New hybrid pyrazole and imidazopyrazole antinflammatory agents able to reduce ROS production in different biological targets

Article abstract of DOI:10.3390/molecules25040899

Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure–activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets.

Full text of DOI:10.3390/molecules25040899

molecules
Article
New Hybrid Pyrazole and Imidazopyrazole
Antinflammatory Agents Able to Reduce ROS
Production in Different Biological Targets
Chiara Brullo 1,
*
, Matteo Massa 1, Federica Rapetti 1, Silvana Alfei 2 , Maria B. Bertolotto 3,
Fabrizio Montecucco 3 , Maria Grazia Signorello 4 and Olga Bruno 1
1
Department of Pharmacy, Section of Medicinal Chemistry, University of Genoa, Viale Benedetto XV 3,
I-16132 Genova, Italy; matteo_massa@outlook.it (M.M.); federica.rapetti@edu.unige.it (F.R.);
obruno@unige.it (O.B.)
Department of Pharmacy, Section of Chemistry and Pharmaceutical and Food Technologies, University
2
of Genoa, Viale Cembrano 4, I-16148 Genova, Italy; alfei@difar.unige.it
First Clinic of Internal Medicine, Department of Internal Medicine, and IRCCS Ospedale Policlinico San
3
Martino Genova-Italian Cardiovascular Network, Largo R. Benzi 10, I-16132 Genoa, Italy;
Maria.Bianca.Bertolotto@unige.it (M.B.B.); Fabrizio.Montecucco@unige.it (F.M.)
Department of Pharmacy, Biochemistry Lab., University of Genoa, Viale Benedetto XV 3, I-16132 Genova,
4
Italy; Mariagrazia.Signorello@unige.it
*
Correspondence: brullo@difar.unige.it; Tel.: +39-010-3538368
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 January 2020; Accepted: 16 February 2020; Published: 18 February 2020
Abstract: Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a
large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain
new molecules potentially able to act on different targets involved in inflammation onset we designed
and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to
differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed
antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good
inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in
inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming
their ability to exert an antiinflammatory response by two independent mechanism. Structure–activity
relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole)
and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if
hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones.
In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the
hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized
by a strong antioxidant activity in different biological targets.
Keywords: pyrazole-4-carbohydrazides; imidazopyrazole-7-carbohydrazides; reactive oxygen
production inhibition; platelets; neutrophils; phosphodiesterase inhibitors
1. Introduction
Inflammation is a very complex response of mammals’ body to different external injuries, including
traumatic events, contact with irritant substances, pathogens invasion and more. There are different
stages in inflammation evolution: the acute phase includes the movement of leukocytes from the
blood vessel to the tissues and the immune system activation, aiming to damage control. Then, the
prolongation of this initially protective response can lead to a state of chronic inflammation that is
Molecules 2020, 25, 899; doi:10.3390/molecules25040899
www.mdpi.com/journal/molecules

Molecules 2020, 25, 899
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characterized by the intervention of other type of cells (such as macrophages and lymphocytes) and
injured tissues destruction. Several pathologies are caused by chronic inflammation.
Neurodegenerative disorders are typically multi-factorial diseases. Neuroinflammation is one
among the different pathological alteration responsible for their onset and, as other multi-target
pathologies, is difficult to treat by a mono-therapy. To get around this problem a new medicinal
chemistry paradigm suggests the use of multi-targeted compounds, single small molecules able to
act with different mechanism of action against two or more target [1,2]. Polypharmacology with
multitarget compounds looks like a winner strategy as it retains the advantages of the multi-drugs
therapy without the toxicity and compliance problems related to the use of more than one molecule in
combination. Since there are different cellular pathways involved in inflammation progression, and
also in neuroinflammation, a multi-target molecules could be very useful in several pathologies such
as Alzheimer’s disease (AD) or multiple sclerosis (MS).
A multi-target compound can be designed by combining two different molecules (obtaining
“chimera” compound) or by overlapping two pharmacophore fragments (obtaining “fused” or
“hybrid” molecule). Our previous research on the antiinflammatory agent topic allowed us to identify
different pyrazolylureas
towards inflamed tissues (chemotaxis inhibitors) with IC50 values in the pico–micromolar range [
Pyrazolylureas inhibited only interleukin 8 (IL8)-induced chemotaxis while imidazopyrazoles
inhibited only formylmethyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. The molecular
stiffening of previous compounds generates a new class of imidazo-pyrazole derivatives where the
urea group is in part inserted in the cycle and in part on the lateral chain (compounds , Figure 1). The
new class of compounds obtained by overlapping and was also considered a successful example
of hybrid derivatives. Indeed, they showed a good dual activity against both IL-8 and fMLP-induced
chemotaxis, with IC50 values in the nanomolar range [ ], by inhibiting p38 mitogen-activated protein
1
and imidazopyrazoles
2
(Figure 1) able to inhibit the neutrophil movement
3–5
].
1
2
1
3
3
1
2
6
kinase (MAPK) phosphorylation, while no inhibition was observed on superoxide anion production
and lysozyme release [7].
N
N
N
OH
EtOOC
O
N
R
NH
N
H
HN
1
R
2
R = alkyl, cycloalkyl,
benzyl
R = COOH, COOEt,
CO-NR'R"
N
N
EtOOC
N
O
NH
R
3
Figure 1. General structure of chemotaxis inhibitors 1, 2, 3.
In the same years we developed a large library of phosphodiesterase type 4D inhibitors (PDE4DIs).
Among them many compounds (particularly GEBR-54 [8] and GEBR-32a [9], Figure 2) showed
interesting PDE4D3 selective inhibition, and in vitro and in vivo assays evidenced their great potential
as memory and cognitive performance enhancers (particularly in AD) without causing side-effects,
such as sedation or emesis, in comparison with other PDE4Is [8–10]. On the other hand, PDE4Is have
been largely studied for their anti-inflammatory effects, particularly in autoimmune diseases such

Molecules 2020, 25, 899
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as psoriasis [11], asthma and chronic obstructive pulmonary disease (COPD) [12
,13]. More recently,
several evidences suggested PDE4Is also for neurodegenerative inflammatory diseases [
first series of PDE4Is synthesized by our research group showed a great anti-inflammatory activity: in
particular, compound GEBR 4a (Figure 2) and some analogues were able to reduce the superoxide
9,14]. The
-
anion and to increase cAMP intracellular level in a dose dependent manner, activity strictly related to
PDE4 inhibition [15].
O
CH3
R
O
O
N
HO
N
N
O
O
N
O
N
H
OH
O
R = CH3 GEBR-54 PDE4D3 IC50 = 7.6 μM
R = CHF2 GEBR-32a PDE4D3 IC50 = 2.4 μM
GEBR-4a PDE4D3 IC50 = 2.8 μM
Figure 2. Structures of PDE4D Inhibitors with memory enhancing and anti-inflammatory activities.
Corresponding IC50 values relative to PDE4D3 inhibition are reported.
Supported by the interesting results obtained with hybrid derivatives 3, and also having in mind
the potential role of PDE4Is in inflammation, we designed new hybrid compounds to obtain molecules
able to act on inflammation through different mechanism. The pyrazole and imidazo-pyrazole scaffolds
present in compounds 1, 2 and 3 were linked, through an acylhydrazone chain, to a substituted catechol
portion that characterized our previous PDE4Is. The acylhydrazone linker was suggested by published
analogue PDE4Is [16]. Different alky or aryl substituents were inserted on the catechol oxygens in
the new compounds 4 and 5, to increase the molecular diversity and to obtain more information for
structure-activity-relationship (SAR) studies; in many of the new synthesized compounds the OCHF2
group has been inserted instead of OCH3, in analogy to the more active previous GEBR compounds.
Once, also a trifunctionalized phenolic scaffold was used (Figure 3, Table 1). In compound 4k a pyrrole
was inserted in position 5, to verify if a free NH2 is necessary for the activity. For the same reason, the
NH in position 1 of the imidazopyrazole 5d was acetylated, thus obtaining compound 5k.
Figure 3. Structures of new hybrid compounds 4a–k, 5a–k and building block 8a–m used in
their preparation.
Compounds 4d and 5d, selected on the basis of their similarity with the parental GEBR molecules,
were preliminary tested to evaluate the chemotaxis inhibition and H2O2 production in human
neutrophils fMLP stimulated, in order to compare them with compounds 3. Surprisingly, the
compounds showing very low or absent chemotaxis (data not shown), whereas a potent antioxidant
activity emerged, as reported in the Results section.

