factors for
haemophili (25 lg ml-1 of NAD and 15 lg ml-1 of hema-
tin) were used. Chocolate agar is blood agar medium that has
been heated to open the pyrrole ring, forming haemin (a
required growth factor for bacteria lacking hemolysins),
providing optimal growth conditions for H. influenzae and
other fastidious bacteria (Rennie et al., 1992; Han et al.,
2006). In clinical microbiology, the TSB medium is used in a
variety of procedures, e.g., for the microbiological test pro-
cedure of culture media according to the standards (NCLSI,
2000, 2004). However, according to our results, TSB sup-
plemented with HTMS is good as a primary enrichment
medium directly inoculated with the various bacteria (Kos-
ikowska and Malm, 2009). The standardized bacterial sus-
pensions with an optical density of 0.5 McFarland
standard––150 9 106 colony-forming units ml-1 in sterile
0.85 % NaCl were prepared. A stock solutions of
N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyra-
zole-1-carbothioamide derivatives at a concentration of
50 mg ml-1 in dimethyl sulfoxide (Sigma) were prepared.
The medium with DMSO at a final concentration and without
the tested compounds served as control––no microbial
growth inhibition was observed.
Preliminary in vitro antibacterial activity of compounds N-
ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-
carbothioamide,
N-(4-metoxyphenyl)-3-amino-5-oxo-4-
phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide, and N-
cyclohexyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyr-
azole-1-carbothioamide was screened by the broth micro-
dilution method using 96-well polystyrene microplates
(NUNC, TC MICROWELL 96F Nunclon D) on the basis
of MIC (minimal inhibitory concentration), usually defined
as the lowest concentration of the compounds at which
there was no visible growth of microorganisms. The anti-
bacterial activity was tested according to EUCAST (2003)
procedure with some modifications.
In order to assay the influence of the tested pyrazole
derivatives on the growth of haemophili rods, 198 ll of
TSB?HTMS medium without (control) and with a series of
twofold dilution of the tested compounds in the range of
final concentration from 1,000 to 62.5 lg ml-1 was inoc-
ulated with 2 ll of the standardized microbial suspension
(total volume per each well ––200 ll), and then incubated
for 18 h at 35 °C in the presence of about 5 % CO2. Then in
order to assay the influence of the tested N-ethyl-3-amino-5-
oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide
with highest inhibitory effect against the planktonic cells of
Haemophilus spp., 198 ll of TSB?HTMS medium without
(control) and with a series of twofold dilution of the tested
compound in the range of final concentration from 0.12 to
31.25 lg ml-1 was inoculated with 2 ll of the standardized
microbial suspension (total volume per each well––200 ll),
and then incubated for 18 h at 35 °C in the presence of
about 5 % CO2. After incubation, spectrophotometric
measurements of optical density at wavelength k = 570
The bacterial cultures in TSB?HTMS medium with
addition of 30 % glycerol were stored at -70 °C. Before
each experiment, bacterial strains were subcultured on
HAEM medium and incubated overnight at 35 °C in about
5 % CO2 atmosphere.
123

Med Chem Res (2014) 23:1057–1066
1063
Cytotoxicity assay
(OD570) of the bacterial cultures with or without the tested
compound were done by using a microplate reader (ELx800
BioTek) in order to determine MIC. The MIC values were
determined by comparison to the growth of a control
(compound-free) medium. Ampicillin was used as a refer-
ence antimicrobial agent on selected H. parainfluenzae
(penicillinase-positive or penicillinase-negative strains)
isolates at the same conditions. The blank control wells
without or with twofold dilution of the tested compounds
added to TSB?HTMS broth without bacterial suspension
were incubated under the same conditions. The experiments
were performed in triplicate.
