Discovery of New Antiproliferative Imidazopyrazole Acylhydrazones Able To Interact with Microtubule Systems

Article abstract of DOI:10.1002/cmdc.202000122

Even though immunotherapy has radically changed the search for anticancer therapies, there are still many different pathways that are open to intervention with traditional small molecules. To expand our investigation in the anticancer field, we report here a new series of compounds in which our previous pyrazole and imidazopyrazole scaffolds are linked to a differently decorated phenyl ring through an acylhydrazone linker. Preliminary tests on the library were performed at the National Cancer Institute (USA) against the full NCI 60 cell panel. The best compounds among the imidazopyrazole series were then tested by immunofluorescence staining for their inhibition of cell proliferation, apoptosis induction, and their effect on the cell cycle and on microtubules. Two compounds, in particular 4-benzyloxy-3-methoxybenzyliden imidazopyrazole-7-carbohydrazide showed good growth inhibition, with IC₅₀ values in the low-micromolar range, and induced apoptosis. Both compounds altered the cell-cycle phases with the appearance of polyploid cells. Immunofluorescence analysis evidenced microtubules alterations; tubulin polymerization assays and docking studies suggested the tubulin system to be the possible, although not exclusive, target of the new acylhydrazone series reported here.

Full text of DOI:10.1002/cmdc.202000122

Accepted Article
Title: DISCOVERY OF NEW ANTIPROLIFERATIVE
IMIDAZOPYRAZOLE ACYLHYDRAZONES ABLE TO
INTERACT WITH MICROTUBULE SYSTEM
Authors: Olga Bruno, Chiara Brullo, Federica Rapetti, Silvana Alfei,
Irena Maric, Francesca Rizzelli, Marina Mapelli, Camillo
Rosano, and Maurizio Viale
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000122
Link to VoR: https://doi.org/10.1002/cmdc.202000122
01/2020

10.1002/cmdc.202000122
ChemMedChem
FULL PAPER
DISCOVERY OF NEW ANTIPROLIFERATIVE IMIDAZOPYRAZOLE
ACYLHYDRAZONES ABLE TO INTERACT WITH MICROTUBULE
SYSTEM
Chiara Brullo,[a] Federica Rapetti,[a] Silvana Alfei,[b] Irena Maric,[c] Francesca Rizzelli,[d] Marina Mapelli,[d]
Camillo Rosano,[e] Maurizio Viale,[f] and Olga Bruno*[a]
[a]
Prof. Chiara Brullo; Dr. Federica Rapetti; Prof. Olga Bruno*
Department of Pharmacy,
University of Genoa,
V.le Benedetto XV, 3, 16132 Genova-Italy
E-mail: brullo@difar.unige.it; federica.rapetti@edu.com; obruno@unige.it
Prof. Silvana Alfei
[b]
Department of Pharmacy,
University of Genoa,
V.le Cembrano, 4, 16148 Genova-Italy
E-mail: alfei@difar.unige.it
[c]
[d]
[e]
[f]
Dr Irena Maric
IRCCS Ospedale Policlinico San Martino, U.O. Cellular Oncology,
L.go. R. Benzi, 10, 16132 Genova-Italy
E-mail: thr599ser602@gmail.com
Dr. Marina Mapelli; Dr. Francesca Rizzelli
European Institute of Oncology
Via Adamello 16, 20139 Milan, Italy
E-mail: marina.mapelli@ieo.it; francesca.rizzelli@ieo.it
Dr Camillo Rosano
IRCCS Ospedale Policlinico San Martino, Proteomics and Mass Spectrometry Unit
L.go. R. Benzi, 10, 16132 Genova-Italy
E-mail: camillo.rosano@hsanmartino.it
Dr Maurizio Viale;
IRCCS Ospedale Policlinico San Martino, U.O. Biotherapy
L.go. R. Benzi, 10, 16132 Genova-Italy
E-mail: maurizio.viale@hsanmartino.it
Supporting information for this article is given via a link at the end of the document
Abstract: Despite immunotherapy has radically changed the
anticancer search, there are still many different pathways that are
open to intervention by traditional small molecules. In order to expand
our investigation on anticancer field, we report here a new series of
compounds in which our previous pyrazole and imidazopyrazole
scaffolds are linked to a differently decorated phenyl ring through an
acylhydrazone linker. Preliminary test on the library was performed at
National Cancer Institute (USA) in the full NCI 60 cell panel. The best
compounds among imidazopyrazole series were then tested for the
inhibition of cell proliferation, the apoptosis induction, and the effect
molecules drugs. Moreover, small-molecules offer several
advantages over recombinant protein approaches, such as
access to specific intracellular targets, more options for drug
formulation and pharmacokinetic challenges, lower cost in
production and delivering. In addition, re-examination of classical
antitumor agents (e.g. doxorubicin or cyclophosphamide)
revealed that they promote an immunogenic cells death; other
(such as taxanes) block tumor growth and also affect, as off-target,
immune cell function in the tumor microenvironment. Small
molecules may therefore have a cooperative role in cancer
on the cell cycle and on microtubules by immunofluorescence staining. immunotherapy.
Both compounds, in particular the 4-benzyloxy-3-methoxybenzyliden
imidazopyrazole-7-carbohydrazide showed a good growth inhibition,
with IC50s in the low micromolar range, and induced apoptosis. Both
compounds altered the cell cycle phases with appearance of
In the last ten years, we identified several pyrazole and
imidazopyrazole derivatives as inhibitors of the neutrophil-
mediated inflammatory response, the last being compounds 1
[1,2] (Fig. 1). More recently, our pyrazolyl-urea GeGe-3 (2) (Fig.
1) was found to control the activation of MAPK signaling and to
block angiogenesis and tumor growth both in vitro and in vivo
tests [3,4].
In order to select new small molecules able to exert a similar
activity, in collaboration with the Developmental Therapeutic
Program (DTP) of the National Cancer Institute, Division of
Cancer Treatment Diagnosis (USA), we screened more than two
hundred compounds deriving from our chemical library.
Among the numerous compounds analyzed, the compounds
belonging to the two series 3a–j and 4a–j (Table 1) showed
interesting anti-proliferative activity.
polyploidy
cells.
Immunofluorescence
analysis
evidenced
microtubules alterations; tubulin polymerization assays and docking
studies suggested the tubulin system as possible, while not exclusive,
target of the new acylhydrazone series here reported.
Introduction
The approval of T cell checkpoint inhibitory antibodies as
ipilimumab, pembrolizumab and nivolumab has started the new
era of anticancer immunotherapy. However, there are still many
alternative pathways open to intervention by traditional small
1
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In these molecules our previous pyrazole and imidazopyrazole
scaffolds are linked through an acylhydrazone linker to a
differently decorated phenyl ring. Interestingly, a large literature
investigation evidenced the presence of an acylhydrazone linker
in several small molecules identified as potential new anticancer
drugs (compounds 5–8, Fig. 2) [5–8]. In all these small molecules
the acylhydrazone moiety was an efficient linker between two
different scaffolds, both being able to interact with the target.