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Table 1. Decorations on the hybrid structures 4a–k, 5a–k and the benzaldehydes 8a–m used for
their preparation.
Comp.
4a, 8a
4b, 8b
4c, 8c
4d, 8d
4e, 8e
4f, 8f
4g, 8g
4h, 8h
4i, 8i
R
R1
R2
R3
R4
H
O-CH3
O-CH3
O-CH(CH3)CH2CH3
O-cyclopentyl
O-cyclopentyl
O-C6H5
O-CHF2
H
H
H
H
H
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
O-cyclopentyl
H
H
H
H
H
H
O-CH3
O-CH3
O-CHF2
O-CHF2
O-CHF2
O-CH3
H
H
H
O-CH2C6H5
O-CH3
O-CH3
H
O-C6H5
O-CH2C6H5
O-CH3
O-CH3
4j, 8j
H
H
4k, 8d
5a, 8k
5b, 8b
5c, 8c
5d, 8d
5e, 8e
5f, 8l
H
H
O-cyclopentyl
O-CH3
O-CH3
O-CH3
H
O-CH3
O-CH3
O-CHF2
O-CH3
H
H
H
H
H
H
H
1-pyrrolyl
H
H
H
H
H
H
O-cyclopentyl
O-CH3
H
H
H
H
O-CH(CH3)CH2CH3
O-cyclopentyl
O-cyclopentyl
O-cyclohexyl
5g, 8m
5h, 8h
5i, 8i
5j, 8j
5k, 8d
H
H
O-CH2-O
O-CH3
O-CH3
O-CH3
H
O-CH3
H
H
H
H
O-CH3
H
H
O-CH3
O-C6H5
O-CH2C6H5
H
H
H
O-cyclopentyl
COCH3
Inflammation and oxidative stress are deeply connected since inflammation can lead to reactive
oxygen species (ROS) production. Oxidative stress occurs in response to injury or to altered metabolic
state as a result of an excessive formation of bioactive oxidation products with respect to the
capacity of the endogenous scavenging mechanism. ROS are important cellular and tissue damage
mediators and are implicated in many pathological processes such as atherosclerosis [17], diabetes [18],
neurodegeneration [19], aging [20] and inflammation [17].
As reported before, interesting antioxidant activity was already observed for our PDE4 inhibitor
GEBR-4a and also other rolipram analogues [21]. Therefore, to clarify if the antioxidant activity
revealed by hybrid compounds
4 and 5, was only due to a cAMP increase or to an independent
additional mechanism, we planned to evaluate the ROS production inhibition on human platelets,
instead of neutrophils, as platelets are lacking in PDE4 enzymes but, in addition to their central role in
the haemostatic process, are involved in the inflammatory process and in the ROS production [22,23].
To date inflammation, thrombosis and haemostasis are reorganized as processes with overlapping
steps and connecting pathways. Following injury to blood vessels, such as during inflammation,
platelets adhere to endothelium with subsequent thrombin formation. Platelet response to thrombin
include cytoskeleton reorganization, degranulation and secretion of proinflammatory cytokines, such as
IL-1, tumor growth factor (TGF), platelet derived growth factor (PDGF); therefore thrombin contribute
to modifying endothelial function participating in the spreading of an inflammatory lesion [22,23].
2. Results and Discussion
2.1. Chemistry
Compounds 4a
benzaldehydes 8a
the pyrazolylcarbohydrazides 7, 10 or the imidazopyrazolyl carbohydrazide 12, respectively.
–
k
and 5a
–k (Scheme 1, Table 1) were obtained by refluxing the suitable
–
m solved in a mixture of absolute ethanol/methanol (7:3) in the presence of

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Scheme 1. Synthesis of compounds
4 and 5. Reagents and conditions: (i) hydrazine monohydrate,
120–130 ◦C, 4 h; (ii) abs. EtOH/MeOH (7:3), reflux, 18 h; (iii) 2,5-dimethoxytetrahydrofuran, acetic acid,
reflux, 4 h; (iv) conc. H2SO4, 0 ◦C, 15 min; (v) acetic anhydride, CH3COONa, 40–50 ◦C, 4 h.
Although formation of E/Z mixtures is possible, only one of the two isomers has been always
obtained as confirmed by 1H- and 13C-NMR spectra. Since aldehyde hydrazones generally adopt
C=N E-geometry [24] and E-geometry was always assigned to the predominant or single isomer
obtained [25
Once acquired NMR spectra, the E-geometry was definitively assigned on the basis of the CONH
group proton atom chemical shift ( 11.23 ppm), in accordance to the literature data. In this regard,
–30], for the prepared acyl hydrazones, the E configuration was a priori hypothesized.
δ
to a series of hydrazone derivatives, having signals of the NH group <12 ppm as in our case, the E
configuration was assigned by NOESY experiments and DFT calculations [31]. Furthermore, it was
also reported that NH chemical shift for Z isomers is instead moved to much higher delta values (
15.5 ppm) [32]. H-NMR spectra images are reported in the Supporting Information.
δ
>
1
Intermediate
5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylate 6 [33] (Scheme 1). We obtained the
5-pyrrolyl analogue by refluxing the pyrazolyl ethyl carboxylate in 2,5-dimethoxy-tetrahydrofuran
in the presence of acetic acid. Moreover, starting from the same compound we prepared
ethyl 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylate (11) [ ] by dehydration in
concentrated sulfuric acid. The carbohydrazides 10 and 12 were obtained by the same procedure
used for . To prepare derivatives and , the suitable benzaldehydes 8h and 8m (Table 1) were
7 was prepared by reacting an excess of hydrazine monohydrate with the ethyl
9
6
6
4
7
4
5
purchased, while the others were prepared following well known procedures. In details, the
3-phenoxy-substituted benzaldehydes 8f and 8i were obtained by adding diphenyliodonium chloride
to 3-hydroxy-4-difluoromethoxy-benzaldehyde [34] (for 8f) or 2-hydroxy-3-methoxy-benzaldehyde
(for 8i) previously converted into the salt forms with NaOH aqueous solution (Crowder’s method [35]).
The others intermediates were prepared by alkylation with the appropriate alkyl chloride in the
presence of K2CO3 [36] starting from the corresponding phenolic benzaldehydes: in particular from
3-hydroxy-4-difluoromethoxy benzaldehyde (to obtain 8a
,
e
,
f,
g
), from isovanillin (to prepare 8c
,
d,
l
), from vanillin (for 8k), and from 2-hydroxy-3-methoxybenzaldehyde (compounds 8b
,
8i, 8j).
Finally, compound 5k was obtained from 5d by treatment with acetic anhydride in the presence of
sodium acetate.

