Synthesis and in vitro selective anti-Helicobacter pylori activity of pyrazoline derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.11.042

In order to develop new anti-Helicobacter pylori agents, a series of N1-substituted 3,5-diphenyl pyrazolines P1-P13 was prepared and evaluated for their antibacterial activity. All synthesized compounds showed little or no activity against different species of Gram-positive and Gram-negative bacteria of clinical relevance and against various strains of pathogenic fungi. The same derivatives exhibited a significant degree of activity against a range of H. pylori strains, including those resistant to the reference compound metronidazole. Among the prepared compounds those with an N1-acetyl group and a 4-methoxy substituent in the 5-phenyl ring showed the best activity against H. pylori metronidazole resistant strains in the 1-4 μg/mL MIC range.

Full text of DOI:10.1016/j.bmcl.2004.11.042

Bioorganic & Medicinal Chemistry Letters 15 (2005) 603–607
Synthesis and in vitro selective anti-Helicobacter pylori
activity of pyrazoline derivatives
F. Chimenti,a B. Bizzarri,a,* F. Manna,a A. Bolasco,a D. Secci,a P. Chimenti,a A. Granese,a
D. Rivanera,b D. Lilli,b M. M. Scaltritoc and M. I. Brenciagliab
a
`
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita ‘La Sapienza’,
P.le A. Moro 5, 00185 Rome, Italy
b
`
`
Dipartimento di Scienze di Sanita Pubblica, Universita ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy
c
`
Istituto di Microbiologia, Universita degli Studi di Milano, via Pascal 36, 20133 Milano, Italy
Received 28 September 2004; revised 15 November 2004; accepted 17 November 2004
Available online 20 December 2004
Abstract—In order to develop new anti-Helicobacter pylori agents, a series of N1-substituted 3,5-diphenyl pyrazolines P1–P13 was
prepared and evaluated for their antibacterial activity. All synthesized compounds showed little or no activity against different spe-
cies of Gram-positive and Gram-negative bacteria of clinical relevance and against various strains of pathogenic fungi. The same
derivatives exhibited a significant degree of activity against a range of H. pylori strains, including those resistant to the reference
compound metronidazole. Among the prepared compounds those with an N1-acetyl group and a 4-methoxy substituent in the
5-phenyl ring showed the best activity against H. pylori metronidazole resistant strains in the 1–4 lg/mL MIC range.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
macrolides and quinolones), bactericidal agents (bis-
muth salts) and antiprotozoal agents (metronidazole).
Although it is widely recognized that this therapy plays
a critical role in improving and/or preventing these gas-
tric pathologies, eradication is not always successful and
a few problems have been observed in the use of these
drugs, such as the emergence of drug resistance11,12
and low compliance,13 related to the occurrence of a
number of harmful side effects.14,15
It is well known that Helicobacter pylori, an S-shaped
spiral microaerophilic, Gram-negative bacterium first
isolated in human gastric mucosa in 1982,1–3 is consid-
ered the major causative agent of several gastric pathol-
ogies, such as chronic active gastritis, peptic ulcer
disease and gastric cancer.4–6 Clinical evidence shows
that the eradication therapy of this microorganism,
which is believed to be an important etiological factor
in the pathogenesis of certain malignant peptic diseases,
can significantly reduce the risk of ulcer relapse and may
help prevent mucosa-associated lymphoid tissue
(MALT)-type gastric carcinoma and other gastric can-
cers.7–9 Hence, the World Health Organization (WHO)
has proposed H. pylori as a Class 1 carcinogen in hu-
mans, since it has been demonstrated that chronic infec-
tion is strongly associated with the development of
gastric malignant diseases.10
As a result, there is a need to develop new, alternative
therapeutic agents with highly selective antibacterial
activity against H. pylori, but without the risk of resis-
tance or other untoward effects.16
At the start of this work, we reasoned that known anti-
microbial agents may not be an appropriate therapy,
since they may favour the emergence of resistant colonies
and also present a potential for the disruption of intesti-
nal microbial flora, which is responsible for side effects.
Until recently, the most effective treatment regimens
have included a combination of antibiotics (b-lactams,
Thus, in order to try and overcome these problems, as a
part of a screening program of a number of compounds,
we decided to evaluate a series of pyrazoline derivatives.
Keywords: Pyrazoline; Antibacterial activity; Anti-Helicobacter pylori
activity.
It is worth noting that some pyrazoles17 and several
1,3,5-trisubstituted pyrazolines are reported to exhibit
*
Corresponding author. Tel.: +39 06 49913975; fax: +39 06
49913763; e-mail: bruna.bizzarri@uniroma1.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.042

