Synthesis and biofilm formation reduction of pyrazole-4-carboxamide derivatives in some Staphylococcus aureus strains

Article abstract of DOI:10.1016/j.ejmech.2016.07.030

The ability of several N-phenyl-1H-pyrazole-4-carboxamide derivatives and other pyrazoles opportunely modified at the positions 3, 4 and 5, to reduce the formation of the biofilm in some Staphylococcus aureus strains (ATCC 29213, ATCC 25923 and ATCC 6538) were investigated. All the tested compounds were able, although to a different extent, to reduce the biofilm formation of the three bacterial strains considered. Among these, the 1-(2,5-dichlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carboxamide 14 resulted as the best inhibitor of biofilm formation showing an IC₅₀ranging from 2.3 to 32 μM, against all the three strains of S. aureus. Compound 14 also shows a good protective effect in vivo by improving the survival of wax moth larva (Galleria mellonella) infected with S. aureus ATCC 29213. These findings indicate that 14d is a potential lead compound for the development of new anti-virulence agents against S. aureus infections.

Full text of DOI:10.1016/j.ejmech.2016.07.030

European Journal of Medicinal Chemistry 123 (2016) 58e68
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biofilm formation reduction of pyrazole-4-carboxamide
derivatives in some Staphylococcus aureus strains
,
,
,
Stella Cascioferro a b, Benedetta Maggio a **, Demetrio Raffa a *, Maria Valeria Raimondi a,
Maria Grazia Cusimano a, Domenico Schillaci a, Barbara Manachini a, Fabiana Plescia a,
Giuseppe Daidone a
a Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Via Archirafi, 32, 90123, Palermo, Italy
b IEMEST, Istituto Euromediterraneo di Scienza e Tecnologia, via Emerico Amari, 123, 90139, Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The ability of several N-phenyl-1H-pyrazole-4-carboxamide derivatives and other pyrazoles opportunely
modified at the positions 3, 4 and 5, to reduce the formation of the biofilm in some Staphylococcus aureus
strains (ATCC 29213, ATCC 25923 and ATCC 6538) were investigated. All the tested compounds were able,
although to a different extent, to reduce the biofilm formation of the three bacterial strains considered.
Among these, the 1-(2,5-dichlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carboxamide 14 resulted as
Received 17 May 2016
Received in revised form
14 July 2016
Accepted 15 July 2016
Available online 18 July 2016
the best inhibitor of biofilm formation showing an IC50 ranging from 2.3 to 32 mM, against all the three
strains of S. aureus. Compound 14 also shows a good protective effect in vivo by improving the survival of
wax moth larva (Galleria mellonella) infected with S. aureus ATCC 29213. These findings indicate that 14d
is a potential lead compound for the development of new anti-virulence agents against S. aureus
infections.
Keywords:
N-phenyl-1H-pyrazole-4-carboxamides
Inhibition of biofilm formation
Anti-virulence
© 2016 Elsevier Masson SAS. All rights reserved.
S. aureus
Wax moth larva model
1. Introduction
The correlation between the biofilm formation and the bacterial
persistence has made necessary the development of new potential
Bacterial biofilm formation is one of the common cause of
persistent infection and represents a major health problem as it
plays an important role in device-related infections such as pros-
thetic valves, catheters, orthopedic devices etc. Furthermore, bio-
films are responsible for persistent infections due to Pseudomonas
aeruginosa, Escherichia coli, Mycobacterium tuberculosis and Strep-
tococcus mutans [1]. Such and other medically relevant pathogens
are responsible for biofilm associated disease, such as cystic fibrosis
lung infection (P. aeruginosa, Burkholderia cepacia), burn wound and
trauma infection (Acinetobacter baumanii), urinary and gastroin-
testinal infection (E. coli), respiratory infection (Bordetella pertussis),
catheters and lung infections (Staphylococcus aureus), nosocomial
sepsis and catheter infection (Staphylococcus epidermidis) and
dental caries (S. mutans) [2], very difficult to be treated by con-
ventional antibiotics [3].
therapeutic approaches which combine the current antibiotic
therapy with drugs targeting biofilm formation [1,4,5].
Biofilms are surface bacterial aggregates encased in a synthe-
sized hydrated matrix, the formation of which require the following
four stages: attachment of cells to a surface, formation of micro
colonies, maturation of the micro colonies into an established
biofilm, and dispersal of the bacteria from the biofilm [6]. Three
principal strategies have been developed to counteract biofilm
formation or target different biofilm developmental stages: inhi-
bition of the adhesion of bacteria to living or non-living surfaces [7],
disruption of biofilm architecture during the maturation process,
inhibition of quorum sensing (QS).
The aryl rhodanine derivatives [8], the cis-2-decenoic acid [9],
and the halogenated furanone derivatives [10] respectively are
examples of these three strategies. Other examples of bacterial
biofilm formation inhibitors, for which the mechanism of action
remain to be elucidated, are the 5-alkylidenethiophen-2(5H)-ones
[11], the pyrazole-1-carbothioamides [12], the 1,3,5-triazine-pyr-
azole conjugates [13], the chromen-pyrazole-pyrimidines [14], and
the 4-diazo-pyrazoles [15,16]. Furthermore, the antibiofilm activity,
through diguanylate cyclase inhibition, for compounds bearing the
* Corresponding author.
** Corresponding author.
E-mail addresses: benedetta.maggio@unipa.it (B. Maggio), demetrio.raffa@
unipa.it (D. Raffa).
http://dx.doi.org/10.1016/j.ejmech.2016.07.030
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
59
N-phenyl-1-carboxamide moiety was described [17].
pathogens [29]. In addition, this animal model is very useful
considering that it is a preliminary and alternative infection model
that generate in vivo data quickly and inexpensively. Three essential
features make it useful for this purpose. First, the microbial viru-
lence is similar in the wax moth larva and mammalians; second, the
wax moth model can be run at the human core body temperature of
37 ꢀC and, finally, an accurate inoculum of the pathogen can be
injected directly into the host's body [29].
Prompted by these observations and in combination with our
researches on pyrazole nucleus [15,16,18e23], we synthesized
pyrazole derivatives of type 1, bearing at the position 4 the N-
phenylcarboxamide moiety, with the aim to evaluate their anti-
biofilm activity (Fig. 1).
Furthermore, to study the influence of substituents on the bio-
film formation inhibitory activity, pyrazoles opportunely modified
at the positions 3, 4 and 5 and, in particular, the ethyl 5-amino-1-
phenyl-1H-pyrazole-4-carboxylate 2, the ethyl 5-amino-1-(pyr-
idin-2-yl)-1H-pyrazole-4-carboxylate 3, the ethyl 5-amino-1-(4-
sulfamoylphenyl)-1H-pyrazole-4-carboxylate 4, the ethyl 5-
amino-1-methyl-1H-pyrazole-4-carboxylate 5, the ethyl 5-amino-
1H-pyrazole-4-carboxylate 6, the ethyl 5-amino-3-methyl-1H-
pyrazole-4-carboxylate 7 and the 3-methyl-N,1-diphenyl-1H-pyr-
azole-5-carboxamides 8a,b (Fig. 2) were considered.
