Synthesis and anti-staphylococcal activity of new 4-diazopyrazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.09.041

Several new 4-diazopyrazole derivatives 6a-g and 9a-c were obtained by the reaction of 1-(R-substituted-phenyl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)ureas 5a-g and N-(1,3-dimethyl-1H-pyrazol-5-yl)-2-(R-substituted-phenyl)acetamides 8a-c respectively with a sevenfold excess of nitrous acid in acetic acid solution. The compounds were assayed for their activity against the Staphylococcus aureus reference strains ATCC 25923, ATCC 29213 and ATCC 6538, as well as six veterinary strains. The best anti-staphylococcal profile was showed by [(R-substituted-phenyl)acetyl](4-diazonio-1,3-dimethyl-1H-pyrazol-5-yl)azanides 9a,c. Compound 9c was also able at 3.1 μg mL^-1 to inhibit of 45.7% the biofilm formation of the strains S. aureus ATCC 29213.

Full text of DOI:10.1016/j.ejmech.2012.09.041

European Journal of Medicinal Chemistry 58 (2012) 64e71
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-staphylococcal activity of new 4-diazopyrazole derivatives
,
a
a
a
Maria Valeria Raimondi a, Benedetta Maggio a , Demetrio Raffa , Fabiana Plescia , Stella Cascioferro ,
*
Gabriella Cancemi a, Domenico Schillaci a, Maria Grazia Cusimano a, Maria Vitale b, Giuseppe Daidone a
a Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32,
90123 Palermo, Italy
b Istituto Zooprofilattico Sperimentale della Sicilia, “A. Mirri”, Via Gino Marinuzzi 3, 90129 Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Several new 4-diazopyrazole derivatives 6aeg and 9aec were obtained by the reaction of 1-(R-substituted-
phenyl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)ureas 5aeg and N-(1,3-dimethyl-1H-pyrazol-5-yl)-2-(R-
substituted-phenyl)acetamides 8aec respectively with a sevenfold excess of nitrous acid in acetic acid
solution. The compounds were assayed for their activity against the Staphylococcus aureus reference strains
ATCC 25923, ATCC 29213 and ATCC 6538, as well as six veterinary strains. The best anti-staphylococcal
profile was showed by [(R-substituted-phenyl)acetyl](4-diazonio-1,3-dimethyl-1H-pyrazol-5-yl)aza-
Received 10 August 2012
Received in revised form
19 September 2012
Accepted 21 September 2012
Available online 1 October 2012
nides 9a,c. Compound 9c was also able at 3.1
S. aureus ATCC 29213.
m
g mLꢀ1 to inhibit of 45.7% the biofilm formation of the strains
Keywords:
4-Diazopyrazoles
Anti-staphylococcal activity
Anti-biofilm activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Staphylococcus epidermidis is seen more often in polymer-
biomaterial associated infections [10]. Biofilm associated infec-
tions of indwelling medical devices are usually resolved after
replacement of the device but involve a prolonged hospital stay and
increased healthcare costs. The treatment of these kinds of infec-
tions is complicated because staphylococcal biofilms are typically
highly resistant to conventional antibiotics [11] and considering
also that increasing numbers of elderly patients require indwelling
medical devices like artificial knees and hips, a new generation of
anti-infective agents effective in the prevention or eradication of
biofilms is needful [12].
The threat of staphylococcal strains resistant to most or all
conventional antibiotics could be faced by developing new anti-
bacterial agents with different chemical characteristics and
mechanism of action with respect to current antibiotics.
Some of us previously reported the 4-diazopyrazoles 1 and 2 by
reacting 5-benzamidopyrazoles with a sevenfold excess of nitrous
acid in acetic acid media (see Fig. 1).
Staphylococci have an extraordinary ability to acquire antibiotics
resistance traits, and the rise of community and hospital-acquired
methicillin resistant Staphylococcus aureus (MRSA) is a major
health problem that has created a pressing need for novel thera-
peutic options [1].
The ability to form biofilms, bacterial communities able to grow
on surfaces and surrounded by an extracellular polymeric substance
(EPS) matrix, is probably the most important virulence factor of
staphylococci in the development of the chronic and persistent form
of some infectious diseases in humans, such as otitis media, osteo-
myelitis, endophthalmitis, urinary tract infections, acute septic
arthritis, native valve endocarditis, burn or wound infections and
cystic fibrosis associated infections (CF) [2e8].
In veterinary medicine, Staphylococcus spp. are involved in
inflammatory diseases like mastitis. A correlation on the recurrence
and persistence of the diseases and the capability of the bacterial
strains to organise biofilm inside mammary glands has been
reported [9].
Furthermore, staphylococcal biofilms are commonly isolated
from device-related infections of medical relevance. In fact, S.
aureus is an important cause of metal-biomaterial infections, while
The obtained compounds showed antimicrobial activity against
representative Gram-negative and Gram-positive bacteria. The
highest microbial susceptibility was shown by Gram-positive
bacteria, with minimum inhibitory concentration in the range
0.26e12.5
were active against five clinical methicillin-resistant S. aureus strains
[13] in the range 2e8 g/mL, and against standard staphylococcal
strains such as S. aureus ATCC 29213, methicillin-resistant S. aureus
ATCC 43866 and S. epidermidis RP62A in the range 1.5e12.5 g/mL.
mg/mL[13,14]. Moreover, themostactivecompounds1aec
m
* Corresponding author. Tel.: þ39 91 23891949; fax: þ39 91 6100627.
