Novel 4-(3-phenylpropionamido), 4-(2-phenoxyacetamido) and 4-(cinnamamido) substituted benzamides bearing the pyrazole or indazole nucleus: synthesis, biological evaluation and mechanism of action

Article abstract of DOI:10.1016/j.bioorg.2018.10.055

Based on some common structural features of known compounds interfering with p53 pathways and our previously synthesized benzamides, we synthesized new ethyl 5-(4-substituted benzamido)-1-phenyl-1H-pyrazole-4-carboxylates 26a-c, ethyl 5-(4-substituted benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylates 27a-c and N-(1H-indazol-6-yl)-4-substituted benzamides 31a,b bearing in the 4 position of the benzamido moiety the 2-phenylpropanamido or 2-phenoxyacetamido or cinnamamido groups. A preliminary test to evaluate the antiproliferative activity against human lung carcinoma H292 cells highlighted how compound 26c showed the best activity. This last was therefore selected for further studies with the aim to find the mechanism of action. Compound 26c induces intrinsic apoptotic pathway by activating p53 and is also able to activate TRAIL-inducing death pathway by promoting increase of DR4 and DR5 death receptors, downregulation of c-FLIP_L and caspase-8 activation.

Full text of DOI:10.1016/j.bioorg.2018.10.055

Bioorganic Chemistry 83 (2019) 367–379
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel 4-(3-phenylpropionamido), 4-(2-phenoxyacetamido) and 4-
(
cinnamamido) substituted benzamides bearing the pyrazole or indazole
nucleus: synthesis, biological evaluation and mechanism of action
a,1,⁎
b,1
a,⁎
a
Demetrio Raffa
, Antonella D'Anneo , Fabiana Plescia , Giuseppe Daidone ,
c,2 a,2
Marianna Lauricella , Benedetta Maggio
a
University of Palermo, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), Medicinal Chemistry and Pharmaceutical
Technologies, Via Archirafi 32, 90123 Palermo, Italy
b
University of Palermo, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), Laboratory of Biochemistry, Via del Vespro 129,
9
0127 Palermo, Italy
c
University of Palermo, Department of Experimental Biomedicine and Clinical Neurosciences, Laboratory of Biochemistry, Via del Vespro 129, 90127 Palermo, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Based on some common structural features of known compounds interfering with p53 pathways and our pre-
viously synthesized benzamides, we synthesized new ethyl 5-(4-substituted benzamido)-1-phenyl-1H-pyrazole-
2
2
2
-(3-phenylpropanamido)benzamides
-(2-phenoxyacetamido)benzamides
-cinnamamidobenzamides
4
-carboxylates 26a-c, ethyl 5-(4-substituted benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylates 27a-c and
N-(1H-indazol-6-yl)-4-substituted benzamides 31a,b bearing in the 4 position of the benzamido moiety the 2-
phenylpropanamido or 2-phenoxyacetamido or cinnamamido groups. A preliminary test to evaluate the anti-
proliferative activity against human lung carcinoma H292 cells highlighted how compound 26c showed the best
activity. This last was therefore selected for further studies with the aim to find the mechanism of action.
Compound 26c induces intrinsic apoptotic pathway by activating p53 and is also able to activate TRAIL-inducing
P53
TRAIL-receptors
Apoptosis
death pathway by promoting increase of DR4 and DR5 death receptors, downregulation of c-FLIP
activation.
L
and caspase-8
1
. Introduction
p53 gene is not mutated, the function of the p53 is often inhibited
through other mechanisms, including increased expression of MDM2 or
inactivation of the tumor suppressor protein p14/ARF [8]. Several
strategies can be undertaken to restore the altered p53 pathways in
tumor cells such as reactivation of structural stability, reactivation of
p53 transcriptional activity, inhibition of p53/MDM2 interaction [7],
by small molecules [9]. Sendermetan 1, SJ-172550 2 and RO-2443 3
[9], C646 4 [10], BIRB-796 5 [11,12], HZ00 6 [13], Chidamide (Tu-
cidinostat) 7 [14], and (MS-27–275) 8 [15], Tenovin-1 9 [16], Tenovin-
6 10 [16], Sorafenib 11[17] and compound 12 [18] (Fig. 1), are some
examples of active molecules which are able to restore p53 activity with
different mechanisms.