2. Results and discussion
2.1. Chemistry
The synthesis of 1-(2-R1-3-R2-4-R3-5-R4-phenyl)-5-methyl-N-
phenyl-1H-pyrazole-4-carboxamide derivatives 1a-c,e-h was ach-
ieved according to the Scheme 1.
Compounds of type 1 and 8a,b were synthesized, pyrazoles 2-7
have been largely used in the past as starting material in our lab-
oratories and they were available with us [23e27].
A mixture of ethyl acetoacetate 9 and aniline 10 was reacted in a
microwave oven for 5 min at 480 W to obtain a mixture of 3-oxo-N-
phenylbutanamide 11a [30] and 1,3-diphenylurea 11b [31] which
was separated by flash chromatograpy [32]. Compound 11a was
then refluxed with acetic anhydride and triethyl orthoformate for
All derivatives, thus, obtained were tested for both the plank-
tonic growth inhibitory activity and the inhibition of biofilm for-
mation against the following reference bacterial strains: S. aureus
ATCC 6538, S. aureus ATCC 25923 and S. aureus ATCC 29213. Finally,
the antivirulence activity and the toxicity of the most active com-
pound were also evaluated in vivo by the wax moth larva model
(Galleria melonella, Lepidoptera: Pyralidae). Insects are considered
good model to study the effect of different substances and also the
host pathogens interaction [28]. G. melonella is a model widely used
for studies concerning different aspects of host-entomopathogen
interactions but only recently was considered a suitable host for
assessing the in vivo efficacy of antimicrobial agents against human
24
h
to give pure 2-(ethoxymethylene)-3-oxo-N-phenyl-
butanamide 12. Compound 12 was finally treated with the appro-
priate phenylhydrazine 13a-h. As outlined in the Scheme 1, when
compound 12 was treated with the phenylhydrazines 13a-c,e-h,
pyrazoles 1a-c,e-h were isolated as principal products. When
compound 12 was treated with the 2,5-dichlorophenylhydrazine
13d, the only compound isolated was the 2-((2-(2,5-
dichlorophenyl)hydrazinyl)methylene)-3-oxo-N-phenyl-
butanamide 14d (Scheme1) [33].
Compound 1d was finally obtained by fusion of 14d at 170 ꢀC for
15 min (Scheme 2).
The 3-methyl-N,1-diphenyl-1H-pyrazole-5-carboxamides 8a,b
were synthesized as shown in Scheme 3.
The intermediate ethyl acetopyruvate 17 was obtained in good
yield by adding, in an ice bath, stirred ethanolic solution of sodium,
a solution of acetone 15 and diethyl oxalate 16. Treatment of 17
with phenyl hydrazine in acetic acid under magnetic stirring for
24 h yield a 1:1.5 mixture of the two isomers ethyl 3-methyl-1-
phenyl-1H-pyrazole-5-carboxylate 18a and ethyl 5-methyl-1-
phenyl-1H-pyrazole-3-carboxylate 18b which were separated by
flash chromatography [32]. Compounds 18a,b are known [34] and
this allow us to differentiate the two isomers by means of melting
point, IR and 1H NMR. Compounds 18a,b were then hydrolysed
with an alcoholic solution of potassium hydroxide to give the cor-
responding acids 19a,b and finally treated with thionyl chloride to
give 20a,b. Condensation of aniline with 20a,b in chloroform under
reflux for 6 h afforded the expected 3-methyl-N,1-diphenyl-1H-
pyrazole-5-carboxamides 8a,b.
The structures of the new compounds were determined by
analytical and spectroscopic measurements. In particular, all de-
rivatives 1a-h and 8a,b showed signals attributable to aromatic
protons in the range 6.93e8.44
7.98e9.26 , exchangeable with D2O, for the amidic NH. Derivatives
1a-h and 8a,b showed also a singlet in the range 2.40e2.73
d and a singlet in the range
d
d
attributable to the 5-methyl or 3-methyl pyrazole hydrogens.
1H NMR spectrum of compound 14d showed how, in solution
(DMSO), two tautomeric forms are present (Fig. 3).
Tautomer A showed two singlets at 11.84 and 8.82
doublet at 11.32, exchangeable with D2O, for NHs. The split as
doublet at 11.32 of NH is due to the coupling with the adja-
cent¼CH (doublet at 8.23 ). After exchange with D2O the signal at
11.32 disappears and the doublet at 8.23 collapse into a single
signal. Tautomer B instead, showed three singlets at 16.47 , 11.15
and 9.89 , exchangeable with D2O, for NHs and OH, as well as a
d
d and a
d
d
d
d
d
Fig. 1. Structure of pyrazoles of type 1.
d
d

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S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
Fig. 2. Structures of pyrazoles 2-7 and 8a,b.
Scheme 1. Synthetic pathway for the formation of 1-(2-R1-3-R2-4-R3-5-R4-phenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carboxamide derivatives 1a-c,e-h and 14d.

S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
61
singlet at 8.59
showed a singlet, attributable to the methyl group, one at 2.37
(tautomers A), the other 2.02 (tautomer B); by these last signals,
an approximate percentage of the two form was calculated
(tautomer A ~86% and tautomer B ~14%).
d
attributable to ¼CH. Finally, both tautomers
1a-h, 8a,b and 14d on biofilm formation inhibition, their inhibitory
activity on the planktonic growth of above mentioned strains was
evaluated by determining the minimum inhibitory concentration
(MIC). This preliminary assay is essential to determine if any
possible observed effects caused by the compounds on bacterial
biofilm formation are specific to biofilm formation or could be
related to a possible decline in the population of bacteria due to
biocidal activity.
d
d
2.2. Biology
None of the tested compounds caused any significant planktonic
growth inhibition at 250 mM. In view of the results obtained for
The compounds 1a-h, 8a,b and 14d were tested for their ability
to interfere with the biofilm formation in S. aureus ATCC 6538,
S. aureus ATCC 25913 and S. aureus ATCC 29223 bacterial strains, the
results are shown in Table 1.
planktonic growth inhibition, we can assume that the effect of the
compounds on biofilm inhibition is not related to their biocidal
activity. Moreover, drugs could target the early steps of bacterial
adhesion, essential for the establishment of infections and coloni-
zation, without affecting the bacterial growth, imposing a low se-
lection pressure and thus, avoiding development of resistance.