E-mail address: benedetta.maggio@unipa.it (B. Maggio).
m
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.041

M.V. Raimondi et al. / European Journal of Medicinal Chemistry 58 (2012) 64e71
65
Fig. 1. Chemical structures of known 4-diazopyrazole derivatives.
Finally, compounds 1a,c showed in vitro anti-biofilm activity when
tested at 25 g/mL against the above standard strains, showing
In particular, the starting products 1-(1,3-dimethyl-1H-pyrazol-
5-yl)-3-(R-substituted-phenyl)ureas 5aeg and N-(1,3-dimethyl-
1H-pyrazol-5-yl)-(R-substituted-phenyl)acetamides 8aec were
obtained by reacting 1,3-dimethyl-1H-pyrazol-5-yl-amine 3 with
phenylisocyanate derivatives 4aeg or phenylacetylchloride deriv-
atives 7aec respectively. The 4-diazoderivatives 6aeg and 9aec
were obtained by reacting the respective starting products 5aeg
and 8aec with nitrous acid in acetic acid solution. A nitro group
was unexpectedly introduced at the 2-position of the phenyl ring of
ureas 5eeg affording 6eeg.
The structures of all the new compounds were assigned on
the basis of their satisfactory analytical as well as spectroscopic
data. The IR spectra of compounds 6 showed absorption in the
2135e2161 cmꢀ1 range, attributable to the diazo group, as well as in
the 1624e1653 cmꢀ1 one for the ureidic carbonyl group. For
compounds 9aec the spectra showed bands at about 2160 cmꢀ1 for
the diazo group, and at about 1600 cmꢀ1 for the carbonyl one. The
low wave-number values for the carbonyl band are due to
the contribution of the resonance form II to the structure of both
bioisosters 6 and 9 (see Fig. 3).
m
inhibition percentages in the range 69.7e97.5%, whereas 1b showed
comparable activity only against the S. epidermidis RP62A [15].
As an extension of this previous work and with the aim to obtain
more effective compounds, we thought to be of interest to
synthesize compounds 6 and 9 which bear at the place of the
phenyl ring of compounds 1 and 2 a phenylamino or phenylmethyl
moiety respectively (see Fig. 2).
The new compounds show different structural features than 1
and 2 derivatives, such as increased distance between the rings,
more potentiality for H bonds or hydrophobic interactions, which
are all indicative of valuable chemical diversity than the reference
compounds. The phenyl ring of these moieties is substituted with
different groups or atom in order to obtain a good discrimination
among the compounds as regards hydrophobic, electronic and
steric effects. All the new compounds were tested against nine
S. aureus strains, and the most active compounds were assayed for
staphylococcal antibiofilm activity.
The 1H NMR spectra of compounds 6 showed, among the other
signals, those for the two methyls linked to the pyrazole nucleus and
for the phenyl moiety, whereas the signal for the pyrazole H(4) atom
was lacking. In particular, for compounds 6e,f the 1H NMR spectra
showed the typical pattern of a tri-substituted benzene. In order
to discriminate between the 1,2,4 or 1,3,4 isomers, we compared
the 1H NMR spectra of 6e,f with those of both 2-nitro-4-
2. Results and discussion
2.1. Chemistry
The synthesis of the 4-diazoderivatives 6aeg and 9aec has been
obtained according to Schemes 1 and 2 (only a mesomeric form is
indicated for compounds 9 and 6).
Fig. 2. Chemical structures of the new 4-diazopyrazole derivatives.

66
M.V. Raimondi et al. / European Journal of Medicinal Chemistry 58 (2012) 64e71
Scheme 1. Synthesis of [(R-phenyl)carbamoyl](4-diazonio-1,3-dimethyl-1H-pyrazol-5-yl)azanides 6aeg. Reagents and conditions: (i) THF, r.t., 24 h; (ii) CH3COOH/HCl 37%, KNO2/
H2O, r.t., 24 h, away from light.
metoxyacetoanilide and 3-nitro-4-metoxyacetoanilide taken as
reference models [16]. Compounds 6e,f and 2-nitro-4-
metoxyacetoanilide showed a similar aromatic signals pattern
confirming the 1,2,4 substituted structure for 6e,f. For compound 6g
comparison was not possible as benzyl signals superimposed on the
tri-substituted benzene signals.
13C NMR of some representative compounds confirmed the
assigned structure and showed that the pyrazole C(4) atom in the
4-diazopyrazoles is quite shielded. The 13C NMR spectra of 9a and
9b, taken as a example, showed the signal for the C(4) atom at
73.70 and 73.62
d respectively, whereas for the corresponding
ureas 8a,b the C(4) resonances occurred at 99.56 and 99.88
Scheme 2. Synthesis of [(R-phenyl)acetyl](4-diazonio-1,3-dimethyl-1H-pyrazol-5-yl)azanides 9aec. Reagents and conditions: (i) dry CHCl3, TEA, reflux, 5 h; (ii) CH3COOH/HCl 37%,
KNO2/H2O, r.t., 24 h, away from light.

M.V. Raimondi et al. / European Journal of Medicinal Chemistry 58 (2012) 64e71
67
Fig. 3. Resonance forms for compounds 6aeh and 9aec.
d
respectively, due to the lack of the 4-diazo group. The 13C NMR
Compounds 9aec resulted generally more active than the cor-
responding bioisosters 6a,c,e. Possibly this is due to a higher lip-
ophilicity of compounds 9aec than 6a,c,e. The introduction of
substituents in the phenyl ring of 9a produced essentially
a decrease of activity, despite their opposite electronic effects. As
regards the compounds of series 6, the most active were 6bed.
These compounds bear substituents on the phenyl ring which
exert an electron withdrawing effect (R1 ¼ 4-NO2, 4-Cl, 3,4-Cl2
respectively).