The preliminary test was performed at NCI at a single high dose
(10-5 M) in the full NCI 60 cell panel. Among the compounds of
series 3 and 4 NCI selected 4g (R1 = H, R2 = OCH3, R3 = OC6H5)
and 4i (R1 = H, R2 = OCH3, R3 = OCH2C6H5)] which were
progressed to the full 5-dose assay on the same NCI 60 cell panel.
The IC50 and LC50 values calculated for 4g and 4i made us
consider these compounds worthy of further study. In order to
confirm the antiproliferative activity and clarify the mechanism of
action both 4g and 4i were then tested for cell proliferation
inhibition (in cell lines different than the previously used at NCI),
the apoptosis induction and the effect on the cell cycle. Results
suggested a possible involvement of tubulin system in the
mechanism of cell cycle arrest. Therefore, the effect on
microtubules by immunofluorescence staining were tested first.
Subsequently, the ability of compounds 4g and 4i of inhibiting the
tubulin polymerization in comparison with the inactive compound
3a and nocodazole as negative and positive control, respectively
were verified.
Figure 1. Molecular structures of previously reported compounds 1 and 2.
Finally, to confirm whether the compounds are truly capable of
interacting with microtubules, molecular docking simulation was
performed.
Chemistry
Synthetic procedures to obtain title compounds are reported in
Scheme 1. Briefly, the intermediate 10 was prepared by reacting
an excess of hydrate hydrazine with the ethyl 5-amino-1-(2-
hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylate 9 [9]. Starting
from the same intermediate 9 we obtained the ethyl 2-phenyl-2,3-
Figure 2. Acylhydrazone derivatives endowed with potent antiproliferative
activity.
dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylate 12 [10]
by
dehydration in concentrated sulfuric acid. Then, 12 was converted
in the corresponding carbohydrazide 13 as for 10.
Table 1. Structures of compounds 3a–j, 4a–j and intermediates
11a–k.
To prepare benzylidene derivatives 3 and 4, the needed
benzaldehydes 11a, c, d and 11j were purchased, while the
others were prepared following well known procedures. In details,
the phenoxy substituted benzaldehydes 11b, f, g, and 11k were
obtained by the Crowder's method [11]: the suitable hydroxy
substituted benzaldehyde was salified by NaOH aqueous solution,
then diphenyliodonium chloride was added to obtain, after heating,
the desired phenoxy derivative. The dimethoxy benzaldehyde 11e
and the benzyloxy derivatives 11h and 11i were obtained by the
corresponding hydroxy substituted benzaldehyde by alkylation
with the appropriate alkyl chloride in the presence of K2CO3 [12].
All derivatives have melting or boiling points in accordance with
literature data except for compounds 11f and 11k that were
obtained as solids but oils (see Table 1, Supporting information).
Compounds 3a–j and 4a–j (Scheme 1, Table 1) were then
obtained by refluxing the suitable methoxy, phenoxy or benzyloxy
substituted benzaldehydes 11 solved in a mixture of absolute
ethanol/methanol (7:3) in the presence of 5-amino-1-(2-hydroxy-
2-phenylethyl)-1H-pyrazole-4-carbohydrazide (10) or 2-phenyl-
Comp.
R1
R2
R3
3a, 4a, 11a
3b, 4b, 11b
3c, 4c, 11c
3d, 4d, 11d
3e, 4e[a], 11e
3f, 4f, 11f
3g, 4g, 11g
3h, 4h, 11h
3i, 4i, 11i
3j, 11j
H
OC6H5
H
H
H
OC6H5
H
H
OCH2C6H5
H
H
OCH2C6H5
OCH3
OCH3
OC6H5
OCH3
OCH2C6H5
F
H
OCH3
OC6H5
OCH3
OCH2C6H5
OCH3
OC6H5
OCH3
H
H
2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide
(13),
H
respectively.
Although formation of E/Z mixtures is possible, only one of the two
isomers has been always obtained. The E configuration was a
priori hypothesized and then confirmed by 1H and 13C-NMR
spectra analyses, in analogy with similar acyl hydrazones recently
reported [13]. 1H-NMR spectra image is reported (Fig. 1,
Supporting Information).
H
H
4j[a], 11k
OC6H5
H
[a] Compounds 4e and 4j have been already reported [13].
2
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Scheme 1. Reagents and conditions: a) Hydrate hydrazine, 120–130 °C, 4 h; b) abs. EtOH / MeOH (7:3), reflux, 18h; c) conc. H2SO4, 0 °C, 15 min, then NH4OH.
of them showed also lethal effect in different types of cancer. The
most active compounds were 4b, 4g and 4i that affected almost
Results and Discussion
all the cell lines in the panel and also showed the greatest lethality.
Also compound 4e had a good growth inhibition while its activity
was more specific against colon and breast cancer. Compounds
4a and 4c showed a similar profile being active against the same
cell lines with similar potency: HOP-62 Non-Small Cell Lung
cancer, U251 CNS cancer, OVCAR-4 and OVCAR-8 ovarian
cancer and HS 578T breast cancer. Interestingly 4a and 4c are
both mono-substituted in meta position of the benzylidene ring
with phenoxy and benzyloxy group, respectively. In those
molecules the introduction of a methoxy substituent in para
position causes a strong decrease in antiproliferative activity
(compare 4a with 4f and 4c with 4h). The total inactivity of
compound 4d, that is the -para-benzyloxy analogue of 4b, was
unexpected, particularly taking in consideration that 4b is one of
the most active among the imidazopyrazoles. In our mind the
results of the screening assays might be affected by poor
solubility or diffusion problems.
All synthesized compounds were submitted to the National
Cancer Institute (NCI 60 Cell screen) to obtain preliminary
information about their antiproliferative activity. Compounds 3a–j
and 4a–j were tested firstly at a single high dose (10-5 M) in the
full NCI 60 cell panel (leukemia, lung, colon, CNS, ovarian, renal,
prostate, breast cancer and melanoma). NCI provide the One-
dose data as a mean graph of the percent growth of treated cells
or as a number which expresses growth relative to the no-drug
control, and relative to the time zero number of cells. This allows
detection of both growth inhibition (values between 0 and 100)
and lethality (values less than 0). This is the same as for the 5-
dose assay, described below. The results obtained for compound
3a–j and 4a–j, respectively, in the One dose 60 Cell screen were
reported in Supporting Information (Tables 3 and 4, SI). Only cell
lines relative data relevant for growth inhibition or lethality were
shown. In addition, only growth percent < 50 (corresponding to
50% of growth inhibition) were reported.