The data reported in Table 1 showed that all the tested com-
pounds (1a-h, 8a,b and 14d) were able, although to a different
extent, to reduce the biofilm formation of the three bacterial strains
considered. Among these, compounds 1c,e,h and 14d showed the
best activity against all the three strains of S. aureus with 1c and 14d
being the most active. In general, the ability to reduce the staphy-
lococcal biofilm formation of our compounds is in the range
Previously to the investigation of the effect of the compounds
Table 1
Ability of 1a-h, 8a,b and 14 to reduce the biofilm formation of S. aureus 6538,
S. aureus 25913 and S. aureus 29223 bacterial strains.
Compound
IC50
(mM)
S. a. 6538
S. a. 25923
S. a. 29213
2
3
4
5
6
7
1a
1b
1c
1d
1e
1f
1g
1h
8a
8b
14d
95
91
142
ns
85
82
77
103
30
ns
90
55
ns
81
54
64
32
2
2
222
102
77
159
213
378
112
161
552
92
64
434
64
61
74
2
3
34
31
28
152
83
1
2
1
2
2
2
2.3e434
of biofilm formation reported in the literature such as analogs of
rubolides: 1.3e12.4 M (0.6e5.7 g/mL) [35], analogs of oroidin:
20e90 [36], pyrimido chromen-2-ones: 4.13e194
(2.34e75 g/mL) [14] and triazine-pyrazole coniugates:
23.39e190 M (15.62e125 g/mL) [13].
mM in agreement with the activity of numerous inhibitors
3
2
4
3
2
1
2
2
1
2
1
3
m
m
62
309
169
38
52
46
127
46
89
123
54
2.5 0.2
4
3
m
M
mM
m
1
2
2
1
m
m
3
1
In particular, data of Table 1 revealed how the best activity was
showed by the open structure of the intermediate 14d respect to N-
phenylpyrazoles 1a-h and 8a,b. This is in agreement with the re-
sults obtained by us for new series of phenylhydrazinylidene de-
rivatives of type 21-23, [37,38] structurally related to 14d (Fig. 4).
The activity in compounds 21-23 is due to the presence of a phe-
nylhydrazinylidene moiety which is related to the phenyl-
hydrazinylmethylene group of 14d.
1
1
1
1
2
2
4
1
2
2
1
1
1
4
1
76
2.3 0.1
2
Values are the mean of at least three independent determinations. ns ¼ not
significant.
Scheme 2. Synthetic pathway to obtain the 1-(2,5-dichlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carboxamide 1d.

62
S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
Scheme 3. Synthetic pathway to obtain the 3-methyl-N,1-diphenyl-1H-pyrazole-5-carboxamides 8a,b.
Fig. 3. Tautomeric forms of 14d and differences in 1H NMR signals.

S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
63
Fig. 4. Antibiofilm activity (IC50, mM) of 14d and related compounds 21-23 [37].
A comparation of the antibiofilm activities of these compounds
show the good influence of the phenylhydrazinylmethylene group
which generally leads to a more active compound (14d) respect to
compounds 21-23 [38], especially against S. aureus 25913 and
S. aureus 29223 bacterial strains.
untreated larvae. Significant statistical differences were recorded in
the infected larvae compared with all the other groups. In addition,
statistical differences were recorded also between the two infected
groups with S. aureus highlighting that the compound 14d had a
protective role for the larvae.
On the basis of such results, the antivirulence activity of the
most active compound 14d was also evaluated in vivo by the wax
moth larva model (G. mellonella).
As it can be seen from Fig. 5, the survival after 24 h of infected
G. mellonella larvae was increased by a 1.6 factor when compound
14d was injected 1 h before the inoculation of bacteria thus
proving a good protective effect and the reduction of the bacterial
virulence exerted by the tested compound. The low toxicity of
compound 14d was also demonstrated considering that there is a
slightly lower survival percentage difference between uninfected
larvae and larvae treated with 14d. No sub-lethal effects were
recorded for the larvae injected with the compound 14d. In fact, all
larvae that were kept in starvation, lost weight and no statistical
The survival of wax moth larvae were evaluated on five groups
of larvae: untreated larvae; uninfected larvae treated with the
solvent used to dissolve the tested compound (PBS and 3% DMSO);
uninfected larvae treated with the solvents used to dissolve the
tested compound (PBS and 3% DMSO) plus the compound 14d;
larvae pre-treated with the solvent (PBS and 3% DMSO) 1 h before
the injection of live bacteria; and larvae pre-treated with 1 mg/kg of
14d 1 h before the inoculum of live bacteria.
differences were recorded among the groups (df
p ¼ 0.7119). In addition, the time to the pupation and formation of
¼
3,68,
Fig. 5 shows the effect of the compound 14d on the survival of
the larvae of G. melonella. No mortality was detected in the
the chrysalis was in average 7 days ( 1 day) and no statistical

64
S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
Fig. 5. Effect of pre-treatment with a single dose (1 mg/kg) of compound 14d on survival of Galleria mellonella larvae at 37 ꢀC infected with S.aureus ATCC 29213 Survival was
checked at 24, 48 and 72 h (h) after injection. Results are given as mean (n ¼ 3 replicates) with standard deviation. Data entries with different letter are statistically different at post
hoc Tukey's t-test for multiple comparisons within each character (P < 0.05).
differences were recorded among the three groups that did not
receive the bacterial inoculum.
4. Experimental section
4.1. Chemistry
3. Conclusion
4.1.1. General
Reaction progress was monitored by TLC on silica gel plates
(Merck 60, F254, 0.2 mm). Evaporation refers to the removal of
solvent on a rotary evaporator under reduced pressure. All melting
points were determined on a Büchi 530 capillary melting point
apparatus and are uncorrected. IR spectra were recorded with a
Perkin Elmer Spectrum RXI FT-IR System spectrophotometer, with
compound as a solid in a KBr disc. 1H and 13C NMR spectra (300 and
75.43 MHz respectively) were obtained using a Bruker AC-E
300 MHz spectrometer (tetramethylsilane as internal standard):
Based on the literature on anti-biofilm activity possessed by
compounds in which the core is a pyrazole nucleus, the pyrazolo
derivatives 2-7, 1a-c,e-h, 14d and 8a,b were synthesized and eval-
uated for their ability to inhibit the biofilm formation of three
strains of S. aureus. The synthesized compounds resulted in active
interference with biofilm formation of all the tested bacterial
strains. The most active compound in preventing biofilm formation
of the three bacterial strains considered, was not a pyrazole de-
rivative
but
the
intermediate
3-oxo-N-phenyl-2-((2-
chemical shifts are expressed in
d values (ppm). Merck silica gel
phenylhydrazinyl)methylene) butanamide 14d. In the preliminary
in vivo experiment, we observed that compound 14d, also showed
an interesting protective effect at 1 mg/kg increasing the survival of
wax moth larvae infected with S.aureus ATCC 29231 strain. More-
over, compound 14d had no detrimental effect on larvae as no
differences were recorded in the variation of weight or in the time
pass from the later larva instar to chrysalis. In this study, we
demonstrated that the compound 14d did not affect the bacterial
growth but targeted the virulence of the tested pathogen inhibiting
the biofilm formation in vitro and improving the survival of infected
larvae when administered prior the infection. These observation
are very encouraging. On the basis of results obtained, compound
14d can be considered a good candidate as lead, useful for further
developments as antivirulence agent.
(Kiesegel 60/230-400 mesh) was used for flash chromatography
columns.
Exact mass measurements were carried out by a high resolution
Orbitrap mass spectrometer (Q-Exactive by Thermo Fisher Scien-
tific) via direct infusion through an electrospray interface (HESI)
operating in negative ionization mode using the following opera-
tion parameters: flow rate 5
m
L/min; electrospray voltage ꢁ3.1 KV;
capillary temperature: 320 ꢀC; sheath gas: 6 (arbitrary units); res-
olution 35000.
Microanalyses data (C, H, N) were obtained by an Elemental
Vario EL III apparatus and were within 0.4% of the theoretical
values. Yields refer to purified products and are not optimized. The
names of the products were obtained using the ACD/I-Lab Web

S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
65
service (ACD/IUPAC Name Free 8.05).