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F. Chimenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 603–607
moderate antibacterial and antifungal activity.18–20 Be-
sides, a number of acetylpyrazoline derivatives were
found to possess antibacterial activity.21–23 More re-
cently, some new classes of pyrazole compounds acting
as selective inhibitors of H. pylori, with little or no activ-
ity towards other Gram-positive and Gram-negative
bacteria, have been described.24,25
and the substituted acetophenone, using 20% potassium
hydroxide as catalyst in ethanol (Route 1 in Scheme 1).
Since the potassium salts precipitated because of the
highly alkaline reaction environment due to the synthe-
sis of the 2,4-dihydroxy chalcones L–O, the hydroxyls
had to be protected with 3,4-dihydro-a-pyrane before
the condensation reaction. Of the two hydroxyls only
the one in position-4 is protected, while the one in posi-
tion-2 is not available as it is involved in an intramole-
cular hydrogen bond with the oxygen atom of the
carbonyl group. The acetophenone thus protected reacts
with the aldehyde to form the protected chalcone, which
is subsequently freed by hydrolysis (Route 2 in Scheme
1).
Encouraged by this and pursuing our research,26–28 we
here report on the antimicrobial evaluation of a series
of N1-substituted 3,5-diphenyl pyrazoline derivatives
against the most common pathogens, both bacterial
and fungal, and against H. pylori. In previous studies,
we developed effective routes to synthesize these com-
pounds; here, we carried out studies first to evaluate
the activity of these compounds against Gram-positive
and Gram-negative bacteria and then to investigate
anti-H. pylori activity.
For all the synthesized compounds, except for P1, the
diagnostic infrared absorptions were 3450–3100 cmꢀ1
(OH), 1618 cmꢀ1 (C@O) and 1590 cmꢀ1 (C@N). As re-
gards 1H NMR spectra we always detected the following
typical peaks for the pyrazoline nucleus: 5.53–5.47 (q,
1H, H5), 3.89–3.79 (m, 1H, H04) and 3.28–3.20 (dd, 1H,
H4). For the dihydroxy derivatives, P9–P13, two differ-
ent peaks for the hydroxyl groups were observed as
broad singlets at 10.26 and 10.04 ppm, which disap-
peared on treatment with D2O. For the 2- or 4-hydroxy
derivatives, P1 and P3–P8, we only observed a broad
The 1-(4-Cl-phenyl) and 1-acetyl-3,5-diphenyl-4,5-di-
hydro-(1H)-pyrazole derivatives P1–P13 were prepared
by reacting chalcone with appropriate hydrazine in ace-
tic acid26–28 (Scheme 1 and Table 1).
The chalcones A–I were obtained by direct Claisen-
Schmidt condensation between the aromatic aldehydes
Scheme 1.