The anti-staphylococcal activity decreased significantly when
the phenyl ring of compounds 6 bore an electron donating
substituent together with the nitro group, such as for 6eeg. As the
nitro group should take an advantage for activity, due to its electron
withdrawing properties (see above), we realized that the electron
donating groups, namely OMe, OBz, OBu, afford a decreasing of
activity.
data justify the resonance form III which together with the
form II most closely represent the structure of this class of
4-diazopyrazoles.
2.2. Biological activity
Compounds 6aeg and their bioisosters 9aec, in which a CH2
group is in the place of an NH one, were primarily evaluated for
their anti-staphylococcal activity against three reference strains
of S. aureus, namely ATCC 25923, ATCC 29213, ATCC 6538, and
six veterinary strains of the same species, namely 722, 708, 100,
723, 357, 712 (IZS bacterial collection, see Experimental section
e Microbiology). The minimum inhibitory concentration (MIC)
of the tested compounds as well as of rifampicin, used as
a control, is reported in Table 1. The MIC value for rifampicin
when tested against S. aureus ATCC 29213 is within the range of
A useful strategy to control staphylococcal biofilms is the inhi-
bition of their growth on surfaces. With this aim in mind, the most
the literature data (0.06e0.008 m
g mLꢀ1) [17].

68
M.V. Raimondi et al. / European Journal of Medicinal Chemistry 58 (2012) 64e71
Table 1
Antistaphylococcal activity of 6aeg and 9aec against S. aureus reference strains and field isolates of veterinary interest. Minimum inhibitory concentration MIC expressed in
m
g/mL. RIF ¼ rifampicin.
Comp.
6a
6b
6c
6.2
6.2
25.0
6d
6.2
6.2
6.2
6e
6f
6g
9a
9b
9c
6.2
6.2
6.2
RIF
Reference strains
ATCC 25923
ATCC 29213
ATCC 6538
12.5
12.5
12.5
6.2
6.2
6.2
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
6.2
3.1
3.1
25.0
25.0
6.2
0.015
0.015
0.015
Isolates
722
708
100
723
25.0
25.0
12.5
6.2
6.2
50.0
3.1
3.1
>100.0
100.0
3.1
12.5
25.0
6.2
12.5
6.2
3.1
6.2
3.1
3.1
6.2
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
6.2
6.2
3.1
3.1
1.5
1.5
12.5
12.5
6.2
3.1
1.5
6.2
6.2
3.1
6.2
6.2
0.6
0.07
0.07
1.25
0.015
0.3
357
712
3.1
3.1
25.0
50.0
25.0
active compounds against free living (planktonic) forms, 9a and 9c,
were tested for their ability to prevent biofilm formation of two
reference strains and of the best biofilm forming among the strains
of animal origin, the S. aureus 708. Compound 9c showed at
4.1.1.1. 1-(1,3-Dimethyl-1H-pyrazol-5-yl)-3-phenylurea (5a). Yield: 32.3%;
m.p.: 185e186 ꢂC (ethyl acetate); IR (KBr)
n
(cmꢀ1): 3268 (2 ꢃ NH),
1647 (CO); 1H NMR (DMSO-d6)
d
(ppm): 2.13 (s, 3H, CeCH3), 3.63 (s,
3H, NeCH3), 6.02 (s, 1H, pyrazole H-4), 7.00e7.51 (a set of signals,
5H, aromatic protons), 8.56 (s, 1H, NH, exchangeable), 8.88 (s, 1H,
NH, exchangeable). Elemental analysis (C12H14N4O) C, H, N.
3.1 m
g mLꢀ1 an inhibition of 45.7, 38 and 25% against the strains
S. aureus ATCC 29213, ATCC 25923 and 708 respectively. The above
concentration is below the MICs determined against the mentioned
planktonic strains. Compound 9a showed an inhibition of 45.7% at
4.1.1.2. 1-(1,3-Dimethyl-1H-pyrazol-5-yl)-3-(4-nitrophenyl)urea
sub-MIC concentration of 3.1
m
g mLꢀ1 against S. aureus 25923, but
(5b). Yield: 62.9%; m.p.: 226e230 ꢂC (ethanol); IR (KBr)
n
(cmꢀ1):
d (ppm): 2.10
resulted scarcely effective in preventing the biofilm formation of
the other two tested strains.
3352e3223 (2 ꢃ NH), 1733 (CO); 1H NMR (DMSO-d6)
(s, 3H, CeCH3), 3.60 (s, 3H, NeCH3), 6.00 (s,1H, pyrazole H-4), 7.68e
8.21 (a set of signals, 4H, aromatic protons), 8.84 (s, 1H, NH,
exchangeable), 9.51 (s, 1H, NH, exchangeable). 13C NMR (DMSO-d6)
3. Conclusions
d
(ppm): 14.03 (CH3), 35.22 (CH3), 98.01 (CHpyrazole), 118.08
(2 ꢃ CHAr), 125.57 (2 ꢃ CHAr), 137.17 (CAr), 141.68 (CAr), 146.02 (CAr),
To summarize, we have synthesized new 4-diazopyrazole bio-
isosters 6aeg and 9aec as an extension of our previous work in this
field. Some of the newcompounds showed a better anti-staphylococcal
profile than the reference 4-diazo-5-benzamidopyrazoles 1.
151.92 (CO). Elemental analysis (C12H13N5O3) C, H, N.