1-(4-chlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carbox
amide (1c): Compound 1c was known (CAS number 1134899-50-5),
but no references were found. Yields: 68%, mp 190e194 ꢀC, I.R (KBr)
4.1.2. Synthesis of the 3-oxo-N-phenylbutanamide 11a
cmꢁ1 3269, (NH), 1640 (CO); 1H NMR (Acetone)
d
2.64 (s, 3H, CH3);
7.08e8.20 (a set of signals, 10H, aromatic protons, pyrazole H-3);
9.18 (s, 1H, NH; exchangeable with D2O). 13C NMR(
) (Acetone)
An equimolar mixture (38 mmol) of ethyl acetoacetate 9 and
aniline 10 was irradiated at 480 W for 5 min in a Anton Paar
Multiwave 3000 microwave oven. After this time the reaction
mixture was concentrated under vacuum to give a white solid
which was washed with diethyl ether then chromatographed by
flash chromatography [32]: external diameter of the column
4.5 cm, silica gel (0.040e0.063 mm, 600 g), ethyl acetate/cyclo-
exane 6:4 as eluent; fractions 50 ml each.
The initial twenty fractions were discarded, fractions 21-32
were evaporated under reduced pressure to give pure 1,3-
diphenylurea 11b (1.81 g), and fractions 33-56 were evaporated
under reduced pressure to give pure 3-oxo-N-phenylbutanamide
11a (yields 30%). Spectroscopic and physical data of 11a was iden-
tical (mp, IR, 1H NMR) with the reported data [30].
d
12.22 (CH3), 117.17 (CAr), 120.81 (2XCHAr), 124.21 (CHAr), 127.86
(2XCHAr), 129.47 (2XCHAr), 130.19 (2XCHAr), 134.49 (CAr), 139.02
(CAr), 139.86 (CHAr), 140.40 (CAr), 143.47 (CAr), 162.47 (CO). Anal.
Calc. for C17H14ClN3O: C, 65.49%; H, 4.53%; N, 13.48%.Found: C,
65.66%; H, 4.65%; N, 13.37.
5-methyl-1-(3-nitrophenyl)-N-phenyl-1H-pyrazole-4-carboss
amide (1e): yields 51%, mp 184e187ꢀ, I.R (KBr) cmꢁ1 3418, (NH),
1661 (CO); 1H NMR (Acetone)
d 2.73 (s, 3H, CH3); 7.07e8.44 (a set of
signals, 10H, aromatic protons, pyrazole H-3); 9.26 (s, 1H, NH;
exchangeable with D2O). Anal. Calc. for C17H14N4O3: C, 63.35%; H,
4.38%; N, 17.38%. Found: C, 63.49%; H, 4.12%; N, 17.20.
5-methyl-N-phenyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazo
le-4-carboxamide (1g): yields 68%, mp 155e159ꢀ, I.R (KBr) cmꢁ1
4.1.3. Synthesis of the 2-(ethoxymethylene)-3-oxo-N-
phenylbutanamide 12
3304, (br, NH), 1630 (CO); 1H NMR (Acetone)
d 2.71 (s, 3H, CH3);
7.06e8.25 (a set of signals, 10H, aromatic protons, pyrazole H-3);
9.23 (s, 1H, NH; exchangeable with D2O). Anal. Calc. for
A mixture of 3-oxo-N-phenylbutanamide 11 (5.64 mmol),
triethyl orthoformate (7 mmol) and acetic anhydride (16.92 mmol)
was placed under reflux for 90 min. The reaction mixture was then
evaporated in a rotavapor and, after cooling, by addition of a few
drops of ethanol, the 2-(ethoxymethylene)-3-oxo-N-phenyl-
butanamide 12 precipitates as brown solid. The solid was finally
purified by crystallization with hexane/toluene (1:1). Derivative 12
is known but no characterization was found.
C18H14F3N3O: C, 62.61%; H, 4.09%; N, 12.17%. Found: C, 62.73%; H,
3.92%; N, 12.00.
1-(2,5-difluorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-
carboxamide (1h): yields 50%, mp 115e117ꢀ, I.R (KBr) cmꢁ1 3307,
(NH),1667 (CO); 1H NMR (CDCl3)
d 2.53 (s, 3H, CH3); 7.14e7.65 (a set
of signals, 9H, aromatic protons, pyrazole H-3); 7.98 (s, 1H, NH;
exchangeable with D2O). Anal. Calc. for C17H13F2N3O: C, 65.17%; H,
4.18%; N, 13.41%. Found: C, 65.31%; H, 4.37%; N, 13.14.
2-(ethoxymethylene)-3-oxo-Nꢁ-p1henylbutanamide (12): yields
30%, mp 87e88 ꢀC, I.R (KBr) cm 3071 (NH), 1956 (C]C), 1618
(CO); 1H NMR (DMSO)
d 1.47 (t, 3H, CH3); 2.51 (s, 3H, CH3); 4.38 (q,
2H, CH2); 7.06e7.63 (a set of signals, 5H, aromatic protons); 8.47 (s,
1H, CH); 11.04 (s, 1H, NH; exchangeable with D2O).
4.1.5. Preparation of 2-((2-(2,5-dichlorophenyl)hydrazinyl)
methylene)-3-oxo-N-phenylbutanamide 14d
Derivative 14d was obtained by placing at reflux,1.71 mmol of 2-
(ethoxymethylene)-3-oxo-N-phenylbutanamide 12 in 8.5 mL of
4.1.4. General procedure for preparation of 1-(2-R1-3-R2-4-R3-5-
R4-phenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carboxamides 1a-
c,e-h
Derivatives 1a-c,e-h were obtained by placing at reflux
1.71 mmol of 2-(ethoxymethylene)-3-oxo-N-phenylbutanamide 12
in 8.5 mL of anhydrous ethanol with 1.71 mmol of the appropriate
phenylhydrazine 13a-c,e-h for a variable time of 1e48 h as reported
in Table 2. After this time, The solid which separated off were
collected and purificated as reported in Table 2 to give pure 1b, 1c,
1e, 1g and 1h.
anhydrous
ethanol
with
1.71
mmol
of
the
2,5-
dichlorophenylhydrazine 13d for 24 h. After this time, the solvent
was removed under reduced pressure and the compound 14d, are
thus obtained and purified by crystallization from ethanol; deriv-
ative 14d is known [33] but no characterization was found. 1H NMR
spectrum showed that compound 14d is a mixture of two tauto-
mers (Fig. 3). Owing to the prevalence of tautomer A in the mixture,
the intensity of signals relative to tautomer B were negligible in 13
NMR spectrum.
C
Derivatives 1a and 1f are known [39] and their spectroscopic
and physical data were identical with the reported data.