The vero cell culture from the American Type Culture
Collection (ATCC––84113001) was used in the experi-
ment. The minimum essential medium Eagle (MEM,
Sigma) media were supplemented with 10 % fetal bovine
serum (Sigma), 100 U ml-1 of penicillin, and 0.1 lg ml-1
of streptomycin (Polfa-Tarchomin, Poland). The cell cul-
ture was incubated at 37 °C for 24 h in the 5 % CO2
atmosphere. A stock solution of N-ethyl-3-amino-5-oxo-4-
phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide at
concentration of 50 mg ml-1 was dissolved in DMSO
a
(Sigma). The initial concentration of the examined com-
pound in the MEM medium was 500 lg ml-1
.
100 ll of the vero cell culture prepared was plated into
96-well polystyrene microplates (NUNC) at a cell density
2 9 104 cells per well. After 24 h incubation at 37 °C, the
media were removed and the cells were treated with a solution
ofthetestedcompounddilutedintheMEM mediumincluding
2 % of serum. The following final concentrations were
Biofilm assay
In order to assay the effect of N-ethyl-3-amino-5-oxo-4-
phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide on Hae-
mophilus spp. biofilm formation, the method based on
staining with 0.1 % crystal violet described previously by
Kaplan and Mulks (2005) with some modifications (Kos-
ikowska and Malm, 2009) was used. The activity of the
tested compound against biofilm formation was determined
on the basis of MBIC, defined as the lowest concentration of
the compound at which the biofilm formation was inhibited
applied: 3.15, 6.25, 12.5, 25, 50, 100, 200, and 500 lg ml-1
.
At the same time, the cytotoxicity of solvents was examined.
The control cell culture was supplemented with media
including 2 % of serum only. The cell cultures were incubated
for 48 h at 37 °C in the 5 % CO2 atmosphere. Cytotoxicity of
the tested compound was estimated using the MTT method,
described by Takenouchi and Munekata (1998). The MTT
methodisaquantitativecolorimetrictoxicitytest, basedonthe
transformation of yellow, soluble tetrazolium salts (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to
purple-blue insoluble formazane. This process occurs natu-
rally in mitochondria of living cells. After 48 h incubation
with compounds, cell cultures were supplemented with 10 ll
of 5 mg ml-1 MTT solution per well, and further incubated
for 4 h at 37 °C. Afterwards, 100 ll of water solution,
including 50 % dimethylformamide and 20 % SDS, per well
was added and after the all-night incubation the absorbance
was measured by the 96-well plastic plate reader (Organon
Teknika) at wavelengths of k = 540 and 620 nm. The med-
ium with DMSO at tested concentration range without the
tested compound served as control––it was not toxic to vero
cells line. The experiments were carried out in duplicates.
ˇ
´
(Cernohorska and Votava, 2008). In order to assay the
influence of the tested compound on the biofilm formation by
haemophili rods, 198 ll of TSB?HTMS medium without
(control) and with a series of twofold dilution of the tested
compound in the range of final concentration from 0.12 to
31.25 lg ml-1 was inoculated with 2 ll of the standardized
microbial suspension (total volume per each well––200 ll),
and then incubated at 35 °C in the presence of about 5 %
CO2. After overnight incubation of bacterial culture, the
medium above the culture was decanted and then the plates
were washed extensively several times with distilled water to
remove nonadherent or loosely adherent cells, dried in
inverted position and stained with 200 ll of 0.1 % crystal
violet. The plates were left for 15 min to stain the cells, then
washed extensively under distilled water to remove unbound