4.1.1.3. 1-(4-Chlorophenyl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)urea
(5c). Yield: 61.3%; m.p.: 219e221 ꢂC (ethanol); IR (KBr)
n
(cmꢀ1):
3270 (2 ꢃ NH),1644 (CO); 1HNMR (DMSO-d6)
d(ppm):2.09(s, 3H, Ce
4. Experimental protocols
CH3), 3.58(s, 3H, NeCH3), 5.97 (s,1H, pyrazole H-4), 7.31e7.51 (a setof
signals, 4H, aromatic protons), 8.57 (s,1H, NH, exchangeable), 9.01 (s,
1H, NH, exchangeable). Elemental analysis (C12H13N4OCl) C, H, N.
4.1. General
All commercial chemicals were purchased from Aldrich
(SigmaeAldrich, St. Louis, MO, USA). Reaction progress was moni-
tored by TLC on silica gel plates (Merck 60, F254, 0.2 mm) and
visualization on TLC was achieved by UV light. Organic solutions
were dried over Na2SO4. 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 as
solid in KBr disc. 1H NMR and 13C NMR spectra were recorded in
DMSO-d6 or CDCl3 at 300.13 MHz, using a Bruker AC series
300 MHz spectrometer (tetramethylsilane as the internal stan-
4.1.1.4. 1-(3,4-Dichlorophenyl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)
urea (5d). Yield: 49%; m.p.: 221e222 ꢂC (ethyl acetate); IR (KBr)
n
(cmꢀ1): 3265 (2 ꢃ NH), 1646 (CO); 1H NMR (DMSO-d6)
d (ppm):
2.09 (s, 3H, CeCH3), 3.58 (s, 3H, NeCH3), 5.96 (s, 1H, pyrazole H-4),
7.33e7.87 (a set of signals, 3H, aromatic protons), 8.71 (s, 1H, NH,
exchangeable), 9.13 (s, 1H, NH, exchangeable). 13C NMR (DMSO-d6)
d
(ppm): 13.30 (CH3), 34.45 (CH3), 97.25 (CHpyrazole), 118.14 (CHAr),
119.10 (CHAr), 123.11 (CAr), 130.26 (CHAr), 130.71 (CAr), 136.59 (CAr),
139.35 (CAr), 145.09 (CAr), 151.49 (CO). Elemental analysis
(C12H12N4OCl2) C, H, N.
4.1.1.5. 1-(1,3-Dimethyl-1H-pyrazol-5-yl)-3-(4-methoxyphenyl)urea
dard): chemical shifts are expressed in
d values (ppm). Microanal-
(5e). Yield: 50.8%; m.p.: 209e210 ꢂC (ethanol); IR (KBr)
n
(cmꢀ1):
yses data (C, H, N) were obtained by an Elemental Vario EL III
apparatus and are within ꢁ0.4% of the theoretical values. Yields
refer to products after crystallization. The name of the compounds
was obtained using the ACD/I-Lab Web service (ACD/IUPAC Name
Free 8.05).
3269 (2 ꢃ NH), 1634 (CO); 1H NMR (DMSO-d6)
d (ppm): 2.07 (s, 3H,
CeCH3), 3.56 (s, 3H, NeCH3), 3.71 (s, 3H, OeCH3), 5.93 (s, 1H, pyr-
azole H-4), 6.87e7.35 (a set of signals, 4H, aromatic protons), 8.40
(s, 1H, NH, exchangeable), 8.61 (s, 1H, NH, exchangeable). 13C NMR
(DMSO-d6) d (ppm): 14.08 (CH3), 35.16 (CH3), 55.65 (OeCH3), 97.35
(CHpyrazole), 114.48 (2 ꢃ CHAr), 120.67 (2 ꢃ CHAr), 132.86 (CAr),
138.08 (CAr), 145.78 (CAr), 152.52 (CAr), 155.15 (CO). Elemental
analysis (C13H16N4O2) C, H, N.
4.1.1. General procedure for the synthesis of 1-(R-substituted-
phenyl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)ureas (5aeg)
A solution of equimolar amounts (4.5 mmol) of 1,3-dimethyl-
1H-pyrazol-5-amine 3 [18] and substituted phenylisocyanates
4aeg in THF (12 mL) was stirred at room temperature for 24 h.
The solid residue was filtered, washed with 1 mL of ethyl acetate,
and then crystallized from a suitable solvent.
4.1.1.6. 1-(4-butoxyphenyl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)urea
(5f). Yield: 36.7%; m.p.: 178e180 ꢂC (ethanol); IR (KBr)
n
(cmꢀ1):
3266 (2 ꢃ NH), 1645 (CO); 1H NMR (DMSO-d6)
d (ppm): 0.92 (t, 3H,

M.V. Raimondi et al. / European Journal of Medicinal Chemistry 58 (2012) 64e71
69
CH3), 1.42 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.07 (s, 3H, CeCH3), 3.56
(s, 3H, NeCH3), 3.90 (t, 2H, CH2), 5.94 (s, 1H, pyrazole H-4), 6.83e
7.34 (a set of signals, 4H, aromatic protons), 8.43 (s, 1H, NH,
exchangeable), 8.63 (s, 1H, NH, exchangeable). 13C NMR (DMSO-d6)
room temperature to a magnetically-stirred solution of 1-(R-
substituted-phenyl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)ureas (5aeg)
or 2-(R-substituted-phenyl)-N-(1,3-dimethyl-1H-pyrazol-5-yl)acet-
amides (8aec) (2.7 mmol) in glacial acetic acid (26.42 mL). The
reaction mixture was then stirred overnight away from the light,
after which it was filtered off and poured into 70 mL of cold water.