1-(3-chlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-car
boxamide (1b): yields 23%, mp 97e100ꢀ, I.R (KBr) cmꢁ1 3307, (NH),
2-((2-(2,5-Dichlorophenyl)hydrazinyl)methylene)-3-oxo-N-
phenylbutanamide (14d): yields 79%, mp 145e147 ꢀC, I.R (KBr)
cmꢁ1 3322, 3198-3045 (NH), 1658-1563 (CO); 1H NMR (tautomeric
mixture, Acetone) d: tautomer A 2.33 (s, 3H, CH3); 6.83e7.63 (a set
of signals, 8H, aromatic protons); 8.23 (d, 1H, ¼CH); 8.82 (s, 1H, NH;
exchangeable with D2O), 11.32 (d, 1H, NH; exchangeable with D2O),
11.84 (s, 1H, NH; exchangeable with D2O); tautomer B 2.28 (s, 3H,
CH3); 6.83e7.63 (a set of signals, 8H, aromatic protons); 8.59 (s,
1667 (CO); 1H NMR (Acetone)
d
2.67 (s, 3H, CH3); 7.06e8.21 (a set of
signals, 10H, aromatic protons, pyrazole H-3); 9.20 (s, 1H, NH;
exchangeable with D2O). Anal. Calc. for C17H14ClN3O: C, 65.49%; H,
4.53%; N, 13.48%. Found: C, 65.50%; H, 4.51%; N, 13.51.
Table 2
Time of reaction and purification methods for 1a-c,e-h.
Comp.
Time of reaction
Purification method
1a
1b
1c
1e
1f
6 h
24 h
3 h
48 h
24 h
1 h
Crystallized from ethanol
Crystallized first from ethyl acetate then from ethanol
Crystallized from ethanol
Crystallized first from ethanol then purified by preparative TLC(chloroform as eluent)
Crystallized from ethyl acetate
Crystallized first from ethyl acetate/petroleum ether then from ethanol
Washed with ethyl acetate
1g
1h
24 h

66
S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
1H, ¼CH); 9.89 (s,1H, NH; exchangeable with D2O),11.55 (s,1H, NH;
this time the ethanol was removed under reduced pressure, and the
residue was dissolved in water (3.3 m). The solution was acidified
with HCl, filtered and dried to give acids 19a,b. Spectroscopic and
physical data of 19a and 19b were identical with the reported data
for 3-methyl-1-phenyl-1H-pyrazole-5-carboxylic acid [41], and
ethyl 5-methyl-1-phenyl-1H-pyrazole-3-carboxylic acid [34].
exchangeable with D2O), 16.46 (s, 1H, OH; exchangeable with D2O).
13C NMR(
d) (Acetone) tautomer A 26.70 (CH3), 103.05 (CAr), 114.16
(CHAr), 117.65 (CAr), 120.61 (2XCHAr), 121.87 (CHAr), 124.25 (CHAr),
129.70 (2XCHAr), 131.68 (CHAr), 134.04 (CAr), 139.51 (CAr), 146.10
(CAr), 164.62 (CHAr), 167.55 (CO), 197.74 (CO). Anal. Calc. for
C
17H15Cl2N3O2: C, 56.06%; H, 4.15%; N, 11.54%. Found: C, 56.36%; H,
3.99%; N, 11.26. HR-MS (TOF, HESI): m/z calc. for C17H15Cl2N3O2: [M]
363.0514; found [Mþ1] 362.0468.
4.1.10. Synthesis of 3-methyl-1-phenyl-1H-pyrazole-5-carbonyl
chloride (20a) and 5-methyl-1-phenyl-1H-pyrazole-3-carbonyl
chloride (20b)
4.1.6. Preparation of 1-(2,5-dichlorophenyl)-5-methyl-N-phenyl-
1H-pyrazole-4-carboxamide (1d)
The 2-((2-(2,5-Dichlorophenyl)hydrazinyl)methylene)-3-oxo-
N-phenylbutanamide 14d (mg 750, 2 mmol) was fused a 170 ꢀC for
15 min. After cooling, the solid which was obtained was crystallized
from ethanol to give pure 1d.
Compounds 19a,b were refluxed for 5 h with 15 mL of thionyl
chloride. After this time, the mixture was evaporated under
reduced pressure to give the appropriate chlorides 20a,b that were
used crude for the next reaction.
4.1.11. Synthesis of 3-methyl-N,1-diphenyl-1H-pyrazole-5-
carboxamide (8a) and 5-methyl-N,1-diphenyl-1H-pyrazole-3-
carboxamide (8b)
1-(2,5-dichlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-
carboxamide (1d): yields 83%, mp 145e147ꢀ, I.R (KBr) cmꢁ1 3063,
(NH), 1645 (CO); 1H NMR (Acetone)
d
2.47 (s, 3H, CH3); 7.09e8.42 (a
The crude liquid residue of 20a,b coming from the previous
reaction was dissolved in anhydrous chloroform (135 mL) and
refluxed for 6 h with 0.0296 mol of aniline. After the first hour of
reflux, five portion of triethylamine, 2 mL, 1 mL, 0.5 mL, 0.5 mL and
0.3 mL each, were added at intervals of 1 h. The mixture was
evaporated under reduced pressure and the obtained residue was
washed three times with water (50 mL). Then 10 mL of ethanol was
added and the solution was finally evaporated to dryness. The
residue was then crystallized first twice with ethyl acetate and then
with ethanol to give pure the 3-methyl-N,1-diphenyl-1H-pyrazole-
5-carboxamide 8a and 3-methyl-N,1-diphenyl-1H-pyrazole-5-
carboxamide 8b.
set of signals, 9H, aromatic protons and pyrazole H-3); 9.23 (s, 1H,
NH; exchangeable with D2O). Anal. Calc. for C17H13Cl2N3O: C,
58.98%; H, 3.78%; N, 12.14%. Found: C, 59.06%; H, 3.56%; N, 12.34.
4.1.7. Synthesis of the ethyl acetopyruvate (17) [40]
To an ice bath cooled solution of sodium (0.217 mol) in anhy-
drous ethanol(125 mL), a solution of acetone 15 (200 mmol) and
diethyl oxalate 16 (200 mmol) was slowly added. The reaction
mixture was allowed to stir for 4 h keeping the temperature below
10 ꢀC. Subsequently the mixture was filtered, dissolved in cold
water and acidified to pH 3 with sulfuric acid (20%). After the
acidification the solution was extracted three times with
dichloromethane (each 50 mL), the extracts collected and evapo-
rated under reduced pressure to give the ethyl acetopyruvate 17
(yields 60%).
3-Methyl-N,1-diphenyl-1H-pyrazole-5-carboxamide(8a):yields
95%, mp 128e1131ꢀ, I.R (KBr) cmꢁ1 3381, (NH), 1676 (CO); 1H NMR
(CDCl3)
of signals,10H, aromatic protons); 7.62 (s,1H, NH; exchangeable with
D2O). 13C NMR(
) (CDCl3) 13.43 (CH3), 109.21 (CHAr), 120.02 (CHAr),
d 2.34 (s, 3H, CH3); 6.62 (s,1H, pyrazole H-4); 7.12e7.45 (a set
d
4.1.8. Synthesis of the ethyl 3-methyl-1-phenyl-1H-pyrazole-5-
carboxylate 18a and ethyl 5-methyl-1-phenyl-1H-pyrazole-3-
carboxylate 18b
124.88 (2XCHAr),125.08(CHAr),128.42 (CHAr),129.07 (3XCHAr),137.16
(CHAr), 137.56 (CAr), 139.68 (CAr), 149.13 (CAr), 157.63 (CO). Anal. Calc.
for C17H15N3O: C, 73.63%; H, 5.45%; N, 15.15%. Found: C, 73.36%; H,
5.69%; N, 15.29.