dye. Next, in order to elicit a response to each of the wells,
200 ll of isopropyl alcohol (Color Gram 2 R 3-F,
bioMerieux) was added and the plates were left at room
temperature for 15 min to solubilize the dye. The optical
density of the alcohol–dye solution in each well was read at
wave length k = 570 (OD570) by using a microplate reader
(BioTek ELx800). Ampicillin was used as a reference
compound. The blank control wells without or with twofold
dilution of the tested compound added to TSB?HTMS
broth without bacterial suspension were incubated under the
same conditions. The experiments were performed in
triplicate.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
References
Agrawal A, Murphy TF (2011) Haemophilus influenzae infections in
the H. influenzae type b conjugate vaccine era. J Clin Microbiol
49:3728–3732
123

1064
Med Chem Res (2014) 23:1057–1066
Amer FAA, El-Behedy EM, Mohtady HA (2008) New targets for
antibacterial agents. Biol Rev Camb Philos Soc 3:46–57
Armbruster CE, Hong W, Pang B, Dew KE, Juneau RA, Byrd MS,
Love CF, Kock ND, Swords EW (2009) LuxS promotes biofilm
maturation and persistence of nontypeable Haemophilus influ-
enzae in vivo via modulation of lipooligosaccharides on the
bacterial surface. Infect Immun 77:4081–4091
Darras-Joly C, Lortholary O, Mainardi JL, Etienne J, Guillevin L,
Acar J (1997) Haemophilus endocarditis: report of 42 cases in
adults and review. Haemophilus Endocarditis Study Group. Clin
Infect Dis 24:1087–1094
Das M, Badley AD, Cockerill FR, Steckelberg JM, Wilson WR
(1997) Infective endocarditis caused by HACEK microorgan-
isms. Annu Rev Med 48:25–33
Bassler BL (1999) How bacteria talk to each other: regulation of
gene expression by quorum sensing. Curr Opin Microbiol
2:582–587
Bekhit AA, Abdel-Aziem T (2004) Design, synthesis and biological
evaluation of some pyrazole derivatives as anti-inflammatory-
antimicrobial agents. Bioorg Med Chem 12:1935–1945
Deep A, Chaudhary U, Gupta V (2011) Quorum sensing and bacterial
pathogenicity: from molecules to disease. J Lab Physicians 3:4–11
Donlan RM (2001) Biofilm formation: a clinically relevant microbi-
ological process. Clin Infect Dis 33:1387–1392
Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect
Dis 8:881–890
Bjarnsholt T (2013) The role of bacterial biofilms in chronic
infections. APMIS doi: 10.1111/apm.12099
Bjarnsholt T, Givskov M (2007) Quorum-sensing blockade as a
strategy for enhancing host defences against bacterial pathogens.
Philos Trans Royal Soc Lond B 362:1213–1222
Black CT, Kupferschmid JP, West KW, Grosfeld JJ (1988)
Haemophilus parainfluenzae infection in children with the report
of a unique case. Rev Infect Dis 10:342–346
Bottone EJ, Zhang DY (1995) Haemophilus parainfluenzae biliary
tract infection: rationale for an ascending route of infection from
the gastrointestinal tract. J Clin Microbiol 33:3042–3043
Brackman G, Hillaert U, Van Calenbergh S, Nelis H, Coenye T
(2009) Use of quorum sensing inhibitors to interfere with biofilm
formation and development in Burkholderia multivorans and
Burkholderia cenocepacia. Res Microbiol 160:144–151
Burmølle M, Thomsen TR, Fazli M, Dige I, Christensen L, Homøe P,
Tvede M, Nyvad B, Tolker-Nielsen T, Givskov M, Moser C,
Kirketerp-Møller K, Johansen HK, Høiby N, Jensen PØ,
Sørensen SJ, Bjarnsholt T (2010) Biofilms in chronic infec-
tions––a matter of opportunity––monospecies biofilms in mul-
tispecies infections. FEMS Immunol Med Microbiol 59:324–336
Cardines R, Giufre M, Atti ML, Accogli M, Mastrantonio P, Cerquetti
M (2009) Haemophilus parainfluenzae meningitis in an adult
associated with acute otitis media. New Microbiol 2:213–215