At the end of the preliminary screening, only compounds (4g and
4i) satisfying pre-determined threshold inhibition criteria in a
minimum number of cell lines, were progressed to the full 5-dose
assay. The threshold inhibition criteria for progression to the 5-
dose screen was selected by NCI to efficiently capture
compounds with anti-proliferative activity based on careful
analysis of historical DTP screening data. Both compounds
showed a similar profile with IC50 values in the low micromolar
range. In Table 2 we report LC50 values (<10 M alone) for
compounds 4g and 4i against the most affected cell lines. Results
evidenced a very similar pharmacological profile for 4g and 4i, the
latter being slightly more active. Both compounds affected in
particular four CNS cancer cell lines, and showed good potency
against NSLC, colon, ovarian, renal, prostate cancer and
melanoma.
On the basis of these results, only 5 compounds among pyrazoles
3 (3c, 3f, 3g, 3h and 3j) reduced the proliferation in few cell lines,
3h and 3j being the most active ones. Compounds showed the
better activity against different leukemia cell lines and type
OVCAR-8 ovarian cancer, while sporadic activity were noted
against UACC-257 melanoma, T-47D breast cancer and KM12
colon cancer. Except for 3c, all the active compounds are bi-
substituted in the benzylidene moiety; the benzyloxy group is
better than the phenoxy one (compare 3h with 3j), while a fluorine
atom increase the activity in respect to a methoxy group (compare
3j with 3f). However, in general the series 3 showed lesser activity
in respect to series 4 and therefore no further investigations were
planned.
On the contrary, imidazopyrazole derivatives 4 showed very high
antiproliferative activity on several cancer cell lines. Except for 4d,
all compounds were able to strongly reduce cell growth and some
3
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Further investigation performed on A2780, A549, T47D, SKBR3,
MDA-MB-231 and IMR-32 cancer cell lines, confirmed the
antiproliferative activity for both 4g and 4i and evidenced a
significant ability to induce apoptosis (Table 3 and Fig. 3). In
particular, it is of note that in all cell lines 4i was from 1.9 to 8.3
times more active than 4g, with IC50s ranging from 0.32 ± 0.04 to
9.79 ± 1.57 M. Nevertheless, no significant differences between
the two molecules were found for apoptosis triggering after
exposure to the equitoxic concentrations IC50 and IC75.
The microphotographs showed that treated cells presented
various kinds of alterations of the microtubular system such as
alterations of the distribution of chromosomes and the presence
of multinucleated cells, as indicated also by the cell cycle analysis.
Furthermore, in some cell tubules appeared fragmented in dots
(Fig. 4C and 4D) similar to those observed after exposure to anti-
microtubule agents, such as colchicine [14] or other experimental
compounds [15]. The presence of multiple centrosomes (Fig. 4B
and 4F) was also observed, in particular after 4g treatment, while,
after 4i treatment various cells showed more clear alterations of
chromosome distribution in daughter cells and multiple nuclei (Fig.
4E and 4F).
Table 2. LC50 values determined for compounds 4g and 4i.
Comp.
4g
4i
Cancer Cell lines
Table 3. Antiproliferative activity of compounds 4g and 4i in the MTT assay.
LC50
LC50
(M)
(M)
Compounds
NSLC-Cancer
Colon Cancer
NCI-H460
HT29
8.61[a]
7.20
>100
6.41
6.08
8.91
9.84
6.34
>10
4.78
5.80
9.86
5.70
6.43
8.31
6.82
9.09
6.18
6.17
8.40
9.13
9.35
Cell lines
4i
4g
SW-620
SF-295
A2780
1.63 ± 0.36[a]
1.87 ± 0.13
6.93 ± 1.41
23.1 ± 4.4
0.48 ±0 .02
1.00 ± 0.18
2.41 ± 0.95
14.9 ± 3.4
0.32 ± 0.04
9.79 ± 1.57
CNS Cancer
A549
SF-539
T47D
SNB-75
U251
SKBR3
IMR32
Melanoma
LOX IMVI
MDA-MB-435
OVCAR-3 -
OVCAR-4
786-0
2.64 ± 0.78
63.3 ± 7.2
MDA-MB-453
Ovarian Cancer
5.44
>10
[a] Data are expressed as LC50 (M) and are the mean ± SD of 4-6 experiments.
Renal Cancer
>10
Prostate Cancer
PC-3
>10
DAPI assay
[a] Values were calculated from the dose response curves obtained by testing
compound 4g and 4i against NCI 60 Panel Cell Lines at five different
concentrations in the range 100–0.01 M. Only results inferior to 10 M are
reported. Data are the mean ±SD of 3 experiments.
4g IC50
4i IC50
4g IC75
4i IC75
Control
The subsequent cell cycle phases analysis showed that both 4g
and 4i were able to modify the cells distribution in the cell cycle.
In particular, both compounds caused the appearance of
polyploid cells (>4n) together with a concomitant decrease of cells
in the other cell cycle phases (Table 4). These effects were
dependent on the applied concentration and time exposure and
were more evident for 4i.
Those results indicated the possible interaction of 4g and 4i with
the microtubular system of cells. To test this possibility, we treated
A549 cells with equitoxic concentrations (IC50 and/or IC75) of both
compounds for 48 hours (Figure 4).
A2780
A549
SKBR3
IMR32
Figure 3. Apoptotic effect of compounds 4g and 4i in the DAPI test. Histograms
represent the mean ±SD of 3-4 data.
4
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Table 4 Effects of compounds 4g and 4i on cell cycle of A549 cells. [a]
4g
4i
Control
24 h
IC50
48 h
IC50
24 h
48 h
IC50
24 h
+
48 h
IC75
IC75
IC50
IC75
IC75
G0/G1 (2n)
S
30.7±7.5
53.9±13.8
15.4±6.9
8.4±1.2
20.5±1.6
52.9±18.2
11.3±7.4
15.3±10.3
6.6±3.1
26.2±2.8
9.0±2.1
58.3±5.9
20.2±0.3
52.1±3.9
27.8±3.8
7.0±5.5
10.4±0.8 4.9±0.2
35.2±3.6 23.8±3.0
16.8±1.9 9.3±0.7
37.6±5.6 62.0±2.1
25.9±3.7
51.6±6.6
22.5±4.1
43.4±20.6
21.5±16.2
22.2±11.8
27.3±10.6
18.6±5.1
47.1±11.0
G2/M (4n)
>4n
[a] Percentage of A549 cells in the different cell-cycle phases after 24–48 h exposure to compounds 4g and 4i at IC50 and IC75, as calculated by MTT.
Data are expressed as mean ±SD of 3-5 experiments.
Figure 4. Microtubular effect of compounds 4g and 4i on A549 cells. Panel A: Untreated control cells. Panel B, C: Cells treated for 48 h with 4g at IC50 and IC75.