An ice bath cooled solution of 63 mmol ethyl acetopyruvate 17
in 23 mL of acetic acid was treated under magnetic stirring with
63 mmol of phenylhydrazine. The solution was put under reflux for
24 h. After this time, the solution was added of water (69 mL) and
extracted four times with diethyl ether (each 30 mL). The ethereal
fractions were collected, washed four times with a 10% solution of
sodium bicarbonate (each 12 mL), dried with anhydrous sodium
sulphate then concentrated under reduced pressure to obtaining a
mixture of ethyl 3-methyl-1-phenyl-1H-pyrazole-5-carboxylate
18a and ethyl 5-methyl-1-phenyl-1H-pyrazole-3-carboxylate 18b
as oil (7.70 g) which was separated by flash chromatography [32]:
external diameter of the column 5.0 cm, silica gel
(0.040e0.063 mm, 137 g), ethyl acetate/cycloexane 3:7 as eluent;
fractions 55 ml each. The initial four fractions were discarded,
fractions 5-7 were evaporated under reduced pressure to give pure
ethyl 3-methyl-1-phenyl-1H-pyrazole-5-carboxylate 18a (1.81 g),
fraction 8 contain a mixture of 18a and 18b (0.5 g), and finally
fractions 9-13 were evaporated under reduced pressure to give
pure ethyl 5-methyl-1-phenyl-1H-pyrazole-3-carboxylate 18b
(2.71 g). Spectroscopic and physical data of 18a and 18b were
identical with the reported data [34].
5-Methyl-N,1-diphenyl-1H-pyrazole-3-carboxamide(8b):yields
98%, mp 105e106ꢀ, I.R (KBr) cmꢁ1 3381, (NH), 1689 (CO); 1H NMR
(CDCl3)
protons, pyrazole H-4); 8.77 (s, 1H, NH; exchangeable with D2O). 13
NMR( ) (CDCl3) 12.48 (CH3), 107.59 (CHAr), 119.67 (2XCHAr), 123.96
d 2.73 (s, 3H, CH3); 6.83e7.71 (a set of signals, 11H, aromatic
C
d
(CHAr), 125.24 (2XCHAr), 128.68 (CHAr), 128.99 (2XCHAr), 129.35
(2XCHAr), 137.94 (CAr), 139.10 (CAr), 141.38 (CAr), 146.62 (CAr), 159.22
(CO). Anal. Calc. for C17H15N3O: C, 73.63%; H, 5.45%; N,15.15%. Found:
C, 73.96%; H, 5.80%; N, 15.47.
4.2. Biology
4.2.1. Microbial strains
The staphylococcal reference strains used were: Staphylococcus
aureus ATCC 6538, Staphylococcus aureus ATCC 25923 and Staphy-
lococcus aureus ATCC 29213, known for its ability to form a biofilm.
4.2.2 Antibacterial activity
MICs were determined by a micro-method described previously
[42].
4.1.9. Synthesis of 3-methyl-1-phenyl-1H-pyrazole-5-carboxylic
acid (19a) and 5-methyl-1-phenyl-1H-pyrazole-3- carboxylic acid
(19b)
A solution of 18a,b (0.0169 mol) in 6.50 mL of ethanol and 1.5 g
of KOH in 1.1 mL of water were put under reflux for 30 min. After
4.2.3. Evaluation of biofilm formation
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 to a suspension with optical density

S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
67
(OD) of about 0.040 at 570 nm corresponding to ~106 CFU/mL.
Polystyrene 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 2 mL of sterile phosphate-buffered
saline (PBS) and stained with 2 mL of crystal violet 0.1% v/v for
30 min. The excess stain was removed by placing the plates under
running tap water.
analysis, collected datasets were compared using analysis of vari-
ance (ANOVA) with homogeneity of variances performed using
Cochran's test prior to ANOVA analysis. The statistical significances
of the parameter analysed 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 (StatSoft, Tulsa, OK,
USA).
Crystal-violet stained adherent bacteria in each well were re-
dissolved to homogeneity in 2 mL of ethanol, and the optical
density (OD) was read at 600 nm. Each assay was performed in
triplicate and repeated at least twice.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2016.07.
030. These data include MOL files and InChiKeys of the most
important compounds described in this article.
4.2.4. Biofilm prevention assay
Procedure described above was used to evaluate the activity of
compounds in preventing biofilm formation. Polystyrene 24-well
tissue culture plates were filled with 2 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
References
[1] L. Chen, Y. Wen, The role of bacterial biofilm in persistent infections and
control strategies, Int. J. Oral Sci. 3 (2011) 66e73, http://dx.doi.org/10.4248/
IJOS11022.
[2] M. Majik, P. Parvatkar, Next generation biofilm inhibitors for Pseudomonas
aeruginosa: synthesis and rational design approaches, Curr. Top. Med. Chem.
14 (2013) 81e109, http://dx.doi.org/10.2174/1568026613666131113152257.
[3] D. Schillaci, The re-discovering of old molecules to face the antibiotic crisis,
0.3
mM of each compound were directly added to the bacterial
suspension at time zero and incubated at 37 ꢀC for 24 h. After that
time the wells were washed and stained with crystal-violet as seen
in biofilm formation assay.
By comparing the average OD 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:
J. Microb. Biochem. Technol.
5948.1000e121.
[4] S. Cascioferro, M. Totsika, D. Schillaci, Sortase A: an ideal target for anti-
virulence drug development, Microb. Pathog. 77C (2014) 105e112, http://
dx.doi.org/10.1016/j.micpath.2014.10.007.
[5] S. Cascioferro, The future of antibiotic: from the magic bullet to the smart
bullet, J. Microb. Biochem. Technol. 6 (2014), http://dx.doi.org/10.4172/1948-
5948.1000e118.
[6] P.Y. Chung, Y.S. Toh, Anti-biofilm agents: recent breakthrough against multi-
drug resistant Staphylococcus aureus, Pathog. Dis. 70 (2014) 231e239, http://
dx.doi.org/10.1111/2049-632X.12141.
7 (2015), http://dx.doi.org/10.4172/1948-
OD600 growth control ꢁ OD600 sample
Inhibition ð%Þ ¼
ꢂ 100
(1)
OD600 growth control
[7] S. Cascioferro, M.G. Cusimano, D. Schillaci, Antiadhesion agents against Gram-
positive pathogens, Future Microbiol. 9 (2014) 1209e1220, http://dx.doi.org/
10.2217/fmb.14.56.
Each assay was performed in triplicate and assays were repeated
at least twice.
[8] T.J. Opperman, S.M. Kwasny, J.D. Williams, A.R. Khan, N.P. Peet, D.T. Moir,
T.L. Bowlin, Aryl rhodanines specifically inhibit staphylococcal and entero-
coccal biofilm formation, Antimicrob. Agents Chemother. 53 (2009)
4357e4367, http://dx.doi.org/10.1128/AAC.00077-09.
4.2.5. Insect
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.
[9] D.G. Davies, C.N.H. Marques, A fatty acid messenger is responsible for inducing
dispersion in microbial biofilms, J. Bacteriol. 191 (2009) 1393e1403, http://
dx.doi.org/10.1128/JB.01214-08.