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2.2. ROS Production and Aggregation Inhibition Evaluated in Human Platelets in the Presence of Compounds
4a–k and 5a–k
Since in the presence of high intracellular ROS platelets demonstrate increased activation by
agonists and ROS elevation leads to platelet aggregation that can in turn worsen the inflammation
status, preliminary we have tested the aggregation inhibiting effect of compounds as detailed in the
Experimental section. Results of aggregation inhibition and ROS production inhibition are reported in
Table 2. Most of the tested compounds were able to inhibit aggregation and ROS production, being 4a
,
4f and 4g the most active with IC50 values around 10 µM. Interestingly, the same 4a, 4f and 4g were
the most active also as platelet aggregation inhibitors.
Table 2. Inhibiting effect of compounds 4a
(ROS) production expressed as IC50 M) values. Reported data are the mean +/- SD obtained in at
least six different experiments each performed in duplicate.
–k and 5a–j on aggregation and reactive oxygen species
(
µ
Compd.
Aggregation Inhibition IC50 (µM)
ROS production Inhibition IC50 (µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
7.6 ± 0.5
64 ± 2.5
57.1 ± 1.6
29.8 ± 3.7
29.4 ± 4.4
9.8 ± 1.7
11.0 ± 1.2
>200
10.1 ± 2.1
20.5 ± 2.5
16.7 ± 1.1
38.3 ± 2.8
32.8 ± 2.8
8.6 ± 2.2
9.5 ± 2.6
>200
>200
>200
4j
>200
>200
4k
5a
5b
5c
5d
5e
5f
5g
5h
5i
86.1 ± 7.8
31.5 ± 5.2
59.1 ± 5.5
70.9 ± 4.0
71.0 ± 6.8
70.5 ± 8.0
61.6 ± 6.5
67.0 ± 6.6
>200
92.7 ± 5.8
37.2 ± 2.5
66.7 ± 5.7
71.4 ± 6.0
70.6 ± 7.4
78.8 ± 5.6
59.9 ± 5.7
77.9 ± 7.1
>200
>200
>200
5j
>200
>200
5k
>200
>200
2.3. Structure-Activity Relationships
SAR analysis clearly indicated that the OCHF2 substituent at position 4 is a pivotal structural
characteristic, while the substituent in position 3 seems not to be determinant for potency. Another clear
evidence is that compounds bearing the pyrazole nucleus are more active than the imidazo-pyrazole
ones. This is particularly evident by comparing 4b, 4c, 4d and 4e to the corresponding imidazopyrazole
derivatives 5b 5c 5d and 5e, the potency of the first group being approximately from two to four
,
,
times higher than the second ones.
In addition, we can point out that two substituents in the positions 3 and 4 of the aromatic ring are
positive for biological activity, differently than the trisubstitution: in fact, both analogues 4h and 5h are
inactive. A bulky substituent such as a cyclopentyl in position 2 instead of 3 is well tolerated: indeed,
we observed a slight increase of activity in the pyrazole series (compare 4b with the regioisomer 4d
or no change in the imidazopyrazole series (compare 5b with 5d); whereas, aromatic substituents
such benzyloxy or phenoxy in position 2 are detrimental for the activity; in fact, 4i 4j 5i and 5j are
)
,
,
completely inactive. A slight reduction of activity is also evident when the meta and para substituents
are closed in a ring (see compound 5g).

Molecules 2020, 25, 899
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Moreover, it is noteworthy that the presence of a free amino group at position 5 of the pyrazole
ring is fundamental to block ROS production, as the 5-pyrrolyl compound 4k showed lower activity
compared to the 5-NH2-substituted analogue 4d (IC50 = 90
obtained by acetylating the NH group in position 1 of the imidazopyrazole ring, being the acetyl
derivative 5k completely inactive, while the corresponding 5d showed an IC50 value of 70.6 M. This
µM versus 40 µM). A similar result was
µ
observation suggests that, even if the imidazopyrazoles are in general less active than the pyrazole
ones, the effect on ROS production could be due to the same mechanism, which probably requires an
H-bond donor group and a good degree of molecule flexibility.
Finally, we also tested in the same assay the intermediates
ROS production inhibition of compounds and is due to the molecule hybridization and not to
the pyrazole or imidazopyrazole scaffold alone. As we hypothesized, both and 12 were completely
inactive towards ROS production in platelets, as well as in neutrophils (data not shown). For the same
7 and 12 in order to confirm that the
4
5
7
reason we tested on platelets also some compounds among the previous series
as well (data not shown).
3 that resulted inactive
2.4. Flow Cytometric Evaluation of ROS Production on Human Neutrophils in the Presence of Compounds 4d
and 5d
As reported in the introduction, to verify if the pharmacological activities already evidenced
in previous compounds was maintained, preliminary tests were performed on some of the new
hybrid compounds (in particular 4d and 5d, bearing the catechol structure typical of previously
synthesized PDE4DIs). Firstly, the ability of 4d and 5d in inhibiting neutrophil chemotaxis were
evaluated in order to compare the results with compound GEBR-4a. Unexpected negative results (data
not shown) suggested that newly synthesized compounds should have a different target. Consequently,
the ability to inhibit ROS production was also evaluated on neutrophils fMLP activated using flow
cytometric analysis, according to literature method [37]. Interestingly compounds 4d and 5d at 10 µM
concentration were able to inhibit ROS production with an inhibition percentage of 68% and 63%
respectively, using the 20,70-dichlorofluorescein diacetate method (DCFH-DA), a fluorogenic dye that
measures hydroxyl, peroxyl and other reactive oxygen species.
In Figure 4 we report the intracellular ROS level inhibition (4A) and the corresponding
dose-dependent effect (4B) of 4d alone, since results were very similar for 5d. In the same test
also the intermediates
the same time, using propidium iodide (PI) method [38], we assessed cell vitality and no cytotoxic
action of both compounds at 10 M concentration. Propidium iodide is able to penetrate only
7 and 12 were evaluated and resulted inactive (data not shown). In addition, at
µ
the compromised membrane of plasma cells and therefore causes red fluorescence by binding to
double-stranded DNA. On the contrary it don’t cross intact plasma cell membrane, therefore no red
fluorescence is present in vital cells, as evidenced in Figure 4C. Cell viability was over than 95%.
2.5. Enzymatic Assays on PDE4
Compounds 4d and 5d were tested on enzymatic assay to verify the ability to inhibit PDE4D3 and
PDE4B2, the isoforms most investigated in our previous studies (see experimental section). Results
indicated that hybrid compounds 4d and 5d are able to strongly inhibit both isoforms and no differences
were evidenced between pyrazole or imidazopyrazole scaffold. Indeed, 4d showed IC50 values of
1.05
0.55
µ
M on PDE4D3 and 0.55
µ
M on PDE4B2; similarly, 5d showed IC50 of 1.22
µM on PDE4D3 and
µM on PDE4B2. Preliminary evaluation of compound 4d (at 10
µM concentration) on PDE1, PDE2,
PDE3, PDE5 and PDE7 was already performed and no inhibition was observed (data not shown).