Note the presence of multiple centrosomes (B) and microtubular dots (C, in the square insets enlargement of the same image of centrosomes enclosed in the
rectangle). Panel D, E: Cells treated for 48 h with 4i at IC75. In this case also note the presence of microtubular dots in Panel D (square insets), while in panel E and
F note the presence of alteration of chromosome distribution (square insets) and multiple centrosomes (arrows), respectively. Magnification 4 X. The figure was
rendered by Adobe Photoshop 5.0.2.
5
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and 4i, respectively. More in details, molecule 4g is at the correct
position to form hydrogen bonds with residues Lys126. His129,
Glu132 of Stathmin4 and residues His192, Gln193 and Asp414,
belonging to the tubulin  subunit. Molecule 4i has its
imidazopyrazole unit hold by a clamp made by the carboxyl side
chains of Glu429 and Glu433 belonging to the tubulin  chain
while, on the other side, a benzene ring of 4i form hydrophobic
interactions with residues Leu47, Phe49, Tyr64, Ala68 and
Gln313 of the Tubulin tyrosine ligase.
Therefore, to examine the effect of 4g and 4i on tubulin nucleation
and polymerization we performed
a
cell-free tubulin
polymerization assay in vitro (Fig. 5 and Fig. 2, SI). Polymerization
of tubulin was determined by turbidity (absorbance measured at
340 nm) in GTB buffer and in the presence of 10 M 4g, 4i, the
inactive compound 3a as negative control, or 10M nocodazole
as positive control (Fig. 5A). Under these conditions, compounds
4g and 4i showed not effect on tubulin polymerization rate with a
Vmax of 21 mOD/min. In particular, reactions conducted with
compounds 4g and 4i showed unperturbed nucleation, growth
and steady state phases compared to polymerization curves in
GTB buffer or in the presence of compound 3a. We therefore
tested the effect of adding 20 M of each compound dissolved in
GTB buffer containing 0.9 % DMSO (Fig. 5B). Under these
conditions, compound 4g displayed the lowest Vmax, of 16
mOD/min, compared to a Vmax of 19 mOD/min in the control
reaction and Vmax of 21 mOD/min measured with compound 3a.
To assess whether the mild effects observed on tubulin
polymerization were caused by low Kon rates of the compounds
towards tubulin dimers, we repeated the polymerization assays
with 20 M compounds after a 30 minutes incubation on ice of the
compounds with tubulin (Fig. 5C). Under these conditions, the
polymerization rate in the presence of compound 3a was
unchanged, while the Vmax of compounds 4g and 4i were
reduced to 6 mOD/min and 8 mOD/min respectively. Analysis of
the polymerization curves of compounds 4g and 4i revealed a
delay in tubulin nucleation compared to the control reaction. We
conclude that compounds 4g and 4i associate with slow kinetics
to tubulin dimers and interfere significantly with microtubule
assembly only at 20 M concentration after incubation with tubulin
dimers.
A
Ctr Vmax 21 mOD/min
3a Vmax 22 mOD/min
4g Vmax 23 mOD/min
4i Vmax 24 mOD/min
Noco Vmax 2 mOD/min
10 M compounds
Time (sec)
20 M compounds
Ctr Vmax 19 mOD/min
3a Vmax 21 mOD/min
4g Vmax 16 mOD/min
4i Vmax 18 mOD/min
Noco Vmax 2 mOD/min
B
Time (sec)
20 M compounds, 30’ incubation
Ctr Vmax 19 mOD/min
C
3a Vmax 18 mOD/min
4g Vmax 6 mOD/min
4i Vmax 8 mOD/min
Noco Vmax 1 mOD/min
In order to verify the possible interaction of compounds 4i and 4g
with tubulin system we performed different docking simulations
using as a target the atomic structures of the polymeric complex
formed between tubulin , tubulin , stathmin4 and the tubulin
tyrosine ligase [17; PDB code 5J2T]. The binding modes of 4g
and 4i were compared to the ones of the known ligand colchicine
and vinblastine. For all the ligand tested we performed a “blind
docking”: i.e. the docking of small molecules to their targets was
done without a priori knowledge of the binding mode of the ligand
in the active site of the protein. Our procedure was firstly validated
using by correctly positioning both colchicine and vinblastine in
their binding site, with a RMSD of less than 0.3 Å with respect to
the ones determined by X-ray crystallography.
Time (sec)
Figure 5. Effect of 3a, 4g and 4i on tubulin polymerization reactions. (a) Tubulin
polymerization kinetics determined by turbidity at 340 nm in the presence of 10
M Nocodazole (black), 10 M 3a (blue), 10 M 4g (green) or 10 M 4i (orange).
The control polymerization reaction curve in unperturbed conditions is plotted in
red. For each reaction, the maximum velocity (Vmax) was calculated as the
highest value of the first derivative. (b) Tubulin polymerization assays conducted
as in (a) at 20 M concentration of each compounds and nocodazole. The
kinetic in General Tubulin Buffer added with 0.9 % DMSO was used as control
of unaltered polymerization rate. (c) Polymerization curves as in (b) conducted
after 30 min incubation of tubulin with the compounds
The unusual binding modes to their respective targets of both 4g
and 4i were identified by our docking simulations. While 4g was
found bound the subunit  in proximity of stathmin4, 4i seemed to
have a good affinity for a site at the interface between tubulin
 subunit and the tubulin tyrosine ligase protein [Fig. 6, panels A-
C]. The positions of the two molecules seem to prevent the correct
formation of the assembly between the different tubulin subunits,
stathmin4 and tubulin tyrosine ligase, therefore preventing the
correct functioning of the oligomer.
Using the program Autodock 4.2.2 we were able to calculate the
ligand binding Energies and therefore, to estimate an “affinity
constant”. Particularly we found Ki of 52.6 nM and 4.7 nM for 4g
6
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Figure 6. Panel A, ribbon representation of the assembly formed by a tubulin tetramer (−−−), stathmin4, and tubulin tyrosine ligase (green). The docking poses
of 4g and 4i in their binding sites are drawn as green and pink balls, respectively. Panel B, compound 4g (green sticks) is positioned at the interface between tubulin
 subunit (light brown ribbons) and stathmin (pale yellow ribbons), possibly breaking the interactions between residues of the two proteins. Panel C, as above, 4i
(drawn as pink sticks) looks like breaking the interactions between the tubulin  subunit (light brown ribbons) and the tubulin tyrosine ligase (green ribbons). Protein
residues involved in the interactions are drawn as sticks, coloured corresponding to the protein subunit and properly labelled.
together with the simple observation after treatment of frequent
cells with bigger volumes, suggested an interaction with the
Conclusion
microtubular system, therefore altering cell division events. In
particular, cell cycle analysis showed an accumulation of
polyploid cells with a number of chromosomes higher than 4n,
which was roughly concentration- and exposure time-dependent,
coupled with an evident decrease of the percentage of cells in the
other cell cycle phases. On the other hand, immunofluorescence
analysis demonstrated the presence of various alterations of the
microtubular system involving the presence of multiple
centrosomes, the distribution of chromosomes in daughter cells,
and the presence of multinucleated cells and microtubules
fragmented in dots.