ꢀ
[10] M. Sabbah, M. Bernollin, A. Doutheau, L. Soulere, Y. Queneau, A new route
towards fimbrolide analogues: importance of the exomethylene motif in LuxR
dependent quorum sensing inhibition, MedChemComm 4 (2013) 363e366,
http://dx.doi.org/10.1039/C2MD20298K.
4.2.6. Pre-treatment of larvae with antivirulence agents
To evaluate the property of compound 14d to protect against
subsequent S. aureus ATCC 29213 infection, a single treatment dose
of 1 mg/kg of the compound was given to each larva 1 h before the
[11] T. Benneche, G. Herstad, M. Rosenberg, S. Assev, A.A. Scheie, Facile synthesis of
5-(alkylidene)thiophen-2(5H)-ones. A new class of antimicrobial agents, RSC
Adv. 1 (2011) 323e332, http://dx.doi.org/10.1039/C1RA00254F.
[12] U. Kosikowska, A. Malm, M. Pitucha, B. Rajtar, M. Polz-Dacewicz, Inhibitory
inoculation of 10
m
L of live bacteria (2.8 ꢂ 106 cfu). In the experi-
effect
of
N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-
ments there were two negative (uninfected) group controls: one
untreated and the other was injected with PBS containing 3%
DMSO, the same solvents used to dissolve the tested compound.
Three replicates of four larvae for each group were disposed. A
positive group control was pre-treated with PBS containing 3%
DMSO and infected as previously seen for the tested compound
14d. Inoculations were given into alternate prolegs moving up the
body toward the head such that each proleg was injected only once.
Moreover an untouched group of larvae (called untreated) was also
used as control.
carbothioamide on Haemophilus spp. planktonic or biofilm-forming cells,
Med. Chem. Res. Int. J. Rapid Commun. Des. Mech. Action Biol. Act. Agents 23
(2014) 1057e1066, http://dx.doi.org/10.1007/s00044-013-0700-7.
[13] B. Singh, H.R. Bhat, M.K. Kumawat, U.P. Singh, Structure-guided discovery of
1,3,5-triazineepyrazole conjugates as antibacterial and antibiofilm agent
against pathogens causing human diseases with favorable metabolic fate,
Bioorg. Med. Chem. Lett. 24 (2014) 3321e3325, http://dx.doi.org/10.1016/
j.bmcl.2014.05.103.
[14] N.R. Emmadi, K. Atmakur, C. Bingi, N.R. Godumagadda, G.K. Chityal,
J.B. Nanubolu, Regioselective synthesis of 3-benzyl substituted pyrimidino
chromen-2-ones and evaluation of anti-microbial and anti-biofilm activities,
Bioorg. Med. Chem. Lett. 24 (2014) 485e489, http://dx.doi.org/10.1016/
j.bmcl.2013.12.038.
Larvae were stored in Petri dishes in the dark at 37 ꢀC for 72 h in
dark conditions. Larvae were inspected every 24 h and the survival
rate and the weight was observed. Moreover, to detect potential
deleterious effect of the compound 14d, the variation in larval
weight and the time pass from the inoculum at the later larval stage
to the pupation were also checked. For safety reasons only larvae
without bacterial inoculum were used for this bioassay.
[15] M.V. Raimondi, B. Maggio, D. Raffa, F. Plescia, S. Cascioferro, G. Cancemi,
D. Schillaci, M.G. Cusimano, M. Vitale, G. Daidone, Synthesis and anti-
staphylococcal activity of new 4-diazopyrazole derivatives, Eur. J. Med.
Chem. 58 (2012) 64e71, http://dx.doi.org/10.1016/j.ejmech.2012.09.041.
[16] D. Schillaci, B. Maggio, D. Raffa, G. Daidone, S. Cascioferro, M.G. Cusimano,
M.V. Raimondi, 4-Diazopyrazole derivatives as potential new antibiofilm
agents, Chemother 54 (2008) 456e462, http://dx.doi.org/10.1159/000159271.
[17] K. Sambanthamoorthy, R.E. Sloup, V. Parashar, J.M. Smith, E.E. Kim,
M.F. Semmelhack, M.B. Neiditch, C.M. Waters, Identification of small mole-
cules that antagonize diguanylate cyclase enzymes to inhibit biofilm forma-
tion, Antimicrob. Agents Chemother. 56 (2012) 5202e5211, http://dx.doi.org/
10.1128/AAC.01396-12.
4.2.7. Statistical analysis
For survival and weight variation of G. mellonella larvae data
[18] D. Raffa, B. Maggio, M.V. Raimondi, S. Cascioferro, F. Plescia, G. Cancemi,

68
S. Cascioferro et al. / European Journal of Medicinal Chemistry 123 (2016) 58e68
G. Daidone, Recent advanced in bioactive systems containing pyrazole fused
j.bmcl.2012.02.101.
with a five membered heterocycle, Eur. J. Med. Chem. 97 (2015) 732e746,
http://dx.doi.org/10.1016/j.ejmech.2014.12.023.
[19] B. Maggio, M.V. Raimondi, D. Raffa, F. Plescia, S. Cascioferro, G. Cancemi,
M. Tolomeo, S. Grimaudo, G. Daidone, Synthesis and antiproliferative activity
[31] Y. Wei, J. Liu, S. Lin, H. Ding, F. Liang, B. Zhao, Acetoacetanilides as masked
isocyanates: facile and efficient synthesis of unsymmetrically substituted
ureas - Org. Lett. ACS Pub., Acetoacetanilides Masked Isocyanates Facile Effic.
Synth. Unsymmetrically Substituted Ureas - Org. Lett. ACS Publ. 12 (n.d.)
4220e4223.
of
3-(2-chloroethyl)-5-methyl-6-phenyl-8-(trifluoromethyl)-5,6-
dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one, Eur. J. Med. Chem. 96
(2015) 98e104, http://dx.doi.org/10.1016/j.ejmech.2015.04.004.
[32] W.C. Still, M. Kahn, A. Mitra, Rapid chromatographic technique for preparative
separations with moderate resolution, J. Org. Chem. 43 (1978) 2923e2925,
http://dx.doi.org/10.1021/jo00408a041.
[20] B. Maggio, D. Raffa, M.V. Raimondi, M.G. Cusimano, F. Plescia, S. Cascioferro,
G. Cancemi, M. Lauricella, D. Carlisi, G. Daidone, Synthesis and anti-
proliferative activity of new derivatives containing the polycyclic system 5,7:
7,13-dimethanopyrazolo[3,4-b]pyrazolo[30,40:2,3]azepino[4,5-f]azocine, Eur.
J. Med. Chem. 72 (2014) 1e9, http://dx.doi.org/10.1016/j.ejmech.2013.11.016.
[21] B. Maggio, D. Raffa, M.V. Raimondi, G. Daidone, Non-classical transformation
of benzendiazonium hydrogen sulfates. access to 1,3-dimethylisochromeno
[4,3-c]pyrazol-5(1h)-one, a potential benzodiazepine receptor ligand, Mole-
[33] A.M. Hussein, A.A. Harb, I.A. Mousa, b-Oxoanilides in heterocyclic synthesis:
an expeditious synthesis of new polyfunctionally substituted pyridine and
pyrazole derivatives, J. Heterocycl. Chem. 45 (2008) 1819e1823, http://
dx.doi.org/10.1002/jhet.5570450641.