Molecules 2020, 25, 899
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Figure 4. Modulation of neutrophil oxidative metabolism and cell vitality assessed by flow cytometric
analysis in the presence of propidium iodide (PI). Representative images relative to evaluation of
compound 4d in formyl-methyl-leucyl-phenylalanine (fMLP)-activated neutrophils. ( ): reactive
oxygen species (ROS) intracellular level reduction ( ): dose-dependent effect of compound on ROS
reduction ( ): cell vitality determined by PI method. Data are expressed as mean SD and are
representative of four separate experiments.
A
B
C
±
3. Experimental
3.1. General Information
All chemicals were purchased from Chiminord and Aldrich Chemical (Milan, Italy). Solvents were
reagent grade. Unless otherwise stated, all commercial reagents were used without further purification.
Aluminium- backed silica gel plates (DC-Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany),
were used in thin-layer chromatography (TLC) for routine monitoring the course of reactions. Detection
of spots was made by UV light. Merck silica gel, 230–400 mesh, was used for chromatography. Melting
points are not “corrected” and were measured with an M-560 instrument (Buchi instruments, Flawil,
1
Switzerland). IR spectra were recorded with a 398 spectrophotometer (Perkin-Elmer, Milan, Italy). H
NMR spectra were recorded on a Gemini 200 (200 MHz, Varian Gemini, Palo Alto, CA, USA) and a
DPX-300 (300 MHz) instrument (Bruker, Billerica, MA, USA); chemical shifts are reported as δ (ppm)
relative to tetramethylsilane (TMS) as internal standard; signals were characterized as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), br s (broad signal), n t (near triplet); J in Hz. Elemental
analyses were determined with an elemental analyzer EA 1110 (Fison-Instruments, Milan, Italy) and
the purity of all synthesized compounds was >95% (see Supplementary Materials). Flow cytometric
assays were performed by flow cytometry (FC500, Beckman Coulter, Hialeah, FL, USA).

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3.2. Synthesis
3.2.1. General Procedure for 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (
and 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (12)
7)
A mixture of 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl ester (6,
0.81 g, 3.1 mmol) or 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylic acid ethyl ester
◦
(
11, 0.8 g, 3.1 mmol) and hydrazine monohydrate (5 mL, 100 mmol) was stirred at 120 C for 8 h.
After cooling to room temperature, water (10 mL) was added and the obtained solids were purified
by Florisil column cromatography using a diethyl ether/methanol mixture (9:1) as the eluent. The
obtained solids were then recrystallized by ethanol/methanol (2:1).
5-Amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (
7
)
.
White solid. Yield 48%. Mp:
3.90–4.08
183–184 ◦C. IR (KBr) cm 1: 3513, 3389 (NH2), 3316 (OH), 1603 (C=O). 1H-NMR (CDCl3):
−
δ
(m, 2H, CH2N), 4.21 (br s, 2H, NH2 disappears with D2O), 4.86–5.03 (m, 1H, CHOH), 5.72 (d, J = 4.6,
1H, OH, disappears with D2O), 6.04 (br s, 2H, NH2, disappears with D2O), 7.21–7.47 (m, 5H, Ar), 7.66
(s, 1H, H3 pyraz.), 8.96 (br s, 1H, CONH, disappears with D2O). Analysis (%) calcd. for C12H15N5O2.
2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (12). White solid. Yield 66%. Mp
250–252 ◦C. IR (KBr) cm 1: 3296 (NH2), 1627 (C=O). 1H-NMR (DMSO-d6)
δ: 3.77 (t, J = 8.0, 1H, H3),
−
4.26 (s, 2H, NH2, disappears with D2O), 4.57 (t, J = 8.0, 1H, H2), 5.42 (dt, J = 4.0, J = 8.0, 1H, H3),
7.04 (br s, 1H, NH, disappears with D2O), 7.26–7.52 (m, 5H, Ar), 7.67 (s, 1H, H6), 8.80 (s, 1H, CONH,
disappears with D2O). Analysis (%) calcd. for C12H13N5O.
3.2.2. Preparation of Ethyl 1-(2-hydroxy-2-phenylethyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-
4-carboxylate (9)
To a solution of 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl ester
(6
) [33] (0.27 g, 1 mmol) in glacial acetic acid (3 mL), 2,5-dimethoxytetrahydrofuran (0.15 g, 1.1 mmol)
was added and the mixture was stirred at reflux temperature for 2 h. After cooling to room temperature,
the mixture was diluted with water (10 mL) and extracted with ethyl acetate (3 10 mL). The organic
phase was washed with sodium hydrogen carbonate saturated solution (1 10 mL), water (1 10 mL),
×
×
×
dried (MgSO4) and concentrated under reduced pressure to give an oil that was purified by silicagel
column chromatography using as the eluent firstly dichloromethane, then diethylether. The product
was obtained as a white solid. Yield: 55%. Mp: 103–104 ◦C. IR (KBr) cm−1: 3318 (OH), 1687 (C=O).
1H-NMR (CDCl3):
δ 1.22 (t, J = 6.4, 3H, CH3), 2.90 (br s, 1H, OH disappears with D2O), 4.03–4.29 (m,
4H, CH2O + CH2N), 5.12–5.22 (m, 1H, CHOH), 6.35 (s, 2H, H2 pyrr.), 6.58 (s, 2H, H3 pyrr.), 7.17–7.52
(m, 5H, Ar), 8.09 (s, 1H, H3 pyraz.). Analysis (%) calcd. for C18H19N3O3.
3.2.3. Preparation of 1-(2-hydroxy-2-phenylethyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-
4-carbohydrazide (10)
Ethyl 1-(2-hydroxy-2-phenylethyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-4-carboxylate (9, 0.50 g,
1.53 mmol) was solved in hydrazine monohydrate (5 mL) and the mixture was stirred at reflux
for 5 h. After cooling to room temperature, the mixture was diluted with water (10 mL) and extracted
with ethyl acetate (3
×
10 mL). The organic phase was washed with brine (2
×
10 mL), dried (MgSO4)
and concentrated under reduced pressure to give an oil that was purified by Silica gel column
chromatography using dichloromethane, as the eluent. The oil residue was crystallized by ethanol
obtaining the final product as a white solid. Yield: 61%. Mp: 145–146 ◦C. 1H-NMR (CDCl3):
δ 3.88 (d,
J = 6.4, 2H, CH2N), 4.29 (br s, 2H, NH2 disappears with D2O), 4.88-4.99 (m, 1H, CHOH), 5.74 (d, J = 4.4,
1H, OH disappears with D2O), 6.24 (s, 2H, H2 pyrr.), 6.78 (s, 2H, H3 pyrr.), 7.04–7.41 (m, 5H, Ar), 8.00
(s, 1H, H3 pyraz.), 9.07 (br s, 1H, CONH disappears with D2O). Analysis calcd. for C16H17N5O2.