The tumor suppressor p53 is a highly regulated transcription factor
that plays a fundamental role in preventing tumorigenesis [1]. In
normal cells p53 levels are tightly controlled via the E3 ubiquitin ligase
MDM2, that binds p53 promoting its ubiquitination and proteasomal
degradation [2]. This represents a regulatory mechanism to maintain in
normal cells low levels of p53 preventing aberrant apoptosis. In re-
sponse to DNA damage or other cellular stress signals p53 is activated
and induces up- or down regulation of a variety of genes involved in cell
cycle arrest [1], DNA repair [3], senescence [4], or apoptosis [5]. The
activity and cellular levels of p53 are regulated through post-tran-
scriptional and post-translational mechanisms, including phosphoryla-
tion, acetylation, sumoylation, and methylation [2,6].
Despite their different mechanisms of action, some structural fea-
tures are common to them. In particular, the molecules have the ability
to assume several conformations due to the free rotation around the
several sigma bonds in the structure; superimposition of the staggered
p53 function is compromised in almost 50% of human cancers
through inactivating mutations in p53 gene [7]. In cancers in which the
⁎
Corresponding authors.
E-mail addresses: demetrio.raffa@unipa.it (D. Raffa), fabiana.plescia@unipa.it (F. Plescia).
These authors contributed equally to this work.
1
2
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2018.10.055
Received 17 August 2018; Received in revised form 3 October 2018; Accepted 27 October 2018
Available online 29 October 2018
0045-2068/ © 2018 Elsevier Inc. All rights reserved.

D. Raffa et al.
Bioorganic Chemistry 83 (2019) 367–379
Fig. 1. Examples of compounds interfering with p53 pathways.
conformation of these molecules shows a chain length ranging between
pyrazole, ethylene, vinylene and phenoxyacetamido (Fig. 2).
1
1 and 20 Å. All the structures present two or more electronegative
In the last decade we have dealt with studying the antitumor ac-
tivity of several new compounds trying to elucidate the mechanisms of
action underlying their activity [19–24]. We also studied the
atoms able to form hydrogen bonds. Finally, an overview of the struc-
tures in Fig. 1 shows the presence of some typical moieties such as:
Fig. 2. Structural common features of compounds interfering with p53 pathways.
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Bioorganic Chemistry 83 (2019) 367–379
Fig. 3. Previously synthesized anthranilamides 13–19.
relationship between the mechanism of action and the scaffold bound to
the benzamido moiety of some anthranilamide derivatives (Fig. 3,
compounds 13–19) [25–28].
molecules having the common features of compounds interfering with
p53 pathways (Fig. 4).
In particular, first we have selected anthranilamides bearing the
phenylpropanamido, cinnamamido and 2-phenoxyacetamido moieties
then, we have moved the carboxyamido group from the ortho to the
On that basis, in this paper, our strategy was to make some struc-
tural modification on our anthranilamides in order to make new
Fig. 4. Structural modification on anthranilamides.
369

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Bioorganic Chemistry 83 (2019) 367–379
para position. Also, we have lengthened the molecules by adding an N-
phenylpyrazole nucleus presents in some compounds interfering with
p53 pathways (Fig. 4).
Furthermore, as reported in Scheme 2, the reaction of the 4-ni-
trobenzoyl chloride 20 with 6-aminoindazole 28 in pyridine at 0–5 °C
gave the nitrobenzamide 29 which, in turn, was transformed by hy-
drogenation in presence of palladium on charcoal to give the 4-ami-
nobenzamide 30. Derivatives 31a,b were finally obtained by reacting,
in pyridine at 0–5 °C, the compound 30 and the acyl chlorides 25a,b.
Attempt to obtain the 4-nitrobenzamide 33 with the same way,
failed. The reaction of the 4-nitrobenzoyl chloride 20 with 1H-indazol-
3-amine 32 gave the unexpected derivative 34 instead of the compound
33 (Scheme 3).