[34] A. Schmidt, T. Habeck, M.K. Kindermann, M. Nieger, New pyrazolium-
carboxylates as structural analogues of the pseudo-cross-conjugated
betainic alkaloid nigellicine, J. Org. Chem. 68 (2003) 5977e5982, http://
dx.doi.org/10.1021/jo0344337.
cules
18
(2013)
13096e13110,
http://dx.doi.org/10.3390/
molecules181013096.
[35] U.A. Pereira, L.C.A. Barbosa, C.R.A. Maltha, A.J. Demuner, M.A. Masood,
[22] B. Maggio, D. Raffa, M.V. Raimondi, F. Plescia, M.L. Trincavelli, C. Martini,
F. Meneghetti, L. Basile, S. Guccione, G. Daidone, Synthesis, benzodiazepine
receptor binding and molecular modelling of isochromeno[4,3-c]pyrazol-
5(1H)-one derivatives, Eur. J. Med. Chem. 54 (2012) 709e720, http://
dx.doi.org/10.1016/j.ejmech.2012.06.028.
[23] D. Raffa, O. Migliara, B. Maggio, F. Plescia, S. Cascioferro, M.G. Cusimano,
G. Tringali, C. Cannizzaro, F. Plescia, Pyrazolobenzotriazinone derivatives as
COX inhibitors: synthesis, biological activity, and molecular-modeling studies,
Arch. Pharm. Weinh. 343 (2010) 631e638, http://dx.doi.org/10.1002/
ardp.200900317.
[24] G. Daidone, D. Raffa, F. Plescia, B. Maggio, A. Roccaro, Synthesis of pyrazole-4-
carbohydrazide derivatives of pharmaceutical interest, Arkivoc 2002 (2002)
227e235.
[25] D. Raffa, G. Daidone, B. Maggio, S. Cascioferro, F. Plescia, D. Schillaci, Synthesis
and antileukemic activity of new 3-(1-phenyl-3-methylpyrazol-5- yl)-2-
styrylquinazolin-4(3H)-ones, Farmaco 59 (2004) 215e221, http://dx.doi.org/
10.1016/j.farmac.2003.10.004.
A.L. Pimenta, g-Alkylidene-g-lactones and isobutylpyrrol-2(5H)-ones ana-
logues to rubrolides as inhibitors of biofilm formation by Gram-positive and
Gram-negative bacteria, Bioorg. Med. Chem. Lett. 24 (2014) 1052e1056,
http://dx.doi.org/10.1016/j.bmcl.2014.01.023.
ꢁ
ꢁ
ꢁ
ꢂ
ꢂ
[36] Z. Hodnik, J.M. Łos, A. Zula, N. Zidar, Z. Jakopin, M. Łos, M. Sollner Dolenc,
ꢁ
ꢁ ꢁ
J. Ilas, G. We˛grzyn, L. Peterlin Masic, D. Kikelj, Inhibition of biofilm formation
by conformationally constrained indole-based analogues of the marine alka-
loid oroidin, Bioorg. Med. Chem. Lett. 24 (2014) 2530e2534, http://dx.doi.org/
10.1016/j.bmcl.2014.03.094.
[37] B. Maggio, D. Raffa, M. Raimondi, S. Cascioferro, F. Plescia, D. Schillaci,
M. Cusimano, A. Leonchiks, D. Zhulenkovs, L. Basile, G. Daidone, Discovery of a
new class of sortase
pathogens: 2-(2-Phenylhydrazinylidene)alkanoic acids and related de-
rivatives, Molecules 21 (2016) 241, http://dx.doi.org/10.3390/
molecules21020241.
a transpeptidase inhibitors to tackle gram-positive
[38] S. Cascioferro, B. Maggio, D. Raffa, M.V. Raimondi, M.G. Cusimano, D. Schillaci,
B. Manachini, A. Leonchiks, G. Daidone, A new class of phenylhydrazinylidene
derivatives as inhibitors of Staphylococcus aureus biofilm formation, Med.
Chem. Res. 25 (2016) 870e878, http://dx.doi.org/10.1007/s00044-016-1535-
9.
[26] G. Daidone, B. Maggio, S. Plescia, D. Raffa, C. Musiu, C. Milia, G. Perra,
M.E. Marongiu, Antimicrobial and antineoplastic activities of new 4-
diazopyrazole derivatives, Eur. J. Med. Chem. 33 (1998) 375e382, http://
dx.doi.org/10.1016/S0223-5234(98)80004-4.
[39] T.A. Elmaati, S. Said, N.A. Elenein, M. Sofan, N. Khodeir, 1-(N,N-Dimethyla-
[27] G. Daidone, S. Plescia, D. Raffa, M.L. Bajardi, M. Milici, New N-pyr-
azolylsalicylamides with antifungal activity, Farm. Ed. Sci. 40 (1985) 683e694.
[28] M. Mastore, V. Arizza, B. Manachini, M.F. Brivio, Modulation of immune re-
sponses of Rhynchophorus ferrugineus (Insecta: Coleoptera) induced by the
entomopathogenic nematode Steinernema carpocapsae (Nematoda: rhabdi-
tida), Insect Sci. (2014), http://dx.doi.org/10.1111/1744-7917.12141 n/a-n/a.
[29] A.P. Desbois, P.J. Coote, Wax moth larva (Galleria mellonella): an in vivo model
for assessing the efficacy of antistaphylococcal agents, J. Antimicrob. Che-
mother. 66 (2011) 1785e1790, http://dx.doi.org/10.1093/jac/dkr198.
[30] R.K. Yadlapalli, O.P. Chourasia, K. Vemuri, M. Sritharan, R.S. Perali, Synthesis
and in vitro anticancer and antitubercular activity of diarylpyrazole ligated
dihydropyrimidines possessing lipophilic carbamoyl group, Bioorg. Med.
mino)-2-(N-phenylcarbamoyl)-1-buten-3-one as a building block for the
synthesis of heterocyclic compounds, ChemInform 33 (2002), http://
dx.doi.org/10.1002/chin.200243050, 50e50.
[40] Ethyl acetopyruvate. Org. Synth.
orgsyn.006.0040.
6 (1926) 40, http://ex.doi.org:10.15227/
[41] B.A. Tertov, A.S. Morkovnik, Reactions of N-substituted diazoles and their halo
derivatives with naphthyllithium and naphthylsodium, Chem. Heterocycl.
Compd. 11 (1975) 343e346, http://dx.doi.org/10.1007/BF00470085.
[42] D. Schillaci, S. Petruso, V. Sciortino, 3,4,5,30,50-Pentabromo-2-(20-hydrox-
ybenzoyl)pyrrole: a potential lead compound as anti-Gram-positive and anti-
biofilm agent, Int, J. Antimicrob. Agents 25 (2005) 338e340, http://dx.doi.org/
10.1016/j.ijantimicag.2004.11.014.
Chem.
Lett.
22
(2012)
2708e2711,
http://dx.doi.org/10.1016/