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3.2.4. General Procedure for (E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N0-benzylidene-1H-pyrazole-
4-carbohydrazides 4a–k and N0-benzylidene-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-
7-carbohydrazides 5a–j
A solution of a suitable benzaldehyde
5 mL) was added dropwise to 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide
or 1-(2-hydroxy-2-phenylethyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-4-carbohydrazide 10 or
8 (2 mmol) in a mixture of ethanol/methanol (7:3,
(7
)
( )
2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (12) (2 mmol) solved in the same
mixture solvent (15 mL). The reaction mixture was stirred at 100 ◦C for 18 h, cooled to room temperature
and poured into ice water (10 mL). The white solids obtained were simply dried, if pure at TLC check
(Silica gel, diethyl ether/methanol (8:2)), or recrystallized by absolute ethanol or ethyl acetate.
(E)-5-Amino-N0-(4-(difluoromethoxy)-3-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4a). Yield 63%. White solid. Mp: 119–121 ◦C. 1H-NMR (DMSO-d6):
δ 3.91 (s, 3H,
OCH3), 3.94–4.15 (m, 2H, CH2N), 4.94–5.11 (m, 1H, CHOH), 5.72 (s, 1H, OH, disappears with D2O),
6.77 (br s, 2H, NH2, disappears with D2O), 7.10–7.70 (m, 9H, 5Ar + H2 Ar + H5 Ar + H6 Ar + OCHF2),
7.82–8.20 (m, 2H, CH=N + H3 pyraz.), 11.20 (s, 1H, CONH, disappears with D2O). Anal calcd. for
C21H21F2N5O4.
(E)-5-Amino-N0-(2-(cyclopentyloxy)-3-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4b). Yield 46%. White solid. Mp: 180–181 C. IR (KBr) cm−1: 3427 (OH), 3321,
◦
3323, 3139 (NH2 + NH), 1634 (C=O), 1572 (C=N). 1H-NMR (DMSO-d6):
δ 1.40–2.00 (m, 8H, 4CH2
cyclopent.), 3.78 (s, 3H, OCH3), 3.90–4.80 (m, 2H, CH2N), 4.80–4.90 (m, 1H, OCH cyclopent), 4.95–5.05
(m, 1H, CHOH), 5.65–5.75 (m, 1H, OH, disappears with D2O), 6.20–6.50 (br s, 2H, NH2, disappears
with D2O), 6.68 and 7.12 (qAB, J =10.0, 2H, Ar), 7.20–7.50 (m, 6H, Ar), 7.80–8.20 (m, 2H, CH=N + H3
pyraz.), 11.03 (s, 1H, CONH, disappears with D2O). Anal calcd. for C25H29N5O4.
(E)-5-Amino-N0-(3-(sec-butoxy)-4-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4c). Yield 64%. White solid. Mp: 190–193 ◦C. IR (KBr) cm−1: 3427 (OH),
1
3325, 3234, 3196 (NH2 + NH), 1644 (C=O), 1614 (C=N). H-NMR (DMSO-d6):
δ 0.91–0.94 (m, 3H,
CH3CH2), 1.18–1.35 (m, 3H, CH3CH), 1.40–1.80 (m, 2H, CH3CH2), 3.30–3.40 (m, 1H, CHCH3), 3.79 (s,
3H, OCH3), 3.90–4.20 (m, 2H, CH2N), 4.95–5.05 (m, 1H, CHOH), 5.60–5.80 (m, 1H, OH, disappears
with D2O), 6.15–6.55 (m, 2H, NH2, disappears with D2O), 6.90–7.50 (m, 8H, Ar), 7.70–8.30 (m, 2H,
CH=N +H3 pyraz.), 11.01 (s, 1H, CONH, disappears with D2O). Anal calcd. for C24H29N5O4.
(E)-5-Amino-N0-(3-(cyclopentyloxy)-4-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4d). Yield 78%. White solid. Mp: 193–195 ◦C. IR (KBr) cm−1: 3324, 3219 (NH), 2970
1
(OH), 1615 (C=N), 1642 (CONH). H-NMR (CDCl3):
δ 1.48–2.10 (m, 8H, cyclopent.), 3.89 (s, 3H,
OCH3), 4.02–4.33 (m, 2H, CH2N), 4.78–4.99 (m, 1H, OCH cyclopent.), 5.11–5.32 (m, 1H, CHOH), 5.82
(br s, 2H, NH2 disappears with D2O), 6.80–7.01 (m, 1H, H5 Ar), 7.04–7.22 (m, 1H, H6 Ar), 7.22–7.61 (m,
7 H, 6H Ar + H2 Ar), 7.75–8.38 (m, 2H, CH=N + H3 pyraz.), 9.00 (br s, 1H, CONH, disappears with
D2O). Anal calcd. for C25H29N5O4.
(E)-5-Amino-N0-(3-(cyclopentyloxy)-4-(difluoromethoxy)benzylidene)-1-(2-hydroxy-2-phenylethyl)-
1H-pyrazole-4-carbohydrazide (4e). Yield 96%. White solid. Mp: 178–180 ◦C. IR (KBr) cm−1
:
3450–3200 (NH + OH), 1635 (CONH). 1H-NMR (DMSO-d6):
δ 1.51–2.04 (m, 8H, 4CH2 cyclopent.),
3.30–3.40 (m, 2H, CH2N), 3.90–4.21 (m, 1H, CHOH), 4.84–5.10 (m, 1H, OCH cyclopent.), 5.75 (s, 1H,
OH, disappears with D2O), 6.42 (br s, 2H, NH2, disappears with D2O), 7.06–7.60 (m, 9H, 5Ar + H2 Ar +
H5 Ar + H6 Ar + OCHF2), 7.83–8.35 (m, 2H, CH=N + H3 pyraz.), 11.20 (s, 1H, CONH, disappears with
D2O). Anal calcd. for C25H27F2N5O4.
(E)-5-Amino-N0-(4-(difluoromethoxy)-3-phenoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4f). Yield 38%. White solid. Mp: 184–186 ◦C. 1H-NMR (CDCl3):
δ
3.92–4.26 (m, 2H,
CH2N), 5.08–5.23 (m, 1H, H3 pyraz.), 6.60–6.78 (br s, 2H, NH2, disappears with D2O), 7.17–7.38 (m,