In addition, tubulin polymerization assays showed that upon
incubation with tubulin dimers, the compounds 4g and 4i reduce
tubulin nucleation and microtubule formation, resulting in Vmax of
about 31% and 42% compared to unperturbed polymerization
reaction. Considering that in cells microtubule polymerization is
modulated by numerous microtubule and tubulin-associated
factors such as MAPs [16] these compounds might display more
penetrant depolymerizing phenotypes as the one measured in
vitro. However, it is noteworthy that all these actions appear at
high concentration (20M), while IC50 values in the low
We report here synthesis and biological evaluation of new
acylhydrazone derivatives, identified among a large library of
pyrazole and imidazopyrazole compounds as possible
antiproliferative drugs.
In a preliminary screening performed at National Cancer Institute
on a panel of 60 different type of cancer cells, several
imidazopyrazoles were endowed with strong ability in reducing
cell growth and some of them showed also lethal effect in different
types of cancer. On the contrary, pyrazole analogues were lesser
active. SAR analyses provided important information about the
structural requirements to obtain good antiproliferative activity.
The best compounds (4g and 4i) were selected to perform further
analyses in order to confirm the anticancer activity, and also to
obtain information about the possible mechanism of action.
Our in vitro data demonstrated that imidazopyrazoles 4g and 4i
possess good antiproliferative and apoptotic activities with IC50s
mainly ranging in the low micromolar range of concentrations. 4i
showed the best antiproliferative activity, while the apoptotic
effect was similar for both compounds.
As concerns the possible mechanism of action of those molecules
both the cell cycle analysis and the immunofluorescence staining,
7
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10.1002/cmdc.202000122
ChemMedChem
FULL PAPER
micromolar range were observed in the preliminary
antiproliferative screening.
Finally, we cannot rule out the possibility that compounds 4g and
4i acts also on molecular targets other than microtubules to
induce synergistically the anti-proliferative effects that we observe
in cells.
In conclusion, all SAR information, together the important
biological assessment, pave the way for further development of
new imidazopyrazole based small molecules and strongly
stimulate further research to establish whether other mechanisms
of action are possible for this new class of potential anticancer
drugs.
On the other hand, molecular docking studies confirmed that
compounds 4g and 4i are able to interact with tubulin system,
even if by different binding modes. Particularly, it seems that while
the shorter chain of 4g allows the molecule to accommodate in a
small cleft formed by tubulin  and stathmin3, reinforcing in this
way the binding between the two proteins, molecule 4i is
positioned in a way that might prevent the correct binding of
tubulin tyrosine ligase to the tubulin  chain.
Experimental Section
Chemistry: General: Chiminord\ and Aldrich Chemical, Milan, Italy
provided all chemicals. Solvents were reagent grade. Unless otherwise
stated, all commercial reagents were used without further purification.
Aluminium backed silica gel plates (Merck DC-Alufolien Kieselgel 60 F254,
Darmstad, 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 a Buchi M-560 instrument. IR spectra were recorded with a Perkin-
Elmer 398 spectrophotometer. 1H NMR spectra were recorded on a Varian
Gemini 200 (200 MHz) and a Bruker DPX-300 (300 MHz) instruments;
chemical shifts are reported as  (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%.
(E)-5-amino-)-1-(2-hydroxy-2-phenylethyl)-N'-(3-
benzyloxybenzylidene)-1H-pyrazole-4-carbohydrazide (3c): White
solid, yield 68 %, m.p. 191–192 °C (ethyl acetate). IR (KBr) cm-1: 3400
(OH), 3306, 3227,3060 (NH2 + NH), 1615 (C=O), 1560, 1533 (C=N), 1260
(CO). 1H-NMR (DMSO-d6) : 3.95–4.10 (m, 2H, CH2), 4.90–5.05 (m, 1H,
CHOH), 5.14 (s, 1H, CH2O), 5.73–5.75 (m, 1 H, OH, disappears with D2O),
6.40 (br s, 2H, NH2, disappears with D2O), 7.03–7.07 and 7.20–7.58 (2m,
14H, Ar), 7.80–8.20 (m, 2H, H3 pyraz + CH=N), 11.10 (s, 1H, CONH,
disappears with D2O).
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(4-
benzyloxybenzylidene)-1H-pyrazole-4-carbohydrazide (3d): White
solid, yield 75%, m.p. 269–270 °C (abs. ethanol). IR (KBr) cm-1: 3400 (OH),
3320, 3230 (NH2 + NH), 1610 (C=O), 1511 (C=N), 1251, 1167 (CO). 1H-
NMR (DMSO-d6) : 3.95–4.20 (m, 2H, CH2N), 4.85–5.10 (m, 1H, CHOH),
5.16 (s, 1H, CH2O), 5.73–5.74 (m, 1 H, OH, disappears with D2O), 6.05–
6.60 (br s, 2H, NH2, disappears with D2O), 7.11 and 7.63 (ABq, J =7.4, 2H,
Ar), 7.19–7.56 (2m, 10H, Ar), 7.80–8.30 (m, 2H, H3 pyraz + CH=N), 11.04
(s, 1H, CONH, disappears with D2O).
General procedure for (E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-
benzylidene-1H-pyrazole-4-carbohydrazides
(3a–j)
and
N'-
benzylidene-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(3,4-
carbohydrazides (4a–j): A solution of suitable aldehydes 11 (2 mmol) in
a mixture of ethanol/methanol (7:3) (5 mL) was added dropwise to 5-
amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide 10 or
2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide 13 (2
mmol) solved in the same mixture solvent (15 mL). The reaction mixture
was stirred at 100 °C for 18 h, cooled and poured into ice water (10 mL).
The white solids were simply dried, if pure at TLC check [Silica gel, diethyl
ether/methanol (8:2)], or re-crystallized by absolute ethanol or ethyl
acetate.
dimethoxybenzylidene)-1H-pyrazole-4-carbohydrazide (3e): White
solid, yield 55%, m.p. 155–159 °C (abs. ethanol). IR (KBr) cm-1: 3642 (OH),
3414, 3337, 3219 (NH2 + NH), 1632 (C=O), 1573 (C=N), 1270, 1236 (CO).
1H-NMR (DMSO-d6) : 3.39 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.92–4.10
(m, 2H, CH2N), 4.90–5.10 (m, 1H, CHOH), 5.70–5.80 (m, 1 H, OH,
disappears with D2O), 6.10–6.55 (br s, 2H, NH2, disappears with D2O), 7.04
and 7.16 (ABq, J = 6.0, 2H, Ar), 7.21–7.50 (m, 6H, Ar), 7.70–8.30 (m, 2H,
H3 pyraz + CH=N), 11.10 (s, 1H, CONH, disappears with D2O).