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 603–607
Table 1. Chemico-physical data of derivatives P1–P13
605
Compd
R
R0
R00
Yield (%)
Mp (°C)
P1
P2
2-OH
H
4-Cl phenyl
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
4-OCH3
4-Cl
4-Cl
66
74
66
58
83
73
93
90
52
70
75
70
60
197–198
110–112
136–138
160–164
214–215
259–260
183–184
217–218
259–260
256–258
253–255
253–255
242–244
P3
P4
2-OH
2-OH
4-OH
4-OH
4-OH
4-OH
2,6-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
3,4-OCH3
2-Cl
4-Cl
P5
P6
P7
P8
2-OCH3
4-OCH3
4-CH3
4-CH3
4-Cl
P9
P10
P11
P12
P13
4-OCH3
2,4-OCH3
singlet at 10.20 ppm, which disappears with D2O. For
all compounds, P1–P13, the aromatic protons appear
as multiplets in the 7.60–6.40 ppm range. The methyl
group of the N1-acetyl was always observed at
2.03 ppm, while for the mono- and di-methoxy deriva-
tives, P1, P4, P7, P8, P12 and P13, a singlet centered
at 3.73 ppm was observed. Finally, the methyl substi-
tuted compounds, P9 and P10, show an additional peak
at 2.35 ppm.
NCTC 11637 and the other three metronidazole resis-
tant strains 8, 19 and 20. The MIC ranges and the
MIC at which 50% (MIC50) and 90% (MIC90) of the
H. pylori tested strains are inhibited are presented in
Table 2, together with the MIC values of the prepared
compounds against the metronidazole resistant strains
of H. pylori.
All the assayed compounds showed interesting activity
against H. pylori strains (MIC 1–8 lg/mL) with the
exception of derivatives P1 and P13, whose MIC values
were in the 32–128 lg/mL range. The pyrazolines P7, P8
and P12, bearing a methoxy group in the ortho- or para-
position in the 5-phenyl ring and 4-hydroxy and 2,4-
dihydroxy groups in the 3-phenyl ring, showed the low-
est MIC against the metronidazole resistant strains, sug-
gesting their importance as substituents. The presence of
a second methoxy group in the 5-phenyl ring did not
positively affect the activity (see P4 and P13 derivatives).
An important requisite is the presence of the acetyl
group on the N1 of the pyrazoline nucleus, which ap-
pears necessary for activity. In fact on comparing the
MIC values of compound P1 and compound P8, respec-
tively, with a phenyl group and an acetyl group on the
N1 of the pyrazoline, it can be observed that the former
The synthesized compounds P1–P13 were first assayed
against different species of Gram-positive and Gram-
negative bacteria of clinical relevance and also against
various strains of pathogenic fungi in order to identify
those with little or no activity as leading compounds.
The data obtained against all the assayed species listed
in the Experimental section were in the range 64 to
>128 lg/mL range. From these results it was possible
to select all the synthesized compounds for subsequent
screening towards H. pylori.
Comparison of activity of the substances with the refer-
ence compound metronidazole was carried out against
17 strains of H. pylori including the reference strain
Table 2. Minimal inhibitory concentration (MIC) of compounds P1–P13 and metronidazole (M) against 17 H. pylori strains, including four
metronidazole resistant strains
MIC (lg/mL)
Compd
R
R0
R00
All strains
MIC50
Metronidazole resistant
Range
MIC90
NCTC 11637
8
19
20
P1
P2
2-OH
H
4-Cl phenyl
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
–C(O)CH3
4-OCH3
4-Cl
4-Cl
32–128
2–8
2–8
64
4
64
8
128
8
128
8
32
8
128
8
P3
P4
2-OH
2-OH
4-OH
4-OH
4-OH
4-OH
2,6-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
4
8
8
8
2
8
3,4-OCH3
2-Cl
4-Cl
2–16
1–8
8
16
8
16
16
16
8
8
8
16
8
P5
P6
2
16
16
4
16
8
0.25–16
4–32
2–16
8–128
2–16
2–8
8
16
16
8
8
P7
P8
2-OCH3
4-OCH3
4-CH3
4-CH3
4-Cl
8
2
2
4
1
4
4
4
>64
P9
32
4
128
16
8
64
4
32
4
>64
16
4
P10
P11
P12
P13
M
16
8
4
4
8
4-OCH3
2,4-OCH3
4–32
2–32
0.125–64
8
8
8
4
4
4
16
0.5
16
64
16
64
16
64
16
64
32
64