Then, the solutionwas basified until pH 6 with aqueous concentrated
NaOH and finally the solid product which separated was filtered and
crystallized from a suitable solvent.
d
(ppm): 14.09 (CH3), 14.13 (CH3), 19.20 (CH2), 31.27 (CH2), 35.16
(CH3), 67.75 (CH2), 97.28 (CHpyrazole), 115.06 (2 ꢃ CHAr), 120.62
(2 ꢃ CHAr), 132.74 (CAr), 138.08 (CAr), 145.77 (CAr), 152.48 (CAr),
154.55 (CO). Elemental analysis (C16H22N4O2) C, H, N.
4.1.1.7. 1-[4-(Benzyloxy)phenyl]-3-(1,3-dimethyl-1H-pyrazol-5-yl)
urea (5g). Yield: 73.9%; m.p.: 204e206 ꢂC (ethanol); IR (KBr)
n
4.1.3.1. (4-Diazonio-1,3-dimethyl-1H-pyrazol-5-yl)(phenylcarbamoyl)
(cmꢀ1): 3274 (2 ꢃ NH), 1646 (CO); 1H NMR (DMSO-d6)
d
(ppm):
azanide (6a). Yield: 10.3%; m.p.: 152e154 ꢂC (ethanol); IR (KBr)
n
2.08 (s, 3H, CeCH3), 3.57 (s, 3H, NeCH3), 5.05 (s, 2H, OeCH2), 5.95
(s, 1H, pyrazole H-4), 6.93e7.45 (a set of signals, 9H, aromatic
protons), 8.45 (s, 1H, NH, exchangeable), 8.66 (s, 1H, NH,
exchangeable). Elemental analysis (C19H20N4O2) C, H, N.
(cmꢀ1): 3261 (NH), 2138 ðN2þÞ, 1638 (CO); 1H NMR (DMSO-d6)
d
(ppm): 2.28 (s, 3H, CeCH3), 3.53 (s, 3H, NeCH3), 6.88e7.63 (a set of
signals, 5H, aromatic protons), 9.22 (s, 1H, NH, exchangeable).
Elemental analysis (C12H12N6O) C, H, N.
4.1.2. General procedure for the synthesis of 2-(R-substituted-
phenyl)-N-(1,3-dimethyl-1H-pyrazol-5-yl)acetamides (8aec).
A solution of equimolar amounts (9 mmol) of 1,3-dimethyl-1H-
pyrazol-5-amine 3 [18] and substituted phenylacetylchlorides 7aec
in dry CHCl3 (30 mL) was refluxed for 5 h. After the first hour, trie-
thylamine (1.25 mL, 9 mmol) was added in four portions (0.6, 0.3, 0.2,
0.15 mL respectively with intervals of 1 h between additions). The
reaction mixturewasevaporated under reduced pressure, theresidue
washed with cold water and crystallized from a suitable solvent.
4.1.3.2. (4-Diazonio-1,3-dimethyl-1H-pyrazol-5-yl)[(4-nitrophenyl)
carbamoyl]azanide (6b). Yield: 87.1%; m.p.: 197e200 ꢂC (ethanol);
IR (KBr)
(DMSO-d6)
n
(cmꢀ1): 3231 (NH), 2145 ðNþ2 Þ, 1644 (CO); 1H NMR
d
(ppm): 2.31 (s, 3H, CeCH3), 3.57 (s, 3H, NeCH3), 7.84e
8.16 (a set of signals, 4H, aromatic protons), 10.02 (s, 1H, NH,
exchangeable). 13C NMR (DMSO-d6)
d
(ppm): 11.98 (CH3), 36.89
(CH3), 72.10 (CAr), 118.00 (2 ꢃ CHAr), 125.17 (2 ꢃ CHAr), 141.94 (CAr),
144.90 (CAr), 146.99 (CAr), 146.61 (CAr), 153.32 (CO). Elemental
analysis (C12H11N7O3) C, H, N.
4.1.2.1. N-(1,3-Dimethyl-1H-pyrazol-5-yl)-2-phenylacetamide (8a).
4.1.3.3. [(4-Chlorophenyl)carbamoyl](4-diazonio-1,3-dimethyl-1H-
Yield: 43.6%; m.p.: 87e90 ꢂC (diethyl ether); IR (KBr)
n
(cmꢀ1): 3252
pyrazol-5-yl)azanide (6c). Yield: 23.5%; m.p.: 151e152 ꢂC (ethanol);
(NH), 1666 (CO); 1H NMR (DMSO-d6)
d
(ppm): 2.06 (s, 3H, CeCH3),
IR (KBr)
n
(cmꢀ1): 3214 (NH), 2145 ðNþ2 Þ, 1638 (CO); 1H NMR
3.53 (s, 3H, NeCH3), 3.67 (s, 2H, CH2), 5.96 (s, 1H, pyrazolic H-4),
7.24e7.33 (a set of signals, 5H, aromatic protons), 10.07 (s, 1H, NH,
(DMSO-d6) d (ppm): 2.28 (s, 3H, CeCH3), 3.53 (s, 3H, NeCH3), 7.25e
7.67 (a set of signals, 4H, aromatic protons), 9.42 (s, 1H, NH,
exchangeable). Elemental analysis (C12H11N6OCl) C, H, N.
exchangeable). 13C NMR (CDCl3)
d (ppm): 13.77 (CH3), 34.94 (CH3),
43.52 (CH2), 99.56 (CHpyrazole), 127.77 (CHAr), 129.22 (2 ꢃ CHAr),
129.30 (2 ꢃ CHAr), 134.08 (CAr), 135.53 (CAr), 147.24 (CAr), 169.73
(CO). Elemental analysis (C13H15N3O) C, H, N.