Drago L, De Vecchi E, Torretta S, Mattina R, Marchisio P, Pignataro L
(2012) Biofilm formation by bacteria isolatedfrom upperrespiratory
tract before and after adenotonsillectomy. APMIS 120:410–416
Erwin AL, Smith AL (2007) Nontypeable Haemophilus influenzae:
understanding virulence and commensal behavior. Trends
Microbiol 15:355–362
European Committee for Antimicrobial Susceptibility Testing
(EUCAST) of the European Society of Clinical Microbiology
and Infectious Diseases (ESCMID) (2003) Determination of
minimum inhibitory concentrations (MICs) of antibacterial agents
by broth dilution. EUCAST discussion document E.Dis 5.1
Farag AM, Mayhoub AS, Barakat SE, Bayomi AH (2008) Synthesis
of new N-phenylpyrazole derivatives with potent antimicrobial
activity. Bioorg Med Chem 16:4569–4578
Frankard J, Rodriguez-Villalobos H, Struelens MJ, Jacobs F (2004)
Haemophilus parainfluenzae: an underdiagnosed pathogen of
biliary tract infections? Eur J Clin Microbiol Infect Dis 23:46–48
`
Galli J, Calo L, Ardito F, Imperiali M, Bassotti E, Fadda G, Paludetti
G (2007) Biofilm formation by Haemophilus influenzae isolated
from adeno-tonsil tissue samples, and its role in recurrent
adenotonsillitis. Acta Otorhinolaryngol 27:134–138
Gilbert P, Allison DG, McBain AJ (2002) Biofilms in vitro and
in vivo: do singular mechanisms imply cross-resistance. J Appl
Microbiol (Suppl.) 92:98s–110s
ˇ
´
˘
¨
¨
Cernohorska L, Votava M (2008) Antibiotic synergy against biofilm-
forming Pseudomonas aeruginosa. Folia Microbiol 53:57–60
Chauhan PMS, Singh S, Chatterjee RK (1993) Antifilarial profile of
substituted pyrazoles––a new class of antifilarial agents. Indian J
Chem 32B:858–861
Gokhan-Kelekc¸i N, Yabanoglu S, Kupeli E, Salgin U, Ozgen O, Uc¸ar
G, Yes¸ilada E, Kendi E, Yes¸ilada A, Bilgin AA (2007) A new
therapeutic approach in Alzheimer disease: some novel pyrazole
derivatives as dual MAO-B inhibitors and antiinflammatory
analgesics. Bioorg Med Chem 15:5775–5786
Chin CL, Manzel LJ, Lehman EE, Humlicek AL, Shi L, Starner TD,
Denning GM, Murphy TF, Sethi S, Look D (2005) Haemophilus
influenzae from patients with chronic obstructive pulmonary
disease exacerbation induce more inflammation than colonizers.
Am J Respir Crit Care Med 172:85–91
Chow AW, Bushkell LL, Yoshikawa TT, Guze LB (1974) Hae-
mophilus parainfluenzae epiglottitis with meningitis and bacter-
emia in an adult. Am J Med Sci 267:365–368
Coenye T, Nelis HJ (2010) In vitro and in vivo model systems to study
microbial biofilm formation. J Microbiol Methods 83:89–105
Comber RN, Gray RJ, Secrist JA (1991) Acyclic analogues of
pyrazofurin: syntheses and antiviral evaluation. Carbohydr Res
216:441–452
Cooney TG, Harwood BR, Meisner DJ (1981) Haemophilus parain-
fluenzae thoracic empyema. Arch Intern Med 141:940–941
Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a
common cause of persistent infections. Science 284:1318–1322
Costerton JW, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlish G
(2003) The application of biofilm science to the study and control
of chronic bacterial infections. J Clin Invest 112:1466–1477
Daines DA, Bothwell M, Furrer J, Unrath W, Nelson K, Jarisch J,
Melrose N, Greiner L, Apicella M, Smith AL (2005) Haemoph-
ilus influenzae luxS mutants form a biofilm and have increased
virulence. Microb Pathog 39:87–96
Graham LP (2001) An introduction to medical chemistry, 2nd edn
edn. Oxford University Press, Oxford
Hall-Stoodley L, Stoodley P, Kathju S, Høiby N, Moser C, Costerton
JW, Moter A, Bjarnsholt T (2012) Towards diagnostic guidelines
for biofilm-associated infections. FEMS Immunol Med Micro-
biol 65:127–145
Han XY, Hong T, Falsen E (2006) Neisseria bacilliformis sp. nov.