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14H, 10Ar + H2 Ar + H5 Ar + H6 Ar + OCHF2), 7.97–8.38 (m, 2H, CH=N + H3 pyraz.), 9.18–9.40 (s, 1H,
CONH, disappears with D2O). Anal calcd. for C26H23F2 N5O4.
(E)-5-Amino-N0-(3-(benzyloxy)-4-(difluoromethoxy)benzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4g). Yield 61%. White solid. Mp: 144–146 ◦C. 1H-NMR (DMSO-d6):
δ 3.95–4.20 (m,
2H, CH2N), 4.92–5.10 (m, 1H, CHOH), 5.26 (s, 2H, CH2Bz), 5.75 (s, 1H, OH, disappears with D2O), 6.40
(br s, 2H, NH2, disappears with D2O), 7.16–7.70 (m, 14H, 10Ar + H2 Ar + H5 Ar + H6 Ar + OCHF2),
7.83–8.35 (m, 2H, CH=N + H3 pyraz.), 11.25 (s, 1H, CONH, disappears with D2O). Anal calcd. for
C27H25F2N5O4.
(E)-5-Amino-1-(2-hydroxy-2-phenylethyl)-N0-(3,4,5-trimethoxybenzylidene)-1H-pyrazole-4-carbohydrazide
(
4h). Yield 34%. White solid. Mp: 176–178 ◦C. IR (KBr) cm−1: 3490 (OH), 3435, 3391, 3341, 3239 (NH2
+NH), 1640 (CO), 1561 (C=N). 1H-NMR (DMSO-d6):
δ 3.36 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.83 (s,
3H, OCH3), 4.40–4.10 (m, 2H, CH2N), 4.95–5.05 (m, 1H, CHOH), 5.70–5.80 (m, 1H, OH, disappears with
D2O), 6.25–6.55 (br s, 2H, NH2, disappears with D2O), 6.98 (s, 2H, Ar), 7.25–7.50 (m, 5H, Ar), 7.80–8.20
(m, 2H, CH=N + H3 pyraz.), 11.18 (s, 1H, CONH, disappears with D2O). Anal calcd. for C22H24N4O5.
(E)-5-Amino-1-(2-hydroxy-2-phenylethyl)-N0-(3-methoxy-2-phenoxybenzylidene)-1H-pyrazole-4-carbohydrazide
(
4i). Yield 66%. White solid. Mp: 207–209 ◦C. IR (KBr) cm−1: 3395 (OH), 3324, 3287, 3206, 3239 (NH2
+NH), 1646 (CO), 1551 (C=N). 1H-NMR (DMSO-d6):
δ 3.69 (s, 3H, OCH3), 3.85–4.15 (m, 2H, CH2N),
4.90–5.05 (m, 1H, CHOH), 5.69 (d, J = 2.0, 1H, OH, disappears with D2O), 6.20–6.50 (br s, 2H, NH2,
disappears with D2O), 6.78 (d, J = 4.0, 1H, Ar), 7.00 (t, J = 4.0, 1H, Ar), 7.15–7.45 (m, 10H, Ar), 7.55 (d,
J = 4.0, 1H, Ar), 7.60–8.40 (2 m, 2H, CH=N + H3 pyraz.), 11.20 (s, 1H, CONH, disappears with D2O).
Anal calcd. for C26H25N5O4.
(E)-5-Amino-N0-(2-(benzyloxy)-3-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4j). Yield 71%. White solid. Mp: 198–200 ◦C. IR (KBr) cm−1: 3415 (OH),
1
3352, 3151, 3034 (NH2 +NH), 1627 (CO), 1564 (C=N). H-NMR (DMSO-d6):
δ 3.87 (s, 3H, OCH3),
3.90–4.20 (m, 2H, CH2N), 4.85–5.10 (m, 1H, CHOH), 4.99 (s, 2H, CH2Bz), 5.71 (d, J = 2.0, 1H, OH,
disappears with D2O), 6.20–6.60 (br s, 2H, NH2, disappears with D2O), 7.00–7.55 (m, 13H, Ar),
7.80–8.10 (br s, 1H, H3 pyraz.), 8.35–8.55 (br s, 1H, CH=N), 11.19 (s, 1H, CONH, disappears with D2O).
Anal calcd. for C27H27N5O4.
(E)-5-Amino-N0-(3-(cyclopentyloxy)-4-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carbohydrazide (4k). Yield 80%. White solid. Mp: 206–207 ◦C. IR (KBr) cm−1: 3364, 3309 (NH), 2951
(OH), 1598 (C=N), 1656 (CONH). 1H-NMR (DMSO-d6):
δ 1.45–2.15 (m, 8H, cyclopent.), 3.88 (s, 3H,
OCH3), 3.97–4.19 (m, 2H, CH2N), 4.75–4.97 (m, 1H, OCH cyclopent.), 5.17–5.33 (m, 1H, CHOH), 6.57 (s,
2H, H2 pyrr.), 6.73 (s, 2H, H3 pyrr.), 6.78–7.18 (m, 2H, H5 + H6 Ar), 7.22–7.61 (m, 7H, 5Ar + H3 pyraz. +
H2 Ar), 8.38 (s, 1H, CH=N), 9.85 (br s, 1H, CONH, disappears with D2O). Anal calcd. for C29H31N5O4.
(E)-N0-(3,4-Dimethoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (5a).
Yield 70%. White solid. Mp: 206–207 ◦C. IR (KBr) cm−1: 3432–3213 (NH), 1700–1600 (CONH + C=N).
1H-NMR (DMSO-d6):
δ 3.70–3.92 (m, 7H, 2 OCH3 + H3), 4.63 (t, J = 8.0, 1H, H2), 5.48 (n t., 1H, H3),
7.02 (d, J = 7.8, 1H, H5 Ar), 7.09 (dd, J = 7.8, J = 1.6, 1H, H6 Ar), 7.24–7.53 (m, 7H, 5 Ar + H2 Ar + H6),
7.60–8.40 (m, 2H, CH=N + NH, 1H disappears with D2O), 11.10 (s, 1H, CONH, disappears with D2O).
Anal calcd. for C21H21N5O3.
(E)-N0-(2-(Cyclopentyloxy)-3-methoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-
7-carbohydrazide (5b). Yield 78%. White solid. Mp: 150–151 ◦C. IR (KBr) cm−1: 3392–3234 (NH), 1644
(CONH), 1594 (C=N). 1H-NMR (DMSO-d6):
δ 1.47–2.20 (m, 8H, cyclopent.), 3.72–3.95 (m, 4H, OCH3 +
H3), 4.62 (n t, 1H, H2), 4.75–4.92 (m, 1H, OCH), 5.48 (n t, 1H, H3), 7.00 (d, J = 10.0, 1H, H5 Ar), 7.18 (dd,
J = 10.0, J =1.8, 1H, H6 Ar), 7.23–7.52 (m, 7H, 5H Ar + H2 Ar + H6), 7.99 (br s, 2H, NH + CH=N, 1H
disappears with D2O), 11.07 (s, 1H, CONH, disappears with D2O). Anal calcd. for C25H27N5O3.