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(4-methoxy-3-phenoxy-
benzylidene)-1H-pyrazole-4-carbohydrazide (3f): White solid, yield
44%, m.p. 192–195 °C (ethyl acetate). IR (KBr) cm-1: 3542 (OH), 3346,
3210 (NH + NH2), 1611 (C=O), 1223 (CO). 1H-NMR (DMSO-d6) : 3.80 (s,
3H, OCH3), 3.81–4.10 (m, 2H, CH2N), 4.95–5.00 (m, 1H, CHOH), 5.70–
5.75 (m, 1 H, OH, disappears with D2O), 6.30 (br s, 2H, NH2, disappears
with D2O), 6.80–7.15 (m, 3H, Ar), 7.20–7.50 (m, 10H, Ar), 7.80–8.20 (m,
2H, H3 pyraz + CH=N), 11.05 (s, 1H, CONH, disappears with D2O).
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(3-phenoxybenzylidene)-
1H-pyrazole-4-carbohydrazide (3a): White solid, yield 57%, m.p. 246–
248 °C (abs. ethanol). IR (KBr) cm-1: 3409 (OH), 3310, 3219 (NH2 + NH),
1642 (C=O), 1551 (C=N), 1268,1214 (CO). 1H-NMR (DMSO-d6) : 3.90–
4.20 (m, 2H, CH2N), 4.95–5.05 (m, 1H, CHOH), 5.65–5.80 (m, 1 H, OH,
disappears with D2O), 6.20–6.55 (br s, 2H, NH2, disappears with D2O),
7.05–7.55 (m, 14H, Ar), 7.86 (s, 1H, H3 pyraz), 8.16 (s, 1H, CH=N), 11.10
(s, 1H, CONH , disappears with D2O).
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(3-methoxy-4-
phenoxybenzylidene)-1H-pyrazole-4-carbohydrazide (3g): White solid,
yield 97%, m.p. 193–194 °C (water). IR (KBr) cm-1: 3413 (OH), 3310 (NH
+ NH2), 1614 (C=O), 1488 (C=N), 1224 (CO). 1H-NMR (DMSO-d6) : 3.81
(s, 3H, OCH3), 3.90–4.20 (m, 2H, CH2N), 4.85–5.10 (m, 1H, CHOH), 5.70–
5.80 (m, 1H, OH, disappears with D2O), 6.20–6.60 (br s, 2H, NH2,
disappears with D2O), 6.85–7.20 (m, 3H, Ar), 7.20–7.55 (m, 11H, 10 Ar +
CH=N), 7.80–8.40 (m, 1H, H3 pyraz), 11.10 (s, 1H, CONH, disappears with
D2O).
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(4-phenoxybenzylidene)-
1H-pyrazole-4-carbohydrazide (3b): White solid, yield 83%, m.p. 248–
250 °C (ethyl acetate). IR (KBr) cm-1: 3394 (OH), 3285, 3211,3055 (NH2 +
NH), 1627 (C=O), 1559 (C=N), 1258 (CO). 1H-NMR (DMSO-d6) : 3.90–
4.10 (m, 2H, CH2N), 4.90–5.15 (m, 1H, CHOH), 5.65–5.80 (s, 1 H, OH,
disappears with D2O), 5.15–5.65 (br s, 2H, NH2, disappears with D2O),
6.95–7.60 and 7.60–7.80 (2m, 15H, 14Ar + H3 pyraz), 7.90–8.30 (m, 1H,
CH=N), 11.10 (s, 1H, CONH, disappears with D2O).
8
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10.1002/cmdc.202000122
ChemMedChem
FULL PAPER
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(3-benzyloxy-4-
methoxybenzylidene)-1H-pyrazole-4-carbohydrazide (3h): White solid,
yield 59%, m.p. 149–150 °C (abs. ethanol). IR (KBr) cm-1: 3402 (OH), 3314,
3184 (NH + NH2), 1635 (C=O), 1513 (C=N), 1265 (CO). 1H-NMR (DMSO-
d6) : 3.81 (s, 3H, OCH3), 3.90–4.20 (m, 2H, CH2N), 4.95–5.05 (m, 1H,
CHOH), 5.13 (s, 2H, CH2O), 5.70–5.75 (m, 1 H, OH, disappears with D2O),
6.20–6.50 (br s, 2H, NH2, disappears with D2O), 7.00–7.20 and 7.27–7.50
(2m, 13H, Ar), 7.70–8.30 (m, 2H, H3 pyraz + CH=N), 11.03 (s, 1H, CONH,
disappears with D2O).
8.40 (m, 2H, CH=N + NH, 1H disappears with D2O), 11.10 (s, 1H, CONH,
disappears with D2O).
(E)-N'-(4-methoxy-3-phenoxybenzylidene)-2-phenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-7-carbohydrazide (4f): White solid, yield 96%,
m.p. 218–220 °C (water). IR (KBr) cm-1: 3261, 3061, 3036 (NH), 1636
(CONH), 1592 (C=N). 1H-NMR (DMSO-d6) : 3.72–3.82 (m, 4H, OCH3 +
H3), 4.58 (n t, 1H, H2), 5.45 (n t, 1H, H3), 6.84–7.50 (m, 13H, Ar), 7.80 (s,
1H, H6), 7.90–8.20 (m, 2H, CH=N + NH, 1H disappears with D2O), 11.19
(s, 1H, CONH, disappears with D2O).
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N'-(4-benzyloxy-3-
methoxybenzylidene)-1H-pyrazole-4-carbohydrazide (3i): White solid,
yield 88%, m.p. 98–100 °C (abs. ethanol). IR (KBr) cm-1: 3658 (OH), 3409,
3323, 3217 (NH + NH2), 1620 (C=O), 1452 (C=N), 1270 (CO). 1H-NMR
(DMSO-d6) : 3.82 (s, 3H, OCH3), 3.90–4.10 (m, 2H, CH2N), 4.95–5.05 (m,
1H, CHOH), 5.14 (s, 2H, CH2O), 5.70–5.75 (m, 1 H, OH, disappears with
D2O), 6.20–6.50 (br s, 2H, NH2, disappears with D2O), 7.06 and 7.19 (ABq,
J = 8.4, 2H, Ar), 7.25–7.55 (m, 11H, Ar), 7.70–8.30 (m, 2H, H3 pyraz +
CH=N), 11.03 (s, 1H, CONH, disappears with D2O).