isolated from human infections. J Clin Microbiol 44:474–479
Hastings JW, Greenberg EP (1999) Quorum sensing: the explanation
of a curious phenomenon reveals a common characteristic of
bacteria. J Bacteriol 181:2667–2668
Hentzer M, Givskov M (2003) Pharmacological inhibition of quorum
sensing for the treatment of chronic bacterial infections. J Clin
Invest 112:1300–1307
Hill SL, Mitchell JL, Stockley RA, Wilson R (2000) The role of
Haemophilus parainfluenzae in COPD. Chest 117:293S
Hulzebos EM, Janssen PAH, Maslankiewicz L, Meijerink MCM,
Muller JJA, Pelgrom SMG, Verdam L, Vermeire TG (2001) The
application of structure-activity relationships in human hazard
assessment: a first approach, RIVM Report No 601516008,
Bilthoven, the Netherlands
Jain A, Gupta Y, Agrawal R, Khare P, Jain SK (2007) Biofilms––a
microbial life perspective: a critical review. Crit Rev Ther Drug
Carr Syst 24:393–443
123

Med Chem Res (2014) 23:1057–1066
1065
Kaplan JB, Mulks MH (2005) Biofilm formation is prevalent among
field isolates of Actinobacillus pleuropneumoniae. Vet Microbiol
108:89–94
Kilian M (2007) Haemophilus. In: Murray PR, Baron EJ, Jorgensen
JH, Landry ML, Pfaller MA (eds) Manual of Clinial Microbi-
ology. American Society of Microbiology, Washington DC,
pp 636–648
Kociolek MG (2009) Quorum-sensing inhibitors and biofilms. Anti-
Infect Agents Med Chem 8:315–326
Kosikowska U, Malm A (2009) The preliminary analysis of the
ability of biofilm formation in vitro under stationary conditions
by Haemophilus parainfluenzae isolates from throat of healthy
people. Sepsis 2:203–206
National Committee for Clinical Laboratory Standards (2004) Quality
control for commercially prepared microbiological culture
media; approved standard—third edition. NCCLS document
M22-A3. NCCLS, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898 USA
Oancea S (2010) An overview of conventional and alternative
strategies for developing new antibacterial agents. Acta Chim
Slov 57:630–642
Pillai A, Mitchell JL, Hill SL, Stockley RA (2000) A case of
Haemophilus parainfluenzae pneumonia. Thorax 55:623–624
Pitucha M, Mazur L, Kosikowska U, Pachuta-Stec A, Malm A,
´
Popiołek Ł, Rza˛czynska Z (2010) Synthesis, structure and
antibacterial evaluation of new N-substituted-3-amino-5-oxo-4-
Kumar P, Chandak N, Kaushik P, Sharma C, Kaushik D, Aneja KR,
Sharma PK (2012) Synthesis and biological evaluation of some
pyrazole derivatives as anti-inflammatory–antibacterial agents.