Finally, the biological profile of derivatives containing the indazole
nucleus, a fused bicyclic pyrazole, instead of N-phenylpyrazole was also
investigated (Fig. 4).
The new synthesized molecules were evaluated both for their anti-
proliferative activity and toxicity on both human normal and cancer
cells. Compounds that showed good activity were further studied to
ascertain if the aforementioned structural modification could make
them able to interfere with p53 pathways. Finally, the best active
compound was used to study in deep the mechanism of action through
which they exert their effects.
2.2. Biology
2
.2.1. Benzamides exerts exert antiproliferative effects on human H292
2
. Results and discussion
lung mucoepidermoid carcinoma cells
Synthesized benzamides 26a-c, 27a-c and 31a,b were initially
tested in vitro for their antiproliferative activity against the human lung
mucoepidermoid carcinoma H292 cell line. The percent growth in-
hibition at a screening concentration of 10 μM at 48 h of treatment for
compounds are shown in Table 1.
2.1. Chemistry
A series of pyrazole-4-carboxylates 26a-c, 27a-c and 31a,b were
synthesized as described in Schemes 1 and 2. Crude acyl chlorides 25a-
c were obtained by refluxing the appropriate acids 24a-c with thionyl
chloride. The ethyl 5-(4-nitrobenzamido)-1H-pyrazole-4-carboxylates
The antiproliferative effects of the benzamides were compared with
that exerted in the same cells by SAHA (Vorinostat). SAHA is, a dea-
cetylase inhibitor which has been shown to induce apoptosis in dif-
ferent tumor cell lines by activating p53 [29] and that is currently
employed for clinical application [30]. Compounds 26a, 26c, 27a, and
27c, which showed the greatest efficacy in the preliminary experi-
ments, have been the subject of further studies. To this end, H292 cells
were treated with increasing concentrations of each compound (within
2
2a,b were synthesized by refluxing the 4-nitrobenzoyl chloride 20
with the 1H-pyrazole-4-carboxylates 21a,b in acetonitrile for 8 h
(
Scheme 1). Compounds 22a,b were therefore transformed by hydro-
genation in presence of palladium on charcoal to give the corre-
sponding amines 23a,b. Finally, the reaction of amines 23a,b with acyl
chlorides 25a-c, gave the expected derivatives 26a-c and 27a-c.
Scheme 1. Synthetic pathway to obtain compounds 26 and 27: (i) acetonitrile, reflux, 8 h; (ii) ethanol, H , Pd-C, 20 h; (iii) thionyl chloride, reflux, 5 h; (iv)
2
acetonitrile, reflux, 8 h.
370

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Bioorganic Chemistry 83 (2019) 367–379
Scheme 2. Synthetic pathway to obtain compounds 31: (i) pyridine, 0–5 °C, 24 h; (ii) ethanol, H
2
, Pd-C, 20 h; (iii) thionyl chloride, reflux, 5 h; (iv) pyridine, 0–5 °C,
2
4 h.
the range of 5–40 µM) for 48 h and the viability was evaluated by MTT
assay as reported in Methods. Fig. 5A shows that 26a, 27a and 27c did
not exert a significant cytotoxic effect on H292 cells even at higher used
concentration. In fact, their IC50 at 48 h of treatment were greater than
and untreated and treated cells were maintained in culture for addi-
tional 10 days to allow formation of colonies. The results demonstrated
that 26c suppressed the colony formation in H292 cells in a dose-de-
pendent manner when compared with the untreated groups (Fig. 5C).