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(E)-N0-(3-(sec-Butoxy)-4-methoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-
7-carbohydrazide (5c). Yield 62%. White solid. Mp: 130–131◦C. IR (KBr) cm−1: 3373 (NH),
1631 (CONH), 1594 (C=N). 1H-NMR (DMSO-d6):
δ 0.95 (m, 3H, CH3CH2), 1.25 (d, J = 5.6, 3H, CH3CH),
1.45–1.80 (m, 2H, CH2CH3), 3.60–3.95 (m, 4H, OCH3 + CHCH3), 4.20–4.40 (m, 1H, H3), 4.60 (t, J = 8.0,
1H, H2), 5.40 (t, J = 8.0, 1H, H3), 6.90–7.50 (m, 8H, Ar), 7.95 (br s, 2H, H6 + NH, 1H disappears with
D2O), 11.10 (s, 1H, CONH disappears with D2O). Anal calcd. for C24H27N5O3.
(E)-N0-(3-(cyclopentyloxy)-4-methoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-
7-carbohydrazide (5d). Yield 93%. White solid. Mp: 120–124 ◦C. IR (KBr) cm−1: 3415, 3324 (NH), 1581
(C=N), 1633 (CONH). 1H-NMR (CDCl3):
δ 1.47–2.20 (m, 8H, 4CH2 cyclopent.), 3.90 (s, 3H, OCH3),
4.07 (n t, 1H, H3), 4.62 (n t, 1H, H2), 4.79–4.95 (m, 1H, OCH cyclopent.), 5.50 (n t, 1H, H3), 6.83 (d, J =
8.2, 1H, H5 Ar), 7.09 (dd, J = 8.2, J = 1.6, 1H, H6 Ar), 7.29 (d, J = 1.6, 1H, H2 Ar), 7.32–7.52 (m, 5H Ar),
7.82 (s, 1H, H6 pyraz.), 8.25 (br s, 1H, NH disappears with D2O), 9.18 (s, 1H, CH=N), 9.85 (br s, 1H,
CONH disappears with D2O). Anal calcd. for C25H27N5O3.
(E)-N0-(3-(cyclopentyloxy)-4-(difluoromethoxy)benzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]-pyrazole-
7-carbohydrazide (5e). Yield 62%. Red solid. Mp: 104–105 ◦C. IR (KBr) cm−1: 3228, 2961 (NH), 1642
(CONH), 1599 (C=N). 1H-NMR (DMSO-d6):
δ 1.52–2.00 (m, 8H, 4CH2 cyclopent.), 3.82 (n t, 1H, H3),
4.64 (n t, 1H, H2), 4.87–4.99 (m, 1H, OCH cyclopent.), 5.48 (n t, 1H, H3), 7.07 (t, 1H, J = 70.0, OCHF2),
7.17–7.54 (m, 10H, 8H Ar + H6 + NH, 1 H disappears with D2O), 7.99 (br s, 1H, CH=N), 11.20 (br s, 1H,
CONH, disappears with D2O). Anal calcd. for C25H25F2N5O3.
(E)-N0-(3-(Cyclohexyloxy)-4-(methoxy)benzylidene)-2-phenyl-2,3-d◦ihydro-1H-imidazo[1,2-b]pyrazole-
7-carbohydrazide (5f). Yield 65%. White solid. Mp: 119–120 C. IR (KBr) cm−1: 3584 (NH), 1633
(CONH), 1581 (C=N). 1H-NMR (DMSO-d6):
δ 1.20–2.20 (m, 10H, 5CH2 cyclohex.), 3.80–4.10 (m, 4H,
OCH3 + OCH), 4.25 (n t, 1H, H3), 4.52 (n t, 1H, H2), 5.39 (n t, 1H, H3), 6.60–7.60 (m, 8H, Ar), 7.90 (br s,
1H, CH=N), 8.20 (s, 1H, H6), 10.15 (s, 1H, CONH, disappears with D2O). Anal calcd. for C26H29N5O3.
(E)-N0-(benzo[d][1,3]dioxol-5-ylmethylene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide
(
5g). Yield 93%. White solid. Mp: 230–233 ◦C. IR (KBr) cm−1: 3400, 3227 (NH), 1638 (CONH), 1608
(C=N). 1H-NMR (DMSO-d6):
δ 3.82 (n t, 1H, H3), 4.62 (n t, 1H, H2), 5.43 (n t, 1H, H2), 4.96 (br s, 1H,
NH disappears with D2O), 6.10 (s, 2H, OCH2O), 7.00 (d, J = 8.0, 1H, H5 Ar), 7.14 (dd, J = 8.0, J = 1.6,
1H, H6 Ar), 7.26 (d, J =1.6, 1H, H2 Ar), 7.32–7.52 (m, 6H, 5H Ar + H6), 7.92–8.20 (m, 1H, CH=N), 11.05
(br s, 1H, CONH disappears with D2O). Anal calcd. for C20H17N5O.
(E)-2-Phenyl-N0-(3,4,5-trimethoxybenzylidene)-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (5h).
Yield 74%. White solid. Mp: 120–121 ◦C. IR (KBr) cm−1: 3400–3200 (NH), 1643 (CONH), 1611 (C=N).
1H-NMR (DMSO-d6):
δ 3.60–3.90 (m, 10H, 3OCH3 + H3), 4.60 (n t, 1H, H2), 5.45 (n t, 1H, H3), 6.90–7.00
(s, 2H, Ar), 7.18 (s, 1H, H6) 7.20–7.50 (m, 5H, Ar), 7.90 (br s, 1H, CH=N), 11.10 (br s, 1H, CONH,
disappears with D2O). Anal calcd. for C22H23N5O4.
(E)-N0-(2-(Phenoxy)-3-(methoxy)benzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbo-
hydrazide (5i). Yield 81%. White solid. Mp: 265–266 ◦C. IR (KBr) cm−1: 3261, 3060 (NH), 1629 (CONH),
1580 (C=N). 1H-NMR (DMSO-d6):
δ 3.32 (n t, 1 H, H3), 3.62–3.82 (m, 3H, OCH3), 4.60 (n t, 1H, H2), 5.40
(n t, 1H, H3), 6.75–7.60 (m, 13H, Ar), 7.86 (br s, 1H, H6), 8.28 (br s, 2H, CH=N + NH, 1H disappears
with D2O), 11.19 (s, 1H, CONH disappears with D2O). Anal calcd. for C26H23N5O3.
(E)-N0-(2-(Benzyloxy)-3-(methoxy)benzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-
7-carbohydrazide (5j). Yield 78%. Light yellow solid. Mp 200–202 ◦C. IR (KBr) cm−1: 3602,
3173 (NH), 1644 (CONH), 1578 (C=N). 1H-NMR (DMSO-d6):
δ 3.78–3.96 (m, 4H, OCH3 + H3), 4.61 (n t,
1H, H2), 4.99 (s, 2H, CH2Bz), 5.44 (n t, 1H, H3), 7.00–7.20 and 7.11–7.60 (2 m, 13H, Ar), 7.93 (s, 1H, H6
pyraz.), 8.20–8.50 (br s, 2H, CH=N + NH, 1H disappears with D2O), 11.23 (s, 1H, CONH, disappears
with D2O). Anal calcd. for C27H25N5O3.

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3.2.5. Preparation of (E)-1-acetyl-N0-(3-(cyclopentyloxy)-4-methoxybenzylidene)-2-phenyl-2,3-dihydro-
1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (5k)
A suspension of 5d (0.45 g, 1 mmol) and sodium acetate (0.13 g, 1 mmol) in acetic anhydride
(5 mL) was stirred at 40–50 ◦C for 4 h. After cooling to room temperature, the reaction was poured into
water (20 mL) and extracted with dichloromethane (3
brine (3 10 mL), dried (MgSO4) and concentrated under reduced pressure to give an oil that was
crystallized by absolute ethanol to give a light yellow solid. Yield: 49%. Mp: 140–141 ◦C. IR (KBr)
cm−1: 2959 (NH), 1650 (C=O), 1596 (C=O). 1H-NMR (CDCl3):
1.51–2.20 (m, 8H, 4CH2 cyclopent.),
×
10 mL). The organic phase was washed with
×
δ
3.91 (s, 3H, OCH3), 3.97 (s, 3H, CH3CO), 4.15–4.25 (m, 1H, H3), 4.78–5.03 (m, 2H, H2+ CH cyclopent.),
5.78–5.91 (m, 1H, H3), 6.85 (d, J = 10.0, 1H, H5 Ar), 7.00 (dd, J = 10.0, J = 2.0, 1H, H6 Ar), 7.15–7.25
(m, 1H, H2 Ar), 7.35–7.60 (m, 5H, Ar), 8.15 (s, 1H, H6), 8.30 (br s, 1H, CH=N), 9.87 (s, 1H, CONH,
disappears with D2O). Elemental Analysis (%) calculated for C27H29N5O4.
3.3. Biological Studies
3.3.1. Material and Methods
Compounds
4 and 5 were diluted in saline from a stock DMSO solution immediately before
each experiment. 20,70-Dichlorofluorescein diacetate (DCFH-DA) and thrombin were purchased from
Sigma-Aldrich/Merck Millipore.
3.3.2. Blood Collection and Preparative Procedures
Freshly drawn venous blood from healthy volunteers of the “Centro Trasfusionale, Ospedale San
Martino” in Genoa was collected into 130 mM aqueous trisodium citrate anticoagulant solution (9:1).
The donors claimed to have not taken drugs known to interfere with platelet function during two
weeks prior to blood collection and gave their informed consent. Washed platelets were prepared
centrifuging whole blood at 100
at 1100 g for 15 min. Pellet was washed once with pH 5.2 ACD solution (75 mM trisodium citrate, 42
mM citric acid and 136 mM glucose), centrifuged at 1100 g for 15 min and then re-suspended in pH
×
g for 20 min. The obtained platelet-rich plasma was then centrifuged
×
×
7.4 Hepes buffer (145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose, 10 mM HEPES).
3.3.3. ROS Assay
ROS production was quantified as previously reported [22
probe that yields upon oxidation the fluorescent adduct DCF that is trapped inside the cells. Briefly,
washed platelets (1.0 or
108/mL), pre-incubated with saline, compounds , for 15 min at 37 ◦C, were
,23] by DCFH-DA, a ROS-sensitive
×
4
5
stimulated by 0.1 U/mL thrombin. Incubation was stopped by cooling samples in ice bath and then
samples were immediately analyzed in a Merck Millipore Bioscience Guava easyCyte flow cytometer
(Merk Millipore, Burlington, MA, USA). The reported IC50 value is the molar concentration of the
compound able to obtain 50% inhibition of the maximal aggregation induced by the agonist and is
calculated by the percentage of inhibition that is the inhibition of the maximal aggregation measured in
the presence of the agent compared with that measured in a control sample containing saline, carried
out under the same conditions.
3.3.4. Platelet Aggregation
Platelet aggregation was performed in a Bio-Data Aggregometer (Bio-Data Corporation, Horsham,
PA, USA) according to Born’s method [39] and quantified by the light transmission reached within
6 min at 37 ◦C. Briefly, washed platelets (3.0
×
108/mL) were pre-incubated for 3 min at 37 ◦C with
saline, compound
above detailed.
4 or 5 before the addition of 0.1 U/mL thrombin. The IC50 values were calculated as