Med Chem Res 21:3396–3405
Labandeira-Rey M, Janowicz DM, Blick RJ, Fortney KR, Zwickl B,
Katz BP, Spinola SM, Hansen EJ (2009) Inactivation of the
Haemophilus ducreyi luxS gene affects the virulence of this
pathogen in human subjects. J Infect Dis 200:409–416
Lasa I, Del Pozo JL, Penades JR, Leiva J (2005) Bacterial biofilms
and infection. An Sist Sanit Navar 28:163–175
Lin R, Chiu G, Yu Y, Connolly PJ, Li S, Lu Y, Adams M, Fuentes-
Pesquera AR, Emanuel SL, Greenberger LM (2007) Design,
synthesis, and evaluation of 3,4-disubstituted pyrazole analogues as
anti-tumor CDK inhibitors. Bioorg Med Chem Lett 17:4557–4561
Liu XH, Cui P, Bao-An S, Bhadury PS, Zhu H-L, Wang S-F (2008)
Synthesis, structure and antibacterial activity of novel 1-(5-
substituted-3-substituted-4,5-dihydropyrazol-1-yl)ethanone
oxime ester derivatives. Bioorg Med Chem 16:4075–4082
Macedo AJ, Abraham WR (2009) Can infectious biofilm be
controlled by blocking bacterial communication? Med Chem
5:517–528
phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamides.
Chem 21:215–221
Heteroat
Prakash B, Veeregowda BM, Krishnappa G (2003) Biofilms: a
survival strategy of bacteria. Curr Sci India 85:1299–1307
Raffa RB, Iannuzzo JR, Levine DR, Saeid KK, Schwartz RC, Sucic
NT, Terleckyj OD, Young JM (2005) Bacterial communication
(quorum sensing) via ligands and receptors: a novel pharmaco-
logic target for the design of antibiotic drugs. J Pharmacol Exp
Ther 312:417–423
Raoult D, Drancourt M, Gallais H, Casanova P (1987) Haemophilus
parainfluenzaemeningitis inan adult with aninheriteddeficiencyof
the seventh component of complement. Arch Intern Med 417:2214
Reece RJ, Maxwell A (1991) DNA gyrase: structure and function.
Crit Rev Biochem Mol 26:335–375
Rele M, Giles M, Daley AJ (2006) Invasive Haemophilus parainflu-
enzae maternal-infant infections: an Australasian perspective and
case report. Aust N Z J Obstet Gynaecol 46:258–260
Rennie R, Gordon T, Yaschuk Y, Tomlin P, Kibsey P, Albritton W
(1992) Laboratory and clinical evaluations of media for the
primary isolation of Haemophilus species. J Clin Microbiol
30:1917–1921
Macfarlane S, Dillon JF (2007) Microbial biofilms in the human
gastrointestinal tract. J Appl Microbiol 102:1187–1196
Mahajan N, Havaldar FH, Fernandes PS (1991) Syntheses and
biological activity of heterocycles derived from 3-methoxy-1-
phenyl-1H-pyrazole-5-carboxylate. J Indian Chem Soc 68:245–
246
Sanclement JA, Webster P, Thomas J, Ramadan HH (2005) Bacterial
biofilms in surgical specimens of patients with chronic rhinos-
inusitis. Laryngoscope 115:578–582
Schillaci D, Maggio B, Raffa D, Daidone G, Cascioferro S, Cusimano
MG, Raimondi MV (2008) 4-Diazopyrazole derivatives as
potential new antibiofilm agents. Chemotherapy 54:456–462
Shiroya U, Poshiya A, Patel A, Parikh A, Patel S (2011) DNA-gyrase:
a potential and emerging target for finding novel anti-bacterial
agents. IJAPR 2:480–492
Martin YC, Kofron JL, Traphagen LM (2002) Do structurally similar
molecules have similar biological activity?
45:4350–4358
J Med Chem
Maxwell A (1997) DNA gyrase as a drug target. Trends Microbiol
5:102–108
Steward PS, Costeron JW (2001) Antibiotic resistance of bacteria in
biofilm. Lancet 358:135–138
Mitchell J, Hill S (2000) Immune response to Haemophilus parain-
fluenzae in patients with chronic obstructive pulmonary disease.
Clin Diag Lung Dis 7:25–30
Stoodley HL, Costerton JW, Stoodley P (2004) Bacterial biofilms:
from the natural environment to infectious diseases. Nat Rev
Microbiol 2:95–108
Moxon ER, Sweetman WA, Deadman ME, Ferguson DJ, Hood DW
(2008) Haemophilus influenzae biofilms: hypothesis or fact?