4.1.3.4. (4-Diazonio-1,3-dimethyl-1H-pyrazol-5-yl)[(3,4-
dichlorophenyl)carbamoyl]azanide (6d). Yield: 17.7%; m.p.: 191e194 ꢂC
(ethanol); IR (KBr)
(DMSO-d6) d (ppm): 2.30 (s, 3H, CeCH3), 3.55 (s, 3H, NeCH3), 7.44e
n
(cmꢀ1): 3254 (NH), 2135 ðNþ2 Þ,1638 (CO); 1H NMR
4.1.2.2. 2-(4-Chlorophenyl)-N-(1,3-dimethyl-1H-pyrazol-5-yl)acet-
amide (8b). Yield: 11%; m.p.: 137e140 ꢂC (ethyl acetate); IR (KBr)
n
7.98 (a set of signals, 3H, aromatic protons), 9.62 (s, 1H, NH,
exchangeable). 13C NMR (CDCl3)
(ppm): 12.46 (CH3), 35.02 (CH3),
(cmꢀ1): 3255 (NH), 1664 (CO); 1H NMR (DMSO-d6)
d
(ppm): 2.06 (s,
d
3H, CeCH3), 3.54 (s, 3H, NeCH3), 3.68 (s, 2H, CH2), 5.96 (s, 1H,
pyrazolic H-4), 7.32e7.40 (a set of signals, 4H, aromatic protons),
71.92 (CAr), 117.60 (2 ꢃ CHAr), 119.82 (2 ꢃ CHAr), 125.11 (CAr), 130.28
(CHAr), 132.54 (CAr), 139.41 (CAr), 144.82 (CAr), 154.95 (CAr), 161.23 (CO).
Elemental analysis (C12H10Cl2N6O4) C, H, N.
10.07 (s, 1H, NH, exchangeable). 13C NMR (CDCl3)
d (ppm): 13.76
(CH3), 35.07 (CH3), 42.62 (CH2), 99.88 (CHpyrazole), 129.22
(2 ꢃ CHAr), 130.57 (2 ꢃ CHAr), 132.53 (CAr), 133.69 (CAr), 135.50 (CAr),
147.33 (CAr), 169.35 (CO). Elemental analysis (C13H14N3OCl) C, H, N.
4.1.3.5. (4-Diazonio-1,3-dimethyl-1H-pyrazol-5-yl)[(4-methoxy-2-
nitrophenyl)carbamoyl]azanide (6e). Yield: 15.7%; m.p.: 151e152 ꢂC
(ethanol); IR (KBr)
NMR (DMSO-d6) d (ppm): 2.28 (s, 3H, CeCH3), 3.51 (s, 3H, NeCH3),
n
(cmꢀ1): 3411 (NH), 2161 ðNþ2 Þ, 1627 (CO); 1H
4.1.2.3. N-(1,3-Dimethyl-1H-pyrazol-5-yl)-2-(4-methoxyphenyl)
acetamide (8c). Yield: 14.1%; m.p.: 112e115 ꢂC (ethyl acetate); IR
3.80 (s, 3H, OeCH3), 7.32 (dd, 1H, J ¼ 2.1 Hz, 9.3 Hz), 7.51 (d, 1H,
J ¼ 2.1 Hz), 8.11 (d, 1H, J ¼ 9.3 Hz), 9.54 (s, 1H, NH, exchangeable).
Elemental analysis (C13H13N7O4) C, H, N.
(KBr) n d (ppm):
(cmꢀ1): 3259 (NH), 1663 (CO); 1H NMR (DMSO-d6)
2.06 (s, 3H, CeCH3), 3.53 (s, 3H, NeCH3), 3.58 (s, 2H, CH2), 3.72 (s,
3H, OeCH3), 5.94 (s, 1H, pyrazolic H-4), 6.87e7.25 (a set of signals,
4H, aromatic protons), 9.99 (s, 1H, NH, exchangeable). 13C NMR
4.1.3.6. [(4-Butoxy-2-nitrophenyl)carbamoyl](4-diazonio-1,3-
(CDCl3)
d
(ppm): 13.83 (CH3), 34.97 (CH3), 42.80 (CH2), 55.34 (Oe
dimethyl-1H-pyrazol-5-yl)azanide (6f). Yield: 38.7%; m.p.: 124e
CH3), 99.51 (CHpyrazole), 114.73 (2 ꢃ CHAr), 125.94 (CAr), 130.52
(2 ꢃ CHAr), 135.42 (CAr), 147.31 (CAr), 159.25 (CAr), 169.89 (CO).
Elemental analysis (C14H17N3O2) C, H, N.
125 ꢂC (ethanol); IR (KBr)
n
(cmꢀ1): 3404 (NH), 2154 ðNþ2 Þ, 1651
(CO); 1H NMR (DMSO-d6)
d
(ppm): 0.93 (t, 3H, CH3), 1.43 (m, 2H,
CH2), 1.70 (m, 2H, CH2), 2.28 (s, 3H, CeCH3), 3.51 (s, 3H, NeCH3),
4.01 (t, 2H, CH2), 7.31 (dd, 1H, J ¼ 3 Hz, 9.3 Hz), 7.48 (d, 1H,
J ¼ 3 Hz), 8.12 (d, 1H, J ¼ 9.3 Hz), 9.53 (s, 1H, NH, exchangeable).
Elemental analysis (C16H19N7O4) C, H, N.