(E)-N'-(3-methoxy-4-phenoxybenzylidene)-2-phenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-7-carbohydrazide (4g):. White solid, yield 97%,
m.p. 185–186 °C (water). IR (KBr) cm-1: 3414, 3034 (NH), 1611 (CONH +
C=N). 1H-NMR (DMSO-d6) : 3.78–3.88 (m, 4H, OCH3 + H3), 4.59 (n t, 1H,
H2), 5.45 (n t, 1H, H3), 6.84–7.50 (m, 13H, Ar), 7.80–8.20 (2m, 3H, H6 +
CH=N + NH, 1H disappears with D2O), 11.19 (s, 1H, CONH, disappears
with D2O).
(E)-N'-(3-benzyloxy-4-methoxybenzylidene)-2-phenyl-2,3-dihydro-
1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (4h): White solid, yield
96%, m.p. 126–128 °C (water). IR (KBr) cm-1: 3639, 3063 (NH), 1643
(CONH), 1598 (C=N). 1H-NMR (DMSO-d6) : 3.81 (m, 4H, OCH3 + H3),
4.63 (n t, 1H, H2), 5.14 (s, 2H, CH2Bz), 5.45 (n t, 1H, H3), 7.01–7.50 (m,
14H, 13 Ar + H6), 7.82–8.24 (m, 2H, CH=N + NH, 1H disappears with D2O),
11.05 (s, 1H, CONH, disappears with D2O).
(E)-5-amino-1-(2-hydroxy-2-phenylethyl)-
N'-(4-fluoro-3-
phenoxybenzylidene)-1H-pyrazole-4-carbohydrazide (3j): White solid,
yield 65 %, m.p. 194–195 °C (ethyl acetate). IR (KBr) cm-1: 3451, 3346
(NH + NH2), 3096 (OH), 1621 (C=O), 1505 (C=N), 1287 (CO), 1204 (C-F).
1H-NMR (DMSO-d6) : 3.85–4.20 (m, 2H, CH2N), 4.90–5.05 (m, 1H,
CHOH), 5.65–5.75 (m, 1 H, OH, disappears with D2O), 6.25–6.55 (br s,
2H, NH2, disappears with D2O), 7.00–7.60 (m, 13H, Ar), 8.00–8.35 (m, 2H,
H3 pyraz + CH=N), 11.17 (s, 1H, CONH, disappears with D2O).
(E)-N'-(4-benzyloxy-3-methoxybenzylidene)-2-phenyl-2,3-dihydro-
1H-imidazo[1,2-b]pyrazole-7-carbohydrazide (4i): White solid, yield
69%, m.p. 202–204 °C (water). IR (KBr) cm-1: 3292, 3034 (NH), 1629
(CONH), 1602 (C=N). 1H-NMR (DMSO-d6) : 3.80 (m, 4H, OCH3 + H3),
4.60 (n t, 1H, H2), 5.12 (s, 2H, CH2Bz), 5.44 (n t, 1H, H3), 7.02–7.45 (m,
13H, Ar), 7.62 (s, 1H, H6), 7.82–9.24 (m, 2H, CH=N + NH, 1H disappears
with D2O), 11.04 (s, 1H, CONH, disappears with D2O).
(E)-N'-(3-phenoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-
b]pyrazole-7-carbohydrazide (4a): White solid, yield 77%, m.p. 215–
216 °C (abs. ethanol). IR (KBr) cm-1: 3205, 3035 (NH), 1640 (CONH), 1591
(C=N). 1H-NMR (DMSO-d6) : 3.80 (n t, 1H, H3), 4.58 (n t, 1H, H2), 5.43 (n
t, 1H, H3), 6.98–7.43 (m, 14H, Ar), 7.84 (s, 1H, H6), 7.98–8.10 (m, 2H,
CH=N + NH, 1H disappears with D2O), 11.18 (s, 1H, CONH, disappears
with D2O).
(E)-N'-(3-methoxy-2-phenoxybenzylidene)-2-phenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-7-carbohydrazide (4j): White solid, yield 81%,
m.p. 265–266 °C (water). IR (KBr) cm-1: 3261, 3060 (NH), 1629 (CONH)
1580 (C=N). 1H-NMR (DMSO-d6) : 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).
(E)-N'-(4-phenoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-
b]pyrazole-7-carbohydrazide (4b): White solid, yield 85%, m.p. 226–
228 °C (abs. ethanol). IR (KBr) cm-1: 3171, 3031 (NH), 1635 (CONH), 1580
(C=N). 1H-NMR (DMSO-d6) : 3.80 (n t, 1H, H3), 4.60 (n t, 1H, H2), 5.41 (n
t, 1H, H3), 6.98–7.55 (m, 14H, Ar), 7.67 (s, 1H, H6), 7.70–8.10 (m, 2H,
CH=N + NH, 1H disappears with D2O), 11.10 (s, 1H, CONH, disappears
with D2O).
General procedure for 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-
pyrazole-4-carbohydrazide
imidazo[1,2-b]pyrazole-7-carbohydrazide (13): A mixture of 5-amino-1-
(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl ester
(0.81 g, 3.1 mmol) or 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-
carboxylic acid ethyl ester 12 (0.8 g, 3.1 mmol) and hydrazine hydrate (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 re-crystallized
by ethanol/methanol (2:1).
(10)
and 2-phenyl-2,3-dihydro-1H-
(E)-N’-(3-benzyloxybenzylidene)-2-phenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-7-carbohydrazide (4c): White solid, yield 69%,
m.p. 173–175 °C (abs. ethanol). IR (KBr) cm-1: 3405, 3226 (NH), 1641
(CONH), 1599 (C=N). 1H-NMR (DMSO-d6) : 3.82 (n t, 1H, H3), 4.62 (n t,
1H, H2), 5.16 (s, 2H, CH2Bz), 5.45 (n t, 1H, H3), 7.02–7.52 (m, 14H, Ar),
7.75–8.15 (m, 3H, H6 + CH=N + NH, 1H disappears with D2O), 11.20 ( s,
1H, CONH, disappears with D2O).
9
(E)-N’-(4-benzyloxybenzylidene)-2-phenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-7-carbohydrazide (4d): White solid, yield 87%,
m.p. 188–190 °C (abs. ethanol). IR (KBr) cm-1: 3396, 3227, 3033 (NH),
1640 (CONH), 1610 (C=N). 1H-NMR (DMSO-d6) : 3.82 (n t, 1H, H3), 4.6
(n t, 1H, H2), 5.15 (s, 2H, CH2Bz), 5.42 (n t, 1H, H3), 7.02–7.47 (m, 14H,
Ar), 7.60 (s, 1H, H6), 7.80–8.10 (m, 2H, CH=N + NH, 1H disappears with
D2O), 11.05 (s, 1H, CONH, disappears with D2O).
5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide
(10): White solid, Yield 48 %, m.p. 183–184 °C. IR (KBr) cm-1: 3513, 3389
(NH2), 3316 (OH), 1603 (C=O). 1H-NMR (CDCl3):  3.90–4.08 (m, 2H,
CH2N), 4.21 (br s, 2H, NH2 disappears with D2O), 4.86–5.03 (m, 1H, CH-
OH), 5.72 (d, J = 4.6 Hz, 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).