Trends Microbiol 16:95–100
Murphy TF (2003) Respiratory infections caused by non-typeable
Haemophilus influenzae. Curr Opin Infect Dis 16:129–134
Murphy TF, Brauer AL, Sethi S, Kilian M, Cai X, Lesse AJ (2007)
Haemophilus haemolyticus: a human respiratory tract commensal
to be distinguished from Haemophilus influenzae. J Infect Dis
195:81–89
Musk DJ, Hergenrother PJ (2006) Chemical countermeasures for the
control of bacterial biofilms: effective compounds and promising
targets. Curr Med Chem 13:2163–2177
National Committee for Clinical Laboratory Standards (2000)
Approved standard: M2-A7. Performance standards for antimi-
crobial disk susceptibility tests, 7th ed. National Committee for
Clinical Laboratory Standards, Wayne, Pennsylvania 19087-
1898, USA
Sugiura S, Ohno S, Ohtani O, Izumi K, Kitakimado T, Asai H, Kato K
(1977) Syntheses and antiinflammatory and hypnotic activity of
5-alkoxy-3-(N-substituted carbamoyl)-1-phenylpyrazoles. J Med
Chem 20:80–85
Swords WE (2012) Nontypeable Haemophilus influenzae biofilms:
role in chronic airway infections. Front Cell Infect Microbiol
2:97. doi:10.3389/fcimb.2012.00097
Takenouchi T, Munekata E (1998) Amyloid beta-peptide-induced
inhibition of MTT reduction in PC12h and C1300 neuroblastoma
cells: effect of nitroprusside. Peptides 19:365–372
Tanitame A, Oyamada Y, Ofuji K, Fujimoto M, Suzuki K, Ueda T,
Terauchi H, Kawasaki M, Nagai K, Wachi M, Yamagishi J (2004)
Synthesis and antibacterial activity of novel and potent DNA
gyrase inhibitors with azole ring. Bioorg Med Chem 12:5515–5524
Trollfors B, Brorson JE, Claesson B, Sandberg T (1985) Invasive
infections caused by Haemophilus species other than Haemoph-
ilus influenzae. Infection 13:12–14
123

1066
Med Chem Res (2014) 23:1057–1066
Tse-Dinh YC (2007) Exploring DNA topoisomerases as targets of
novel therapeutic agents in the treatment of infectious diseases.
Infect Disord Drug Targets 7:3–9
Vlastarakos PV, Nikolopoulos TP, Maragoudakis P, Tzagaroulakis A,
Ferekidis E (2007) Biofilms in ear, nose, and throat infections:
how important are they? Laryngoscope 117:668–673
¨
¨
Unal CM, Singh B, Fleury C, Singh K, de Paz LC, Svensater G,
Riesbeck K (2012) QseC controls biofilm formation of non-
typeable Haemophilus influenzae in addition to an AI-2-depen-
dent mechanism. Int J Med Microbiol 302:261–269
Warman ST, Reinitz E, Klein RS (1981) Haemophilus parainfluenzae
septic arthritis in an adult. JAMA 246:868–869
Waters CM, Bassler BL (2005) Quorum sensing: cell-to-cell
communication in bacteria. Annu Rev Cell Dev Biol 21:319–346
Wolcott RD, Ehrlich GD (2008) Biofilms and chronic infections.
JAMA 299:2682–2684
Van Houdt R, Aertsen A, Jansen A, Quintana AL, Michiels CW
(2004) Biofilm formation and cell-to-cell signalling in Gram-
negative bacteria isolated from a food processing environment.
J Appl Microbiol 96:177–184
Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR (2005)
Making ‘sense’ of metabolism: autoinducer-2, LuxS and path-
ogenic bacteria. Nat Rev Microbiol 3:383–396
123