4.1.3. General procedure for the synthesis of [(R-substituted-phenyl)
carbamoyl](4-diazonio-1,3-dimethyl-1H-pyrazol-5-yl)azanides
(6aed), [(4-R-2-nitrophenyl)carbamoyl](4-diazonio-1,3-dimethyl-
1H-pyrazol-5-yl)azanides (6eef), and of [(4-chlorophenyl)
acetyl](4-diazonio-1,3-dimethyl-1H-pyrazol-5-yl)azanides (9aec).
Aqueous 37% hydrochloric acid (1.98 mL) and then potassium
nitrite (1.61 g,18.9 mmol) dissolved inwater (0.85 mL) were added at
4.1.3.7. {[4-(Benzyloxy)-3-nitrophenyl]carbamoyl}(4-diazonio-1,3-
dimethyl-1H-pyrazol-5-yl)azanide (6g). Yield: 13.1%; m.p.: 195e197 ꢂC
(ethanol); IR (KBr)
(DMSO-d6)
n
(cmꢀ1): 3399 (NH), 2141 ðNþ2 Þ, 1653 (CO); 1H NMR
d
(ppm): 2.29 (s, 3H, CeCH3), 3.51 (s, 3H, NeCH3), 5.16 (s, 2H,

70
M.V. Raimondi et al. / European Journal of Medicinal Chemistry 58 (2012) 64e71
OeCH2), 7.34e8.12 (a set of signals, 8H, aromatic protons), 9.53 (s,1H, NH,
exchangeable). Elemental analysis (C19H17N7O4) C, H, N.
each well were re-dissolved to homogeneity in 1 mL of 30% v/v
glacial acetic acid, and the OD was read at 492 nm. Each assay was
performed in triplicate and repeated at least twice.
4.1.3.8. (4-Diazonio-1,3-dimethyl-1H-pyrazol-5-yl)(phenylacetyl)
azanide (9a). Yield: 50.3%; m.p.: 112e114 ꢂC (diethyl ether); IR (KBr)
4.2.4. Biofilm prevention assay
n
(cmꢀ1): 2160 ðN2þÞ,1592 (CO); 1H NMR (DMSO-d6)
d
(ppm): 2.28 (s,
The same safranin procedure was used to evaluate the activity of
the tested compounds in preventing biofilm formation by adding
directly sub-MIC concentrations of compounds to the bacterial
suspension. Comparing the average optical density of the growth
control wells with the sample wells the following formula was used
to calculate the percentages of inhibition for the screening
concentration of the compounds:
3H, CeCH3), 3.53 (s, 3H, NeCH3), 3.63 (s, 2H, CH2), 7.18e7.28 (a set of
signals, 5H, aromatic protons). 13C NMR (CDCl3)
d (ppm): 12.45
(CH3), 35.30 (CH3), 47.12 (CH2), 73.70 (CHpyrazole), 126.34 (CHAr),
128.30 (2 ꢃ CHAr),129.43 (2 ꢃ CHAr), 137.16 (CAr),145.35 (CAr),154.74
(CAr), 181.35 (CO). Elemental analysis (C13H13N5O) C, H, N.
4.1.3.9. [(4-Chlorophenyl)acetyl](4-diazonio-1,3-dimethyl-1H-pyr-
azol-5-yl)azanide (9b). Yield: 7.6%; m.p.: 100e104 ꢂC (ethanol); IR
[(OD growth control ꢀ OD sample)/OD growth control] ꢃ 100.
(KBr)
n
(cmꢀ1): 2154 ðN2þÞ, 1590 (CO); 1H NMR (DMSO-d6)
d (ppm):
All experiments were performed at least in triplicate.
2.29 (s, 3H, CeCH3), 3.49 (s, 3H, NeCH3), 3.63 (s, 2H, CH2), 7.31 (s,
4H, aromatic protons). 13C NMR (CDCl3)
d (ppm): 12.33 (CH3), 35.19
(CH3), 46.37 (CH2), 73.62 (CHpyrazole), 128.30 (2 ꢃ CHAr), 130.78
(2 ꢃ CHAr), 132.12 (CAr), 135.74 (CAr), 145.22 (CAr), 154.89 (CAr),
181.80 (CO). Elemental analysis (C13H12N5OCl) C, H, N.
Acknowledgement
Financial support from MIUR (ex 60%) is greatfully
acknowledged
4.1.3.10. (4-Diazonio-1,3-dimethyl-1H-pyrazol-5-yl)[(4-methoxyphenyl)
acetyl]azanide (9c). Yield: 17.2%; m.p.: 100e105 ꢂC (ethanol); IR (KBr)
n
1
(cmꢀ1): 2160 ðN2þÞ, 1595 (CO); H NMR (DMSO-d6)
d (ppm): 2.28 (s,
Appendix A. Supplementary data
3H, CeCH3), 3.55 (s, 5H, NeCH3, CH2), 3.72 (s, 3H, OeCH3), 6.81e7.53
(a set of signals, 4H, aromatic protons). 13C NMR (CDCl3)
d (ppm): 12.39
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.
09.041.
(CH3), 35.20 (CH3), 46.26 (CH2), 55.25 (OeCH3), 73.77 (CHpyrazole),
113.74 (2 ꢃ CHAr), 128.62 (CAr), 130.37 (2 ꢃ CHAr), 131.19 (CAr), 145.11
(CAr), 158.19 (CAr), 182.87 (CO). Elemental analysis (C14H15N5O2) C, H, N.
References
4.2. Microbiology
[1] K. Ohlsen, U. Lorenz, Novel targets for antibiotics in Staphylococcus aureus,
Future Microbiol. 2 (2007) 655e666.
[2] L. Hall-Stoodley, P. Stoodley, Evolving concepts in biofilm infections, Cell.
Microbiol. 11 (2009) 1034e1043.