(E)-N'-(3,4-dimethoxybenzylidene)-2-phenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-7-carbohydrazide (4e). White solid, yield 70%,
m.p. 206–207 °C (abs. ethanol). IR (KBr) cm-1: 3432, 3213 (NH) 1700,
1600 (CONH + C=N). 1H-NMR (DMSO-d6) : 3.70–3.92 (m, 7H, 2 OCH3 +
H3), 4.63 (n t, 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–
2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carbohydrazide
(13): White solid, Yield 66 %, m.p. 250–252 °C. IR (KBr) cm-1: 3296 (NH2),
1627 (C=O). 1H-NMR (DMSO-d6) : 3.77 (t, J = 8, 1H, H3), 4.26 (s, 2H,
NH2, disappears with D2O), 4.57 (t, J = 8, 1H, H3), 5.42 (dt, J = 4, J = 8,
9
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10.1002/cmdc.202000122
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1H, H2), 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).
DMSO concentration below 1%. Samples were mixed, additionally
incubated for 30 min on ice when specified, and dispensed to a 96-well
plate at 37 C. For each sample, tubulin polymerization was monitored by
fluorescence emission at 340 nm for 60 minutes in 1-minute intervals on
an Infinte 200 PRO-Tecan plate reader.
Antiproliferative activity, by MTT assay, and apoptosis, by DAPI
staining and microscope evaluation: Human cell lines A2780 (ovary,
adenocarcinoma), A549 (lung, carcinoma), T47D (epithelial breast
cancer), SKBR3 (breast cancer), MDA-MB-231 (breast, adenocarcinoma),
and IMR32 (brain, neuroblastoma) were seeded at 104/mL, 105/mL, 0.89
× 104/mL, and 1.9 × 104/mL, respectively in 180 μL RPMI 1640 complete
medium into flat-bottomed microtiter plates. After 6–8 h, 20 L containing
five 1:10 fold compound concentrations diluted in FCS containing 1, 2, or
8% DMSO were added to the culture medium (final DMSO concentrations:
0.1, 0.2, and 0.8%). Seventy-two hours later cells were processed as
described elsewhere [18]. The IC50s values were calculated on the basis
of single concentration–response curves. Each experiment was repeated
3–8 times. The triggering of apoptosis was evaluated on A2780, A549,
SKBR3 and IMR32 cells treated for 24 and 48 h with compounds 4g and
4i at their IC50s and IC75s as evaluated by the MTT assay on target cells.
Floating and adherent cells were harvested and were washed twice with
phosphate buffered saline (PBS), fixed with 100–120 L of 70% ethanol in
PBS, and maintained at 4 °C until analysis. The percentage of apoptotic
cells was evaluated at the fluorescence microscope after adding 6 L of a
DAPI solution at 10 mg/mL in water.
Computational Methods
Docking simulation: Docking simulations were performed using the
program Autodock v.4.2.2. [20]. All the simulations were performed using
the standard defaults; we used a searching grid encompassing the whole
protein surface. The docking experiment consisted of 100 Lamarckian
Genetic Algorithm runs. The generated docking poses were ranked in
order of increasing docking energy values and clustered on the basis of a
RMSD cut-off value of 0.5 Å. From the structural analysis of the lowest
energy solutions of each cluster, we could highlight the protein binding site.
As targets for all the simulations we used: the molecular structure of the
assembly formed by tubulin , tubulin , stathmin4 and the tubulin tyrosine
Ligase [17; PDB code 5J2T]. Figure 5 was drawn with the program
Chimera [21] and rendered by Adobe Photoshop 5.0.2.
Acknowledgements
Cell cycle analysis: The effect of active compounds 4g and 4i on cell
cycle phases was analyzed on A549 cells. To this end cells, treated for 24
and 48 hours with their IC50 and IC75 were harvested, washed with cold
normal saline, and fixed in 70% ethyl alcohol in PBS overnight at −20 °C.
Twenty-four hours later they were centrifuged, washed again with cold
PBS and stained with propidium iodide (PI) staining solution (50 mg/mL PI,
0.05% Triton X-100, 0.2 mg/mL RNase A in PBS) for 20 min, at room
temperature. Cells were finally analyzed for DNA content by means of a
FACSort flow cytometer (BD Bioscences, Mountain. View, CA). The
ModFit LT software was utilized to evaluate the cell cycle distribution.
Authors thanks NCI, Division of Cancer Treatment Diagnosis
(USA), for screening title compounds during the Developmental
Therapeutic Program (DPI). Authors thanks Drs Raggio R.,
Anzaldi M., and Mr Gagliardo O. for technical support. Authors
declare no conflict of interest on publication.
Keywords: antiproliferative agents • apoptosis • cell cycle
inhibition • imidazo[1,2-b]pyrazole acylhydrazones • tubuline
polymerization.
Immunofluorescence staining: The effect on microtubules of
compounds 4g and 4i was verified on A549 cells by immunofluorescence
staining of treated cells. They were cultured on chamber glass slides (BD
Biosciences, Bedford, USA), and treated with the 4g IC50 and IC75, and 4i
IC75 for 48 hours. Once harvested cells were then fixed with methanol:
acetone (1:1) for 10 min at −20 °C, followed by 10 min methanol at −20 °C.
The microtubules and centrosomes were labelled with a mouse anti-β-
tubulin antibody (Sigma-Aldrich, Milano, Italy), and a rabbit polyclonal anti-
γ-tubulin antibody (Sigma-Aldrich), respectively. After 1 h incubation with
primary antibodies at 37°C, cells were washed 3 times in PBS. The slides
were then incubated with fluorescein isothiocyanate (TRITC)-coupled anti-
rabbit secondary antibody (Sigma-Aldrich) and FITC-conjugated anti-
mouse secondary antibody (Sigma-Aldrich) for 30 min at 37 °C. All
antibodies were diluted 1:200 in PBS plus 1% BSA. After 3 times washing
in PBS, cells were counterstained with DAPI and mounted with Moviol.
Preparations were observed using an epifluorescence microscope (Provis
AX70, Olympus, Milan, Italy) with selective single-bandpass filters at 40×
optical magnification. Images were acquired with a digital CCD camera
(C4742 Orca II, Hamamatsu, Japan) driven by Cytovision (Applied
Imaging Corp., Santa Clara, CA, USA).
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Entry for the Table of Contents
▪
Two imidazopyrazole carbohydrazides were identified as potential anticancer drugs by preliminary screening of a large
library of small molecules on NCI 60 panel cell lines. Further analyses evidenced their ability to induce apoptosis and affect
cell cycle phases. Immunofluorescence analysis and tubulin polymerization assays suggest that mechanism of action
involves the microtubule system, hypothesis also supported by docking study.
12
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