606
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 603–607
is not active, while the latter is one of the most active.
The presence in the 5-phenyl ring of an electron-with-
drawing group, such as chlorine, or that of a methyl
group does not affect activity, except for compound P3
against the metronidazole resistant strain 19 (Table 2).
(DMSO). Organisms from routine clinical Gram-posi-
tive (S. aureus, S. epidermidis, Staphylococcus spp., S.
faecium) and Gram-negative isolates (E. coli, K. pneumo-
niae, E. cloacae, P. stuartii) and four Candida species
`
isolates (C. albicans, C. krusei, C. sake and C. parapsilo-
sis) from the respiratory tract were collected from speci-
mens of patients at the ÔAzienda Policlinico Umberto IÕ
of Rome ÔLa SapienzaÕ University. The isolates were
subcultured on qualified medium to ensure purity. Iden-
tification of the isolates was performed by conventional
methodologies; all isolates were subcultured to ensure
optimal growth.
2. Experimental32
2.1. Synthesis
2.1.1. General procedure for the preparation of chalcones
A-I (Route 1). An aqueous solution of potassium
hydroxide (20% 10 mL) was added whilst stirring over-
night to a solution of the appropriate aryl aldehyde
(30 mmol) and appropriate acetophenone (30 mmol) in
ethanol 96% (75 mL) at room temperature. The reaction
mixture was then poured into water (100 mL) and after
neutralization with hydrochloric acid (5%) a yellow solid
was recrystallized from ethanol.
The in vitro antibacterial activities of the compounds
were determined by the broth micro dilution method,
as recommended by the National Committee for Clini-
cal Laboratory Standards29 with Mueller-Hinton II
¨
broth (BBL Microbiology Systems, Cockeysville, MD).
Microtiter plates containing serial dilutions of each anti-
microbial agent were inoculated with each organism to
yield the appropriate density (105/mL) in a 100 lL final
volume; each plate included positive controls (bacteria
without a compound) and a negative control (medium
only). The plates were incubated for 18–22 h at 35 °C.
The Minimal Inhibitory Concentration (MIC) for all
isolates was defined as the lowest concentration of anti-
microbial agent that completely inhibited the growth of
the organism, as detected by an unaided eye.
2.1.2. General procedure for the preparation of 2,4-
dihydroxy-phenyl chalcones L–O (Route 2). A solution of
3,4-dihydro-a-pyran (89.68 mmol) in methylene chloride
(50 mL) was added dropwise to a suspension of 2,4-
dihydroxy-acetophenone (30 mmol) and pyridinium p-
toluene sulfonate (PPTS) (0.72 mmol) in methylene
chloride (150 mL) and stirred at room temperature for
12 h. The reaction mixture was washed with water, the
organic layer dried (Na2SO4) and concentrated in vacuo
to obtain the protected acetophenone. A solution of 2,4-
dihydroxy-4-(tetrahydropyran-2-yloxy) acetophenone
(5 mmol) and the appropriate benzaldehyde (5 mmol)
in ethanol (12 mL) was slowly added to a solution of
barium hydroxide octahydrate (7.43 mmol) in ethanol
(60 mL) and stirred for 24 h at 40 °C. The reaction mix-
ture was concentrated in vacuo, washed with water
(100 mL), neutralized with 1 M HCl and extracted with
ethyl acetate. The organic layer was dried (Na2SO4) and
concentrated in vacuo yielding the crude 2,4-dihydroxy-
4-(tetrahydropyran-2-yloxy) chalcone. The compound
was suspended in ethanol (30 mL) and p-toluene sulf-
onic acid (0.12 mmol) was added. The reaction mixture
was stirred for 12 h at room temperature, diluted with
water (30 mL), neutralized with Na2CO3 and extracted
with ethyl acetate. The organic layer was dried (Na2SO4)
and evaporated in vacuo to give the chalcones L–O.
The in vitro antifungal activities of the compounds were
determined by the broth micro dilution method with
Sabouraud dextrose broth (BBL Microbiology Systems,
Cockeysville, MD) as recommended by the NCCLS.30
Microtiter plates containing serial dilutions of each anti-
microbial agent were inoculated with each organism to
yield the appropriate density (103/mL) in a 100 lL final
volume; each plate included positive controls (fungi
without a compound) and a negative control (medium
only). The plates were incubated for 24 h at 37 °C. The
MIC for all isolates was defined as the lowest concentra-
tion of antimicrobial agent that completely inhibited
growth of the organism as detected by an unaided eye.
Anti-H. pylori activity: Sixteen clinical H. pylori isolates
and the reference strain NCTC 11637 were used. Four of
these strains were metronidazole resistant.
They were maintained at ꢀ80 °C in Wilkins Chalgren
with 10% (v/v) horse serum (Seromed) and 20 % (v/v)
glycerol (Merck) until they were used for the experi-
ments. The bacteria were grown on Columbia agar base
(Difco Laboratories) supplemented with 10% horse ser-
um (Seromed) and 0.25% Bacto yeast extract (Difco)
incubated for 72 h at 37 °C under microaerobic condi-
tions (10% CO2) in a gas incubator (Haereus). Before
use the media were always preincubated under the same
microaerobic conditions for a minimum of 2 h to allow
equilibration and none of the cultures were kept in air
for more than 15 minutes.
2.1.3. General procedure for the preparation of 1-(4-Cl-
phenyl) and 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyr-
azole derivatives P1–P13. A solution of chalcone A–O
(5 mmol) in 30 mL of acetic acid was added dropwise
to 12.5 mmol of the appropriate hydrazine in 10 mL of
absolute ethanol and stirred at 120 °C for 24 h. The mix-
ture was then poured in ice water obtaining the crude
pyrazole derivatives P1–P13, which were crystallized
from ethanol.
2.2. Antimicrobial Activity
2.2.1. Antibacterial and antifungal activity. All synthe-
sized derivatives were evaluated for their antimicrobial
and antifungal activity dissolved in dimethylsulfoxide
The MIC were determined by the agar dilution standard
method31 incubating the bacteria in microaerobic
conditions.

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 603–607
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Acknowledgements
This work was supported by grants from MURST.
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