5
0 µM. Instead, 26c markedly reduced the H292 cell viability compared
with control cells in a dose-dependent manner with an IC50 of 5 μM at
2
.2.2. Apoptotic effects of compound 26c on H292 cells
4
8 h of treatment. Interestingly, 26c exerted on H292 cells an anti-
To determine whether 26c inhibited cell growth by inducing
proliferative effect similar to SAHA, which exhibited on H292 an IC50
of 5 μM at 48 h of treatment (not shown). This data is in accordance
with our previous results obtained in H292 cells [31].
apoptosis, H292 cells were treated with increasing concentrations of the
compound (1–5 µM) for different times and stained with AO/EB as re-
ported in Methods. The results shown in Fig. 6 revealed that untreated
cells appeared viable with bright green nuclei, whereas cells treated
with 26c showed nuclear condensation and DNA fragmentation, char-
acteristic signs of the apoptotic cell death. The effect appeared after
The activity of 26c, which turned out to be the most active, was also
evaluated on HDFα, a normal human fibroblast cell line. Interestingly,
the effect of the compound was less pronounced than in H292 cells
(
IC50 of 20 µM at 48 h of treatment), thus suggesting that this com-
2
4 h of treatment and increased at 48 h.
pound may have limited toxicity in vivo at doses that would limit tumor
growth (Fig. 5B).
2
.2.3. 26c treatment induces the activation of intrinsic and extrinsic
Having demonstrated growth-inhibiting effect of 26c, we next ex-
amined its effect on colony formation by a clonogenic assay. This
procedure determines the ability of a cell to indefinitely proliferate,
thereby retaining its ability to form a large colony or a clone also after
the treatment with an antitumor compound. To this end, H292 cells
were plated with and without the addition of increasing doses of 26c
apoptotic pathways on H292 cells
Then, flow cytometric analyses with propidium iodide (PI) staining
of DNA were performed to investigate the distribution of cells in the
different phases of the cell cycle. These studies showed that 26c in-
creased the amount of cells in subG0/G1 phase, characteristic of
apoptotic cells with fragmented DNA. As shown in Fig. 7, the
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Bioorganic Chemistry 83 (2019) 367–379
Scheme 3. Synthetic pathway to obtain compounds 34: (i) pyridine, 0–5 °C, 24 h..
Table 1
the acetylated form of p53 (Fig. 8A), thus suggesting the activation of
the transcription factor. The effect was more pronounced after 24 h of
treatment with 5 µM 26c (increase of 3.2-fold respect to the control).
It is known that p53 regulates the expression of Bcl-2 family
members which are involved in the control of mitochondrial membrane
integrity [5]. In particular p53 up-regulates the levels of pro-apoptotic
members Bax and PUMA, while down-regulates the expression of the
anti-apoptotic ones such as Bcl-2 and Bcl-XL [34]. These events favor
mitochondrial membrane potential dissipation and activation of apop-
tosis. Our data shown in Fig. 8B demonstrated that H292 cells exposed
to 5 µM 26c exhibited a time-dependent decrease in Bcl-2 expression
compared with control cells. The decrease of Bcl-2 level was evident at
Percent growth inhibition obtained with the
H292 cell line with compounds at 10 μM at 48 h.
Comp.
H292 (10 µM)
2
2
2
2
2
2
3
3
6a
6b
6c
7a
7b
7c
1a
1b
15
11
60
20
12
25
ns
14
60
SAHA
4
8 h of treatment, differently from the increase of p53 and its acetylated
ns not significant (% inhibition < 10%).
form. This different temporal effect is related to the fact that p53 ac-
tivation is an early event of intrinsic apoptotic pathway, which then
regulates Bcl-2 decrease.
proportion of H292 cells in subG0/G1 phase increased from 0.5% in
control cells to 22.9% in 5 μM 26c-treated cells at 48 h. This effect was
time-dependent, in fact prolonging the time of treatment to 72 h the
percentage of 26c-treated cells in subGo/G1 reached 30.5%.