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3.3.5. Isolation of Human Primary Neutrophils
Heparinized (heparin 10 U/mL Vister, PfizerItalia, Borgo San Michele, Latina, Italy) venous blood
was obtained from healthy male volunteers after written informed consent. Neutrophils were isolated
by dextran sedimentation (Dextran 70.000 Plander, Fresenius Kabi Italia, Verona, Italy), and subsequent
centrifugation on a Ficoll-Hypaque density gradient (Lympholyte-I from Cedarlane Laboratories
Ltd. Hornby, ON, Canada), as described [37,40]. Contaminating erythrocytes were removed by
hypotonic lysis. Neutrophils were washed and resuspended in Hanks’balanced salt solution (HBSS;
EuroCLone, Wetherby West, Yorkshire, UK) mixed with Dulbecco’s PBS (EuroClone; HBBS:PBS. Final
cell suspensions contained 97% or more neutrophils and more than 98% viable cells, as evaluated by a
trypan blue test kit (Sigma Aldrich) [38].
3.3.6. Flow Cytometric Assessment of Neutrophil Oxidative Metabolism
Flow cytometric analysis of neutrophil oxidative metabolism was carried out according to
literature method [37]. Briefly, neutrophils, were cultured for 1 h with or without 4d and 5d at different
concentration. Thereafter, neutrophils were pre-treated with 20-70-dichlorofluorescein-diacetate
(DCFH-DA; Sigma, Milan, Italy) (5 mM), During the incubation time, DCFH-DA entered into the
cells, and it was cleaved by intracellular esterases to give non-fluorescence DCFH trapped within
the cells. After washing in PBS, the cells were incubated for 15 min at room temperature in the
presence or absence of N◦-formyl-l-methionin-l-leucyl-l-phenylalanine (fMLP; Sigma, Milan, Italy)
(1 mM) for 30 min at 37 C. During this period, intracellular ROS oxidized DCFH to give a green
fluorescence DCFH. The reaction was stopped by keeping the samples on ice before being analyzed by
flow cytometry (FC500, Beckman Coulter).
In the same test, cell vitality in presence of 4d (10
method [40 42] at the end of the incubation time. In detail, in the same tubes we added PI staining
solution (10 g/mL, Invitrogen ThermoFisher Scientific, Waltham, MA, USA) to detect dead cells.
µM) was assessed by propidium iodide (PI)
–
µ
PI is excited at 488 nm and, emits at a maximum wavelength of 617 nm. Because of these spectral
characteristics, PI can be used in combination with other fluorochromes excited at 488 nm such as
fluorescein isothiocyanate (FITC). Auto-fluorescence levels of sample were measured and subtracted
from each analysis. Data were expressed as mean fluorescence intensity ((MFI), compared with the
expression on cells stimulated with control medium alone (defined as 100%)).
3.3.7. Enzymatic Assays
PDE4 tests were performed by Scottish Biomedical Drug Discovery (Scottish, Biomedical Drug
Discovery, Glasgow, UK) using human recombinant enzyme obtained from Spodoptera frugiperda (Sf9)
cells using a baculovirus expression system (PDE enzyme at 0.5 U/mL in 20 mM Tris.HCl pH 7.4) as
previously reported [8,43]. IMAP technology is based on the high affinity binding of phosphate by
immobilized metal coordination complexes on nanoparticles. The binding reagent complexes with
phosphate groups on nucleotide monophosphate generated from cyclic nucleotides (10 mM cAMP)
through phosphodiesterases. With fluorescence polarisation detection, binding causes a change in
the rate of the molecular motion of the phosphate bearing molecule, and results in an increase in the
fluorescence polarization value observed for the fluorescent label attached to the substrate.
Title compounds 4d and 5d were solved in DMSO at 10−2 M concentration and then diluted with
water to the final suitable concentrations. All our subsequent assays were performed in 3% DMSO
(final). All the compounds were tested preliminary at 10 µM concentration, in duplicate.
In all the experiments, Rolipram as reference compound (at 10 µM for PDE4B2 and 1 µM for
PDE4D3) was tested at nine concentrations, in duplicate, to obtain an inhibition curve in order to
validate this experiment.

Molecules 2020, 25, 899
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Being inhibition >50%, 4d and 5d have been further tested in the same assay at five different
concentrations (5
10−8–10−4 M) and IC50 values were determined by nonlinear regression analysis of
×
the inhibition curve, using Hill equation curve fitting (Graph Pad Prism software, version 5.0a).
4. Conclusions
Starting from previous anti-inflammatory pyrazole and imidazopyrazole scaffolds and PDE4Is,
we designed and synthesized a series of hybrid molecules in order to obtain new antiinflammatory
agents able to act on different biological targets. Preliminary tests evidenced a great antioxidant activity
in neutrophils for compounds 4d and 5d. In addition, the same derivatives showed a good PDE4
inhibition, and also against PDE4B, isoform most involved in inflammation. Results strongly suggested
that the PDE4 inhibition is related to antioxidant activity in neutrophils [15]. In addition, the block of
ROS production on human platelets, that produce high levels of ROS upon thrombin stimulation by
a PDE4-independent mechanism, confirmed that most of tested compounds, except the non-hybrid
intermediates, are able to exert an antioxidant response by two independent mechanisms. Interestingly,
both heterocycle scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety, were
determinant for the pharmacodynamic properties. In particular, hybrid molecules bearing to the
pyrazole series were more active than the imidazopyrazole ones, confirming that this part is involved
in the target interaction. In addition, SAR analyses evidenced a clear relationship between the catechol
substituents and the activity.
In conclusion, the hybridization between pyrazole and imidazopyrazole scaffolds and substituted
catechol ring through an acylhydrazone linker, gave a new series of multitarget antiinflammatory
compounds, characterized by a strong antioxidant activity in different biological system.
Supplementary Materials: The following are available online. Figure S1. 1H-NMR spectrum of compound 5a,
Table S1: Elemental analysis of compounds 4a–k, 5a–k, 7, 9, 10, 12.
Author Contributions: C.B., M.M., F.R., O.B. design, synthesis of compounds, SAR analyses; S.A.; E/Z
configuration; M.B.B., F.M. evaluation of ROS inhibition in neutrophil; M.G.S. evaluation of aggregation inhibition
and ROS production inhibition in platelets. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors thank M. Anzaldi, R. Raggio, F. Tuberoni for spectral recording.
Conflicts of Interest: The authors have declared that there is no conflict of interests.
Abbreviations
AD
Alzheimer’s Disease
COPD
fMLP
IL-1
Chronic obstructive pulmonary disease
Formyl-methyl-leucyl-phenylalanine
Interleukin 1
IL-8
Interleukine-8
MS
Multiple Sclerosis
p38 MAPK
PDE4
PDE4Is
PDGF
ROS
p38 Mitogen-Activated Protein Kinase
Phosphodiesterase type 4
Phosphodiesterase type 4 Inhibitors
Platelet derived growth factor
Reactive oxygen species
SAR
TGF
Structure-activity-relationship
Tumor growth factor
TNFα
Tumor necrosis factor alpha

Molecules 2020, 25, 899
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Sample Availability: Samples of the compounds are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).