[3] R.A. Brady, J.G. Leid, J.H. Calhoun, J.W. Costerton, M.E. Shirtliff, Osteomyelitis
and the role of biofilm in chronic infection, FEMS Immunol. Med. Microbiol. 52
(2008) 13e22.
[4] M.C. Callegan, M.S. Gilmore, M. Gregory, R.T. Ramadan, B.J. Wiskur,
A.L. Moyer, J.J. Hunt, B.D. Novosad, Bacterial endophthalmitis: therapeutic
challenges and host-pathogen interactions, Prog. Retin. Eye Res. 26 (2)
(2007) 189e203.
[5] A. Ronald, The etiology of urinary tract infection: traditional and emerging
pathogens, Am. J. Med. 113 (2002) 14se19s.
[6] M.E. Shirtliff, J.T. Mader, Acute septic arthritis, Clin. Microbiol. Rev. 15 (2002)
527e544.
[7] R.M. Donlan, J.W. Costerton, Biofilms: survival mechanisms of clinically rele-
vant microorganisms, Clin. Microbiol. Rev. 15 (2002) 167e193.
[8] J.C. Davies, D. Bilton, Bugs, biofilms, and resistance in cystic fibrosis, Respir.
Care 54 (5) (2009) 628e640.
[9] M.B. Melchior, H. Vaarkamp, J. Fink-Gremmels, Biofilms: a role in recurrent
mastitis infections? Vet. J. 171 (2006) 398e407.
[10] F. Götz, Staphylococcus and biofilms, Mol. Microbiol. 43 (2002) 1367e
1378.
[11] P. Gilbert, D.G. Allison, A.J. McBain, Biofilms in vitro and in vivo: do
singular mechanisms imply cross-resistance? J. Appl. Microbiol. 92 (2002)
98Se110S.
[12] A.S. Lynch, D. Abbanat, New antibiotic agents and approaches to treat
biofilm-associated infections, Expert Opin. Ther. Pat. 20 (10) (2010)
1373e1387.
[13] G. Daidone, M.L. Bajardi, S. Plescia, D. Raffa, D. Schillaci, B. Maggio, F. Benetollo,
G. Bombieri, One-step synthesis, crystallographic studies and antimicrobial
activity of new 4-diazopyrazole derivatives, Eur. J. Med. Chem. 31 (1996)
461e468.
[14] G. Daidone, B. Maggio, S. Plescia, D. Raffa, C. Musio, C. Milia, G. Perra,
M.E. Marongiu, Antimicrobial and antineoplastic activities of new 4-
diazopyrazole derivatives, Eur. J. Med. Chem. 33 (1998) 375e382.
[15] D. Schillaci, B. Maggio, D. Raffa, G. Daidone, S. Cascioferro, M.G. Cusimano,
M.V. Raimondi, 4-Diazopyrazole derivatives as potential new antibiofilm
agents, Chemotherapy 54 (2008) 456e462.
[16] B. Schimidt, R. Berger, F. Hölter, Functionalized alkoxy arene diazonium salts
from paracetamol, Org. Biomol. Chem. 8 (2010) 1406e1414. Supplementary
Material, S11 and S15.
4.2.1. Bacterial strains
The following reference strains were utilized: S. aureus ATCC
25923, S. aureus ATCC 29213 and S. aureus ATCC 6538. Moreover,
a group of six strains of S. aureus isolated from milk and food
samples were also utilized. The isolates belong to the bacterial
collection of IZS of Sicily [19].
4.2.2. Determination of MICs
Minimum inhibitory concentrations (MICs) against planktonic
strains were determined by a broth dilution micro-method as
previously described [20]. Briefly, a series of solutions with a range
of concentrations from 50 to 0.001
serial dilution) were made in Mueller Hinton broth (Merck) in a 96-
well plate. To each well 10 L of a bacterial suspension obtained
m
g mLꢀ1 (obtained by twofold
m
from a 24 h culture containing w106 cfu mLꢀ1 was added. The plate
was incubated at 37 ꢂC for 24 h. After this time, the MIC values were
determined by a microplate reader (ELX 800, Bio-Tek Instruments)
as the lowest concentration of compound at which the optical
density (OD) at 570 nm of the well was comparable to the negative
control well (broth only).
4.2.3. Biofilm capability evaluation
All the staphylococcal 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 ꢂC in a shaking
bath and then diluted 1:200 to a suspension with optical density
(OD) of about 0.040 at 570 nm. Polystyrene 24-well tissue culture
plates were filled with 1 mL of diluted suspension and incubated for
24 h at 37 ꢂC. Then, the wells were washed three times with 1 mL of
sterile phosphate-buffered saline (PBS) and stained with 1 mL of
safranin 0.1% v/v for 1 min. The excess stain was removed by placing
the plates under running tap water. Plates were dried overnight in
inverted position at 37 ꢂC. Safranin stained adherent bacteria in
[17] National Committee for Clinical Laboratory Standards (NCCLS), Methods for
Diluition Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.

M.V. Raimondi et al. / European Journal of Medicinal Chemistry 58 (2012) 64e71
71
Approved Standard, third ed., vol. 13, NCCLS, Villanova, 1993, NCCLS Docu-
ment M7-A3.
[18] E.C. Taylor, K.S. Hartke, The reaction of malononitrile with substituted
hydrazines: new routes to 4-aminopyrazolo[3,4-d]pyrimidines, J. Am. Chem.
Soc. 81 (1959) 2456e2464.
[19] Istituto Zooprofilattico Sperimentale della Sicilia, “A. Mirri”, Via Gino Mar-
inuzzi 3, 90129 Palermo, Italy.
[20] 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.