TRAIL (Apo2L), a member of the TNF superfamily, is involved in the
activation of apoptotic extrinsic pathway in a number of tumor cell
lines but is ineffective in the majority of normal ones [29,35]. Binding
of TRAIL to its receptors DR4 (TRAILR1) or DR5 (TRAILR2) results in
the trimerization of the receptors with the production of the death-in-
ducing signalling complex (DISC). Within this complex, pro-caspase-8 is
activated by autoproteolytic cleavage with the consequent activation of
effector caspases, such as caspase-3 and caspase-6, and induction of
apoptosis [36].
p53 is a master regulator of the intrinsic apoptotic pathway in re-
sponse to DNA damage or different kind of cellular stress [32]. To ex-
plore the ability of 26c to induce p53 in lung cancer cells, the level of
the protein was analysed by western blotting (Fig. 8A). Densitometric
quantification of bands obtained in repeated experiments showed that
2
6c increased in H292 cells the levels of p53 already after 24 h of
treatment. In particular, in comparison with the control, cells treated
Activation of caspases-8 can be inhibited by the cellular c-FLIP ,
L
with 5 µM 26c showed an increase in the levels of p53 by 4.6-fold and
which prevents the recruitment of the pro-caspase into the DISC com-
plex. To ascertain whether 26c is involved in the activation of TRAIL-
induced apoptosis, we performed western blotting analyses to evaluate
2
.8-fold after 24 and 48 h, respectively.
It has been reported that p53 undergoes to different post-transla-
tional modifications for its regulation and activation [1]. Acetylation of
p53 at carboxyl-terminal lysine residues is an important reversible en-
zymatic change that occurs in response to DNA damage and genotoxic
stress [29]. This event has been shown to enhance the p53 transcrip-
tional activity associated with cell cycle arrest and apoptosis [33]. Our
data showed that in H292 cells 26c treatment induced an increase of
the effects of the compound on TRAIL receptors, caspase-8 and c-FLIP .
L
Our results demonstrated that H292 cells exhibit both DR4 and DR5
receptors and that the level of these death receptors was up-regulated
by 26c treatment (Fig. 9A and B). The effect was more pronounced for
DR4, whose levels increased by 2.5 fold in cells treated with 5 µM 26c
for 48 h respect to the control (Fig. 9A). Our results also demonstrated
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Bioorganic Chemistry 83 (2019) 367–379
3
Fig. 5. Effects of benzamides on H292 cell viability and colony generation ability. H292 cells (5 × 10 ) were incubated in the presence of various doses of 26a, 26c,
2
7a, and 27c for 48 h, (B) The dose dependent effect of 26c compound evaluated in both human lung mucoepidermoid carcinoma H292 cells and normal human
HDFα cells at 48 h of treatment. Then the cytotoxic effect was determined by MTT assay as described in Section 4.2.1. Values are the means of three independent
experiments ± S.E. (*) p < 0.05 compared to the untreated sample. (C) Effect of compound 26c on colony generation ability of H292 cells. Clonogenic assay was
performed seeding a single cell suspension (200 cells) in 6-well plates and after 48 h the treatment was performed with different doses of compounds. The ability of
cells to generate colonies was evaluated after 10 days as reported in Methods. The number of colonies for each condition is reported in the upper panel of the figure,
whereas the number of cells for each colony is reported in the lower panel.
that 26c reduced the levels of c-FLIP
L
as well as decreased the levels of
cytotoxic effect of the compound is due to the activation of apoptosis, as
shown by AO/EB staining and flow cytometric analyses. In particular
our data provided evidence that 26c activated both intrinsic and ex-
trinsic apoptotic pathways. In fact, 26c increased p53 level as well as its
acetylated form, which can be responsible for the decrease in Bcl-2
level. Furthermore, 26c activated TRAIL-inducing apoptosis by up-
pro-caspase-8, thus suggesting the activation of the protease (Fig. 9A).
3
. Conclusion
Our data demonstrated that 26c exerted cytotoxic effects in H292
regulating DR4 and DR5 and down-regulating c-FLIP
L
with the
cells, a human lung carcinoma cell line with an IC50 at 48 h of 5 µM. The
Fig. 6. Apoptotic effect induced by 26c in human lung mucoepidermoid carcinoma H292 cells. The figure describes the effects of 26c treatment on H292 cell
morphology analyzed by fluorescence microscopy, after acridine orange/ethidium bromide double staining, as described in Sections 4.2.1 and 2. Three different
visual fields were examined for each condition. Microphotographs were taken at a magnification of 200×. Results are representative of three independent ex-
periments.
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Bioorganic Chemistry 83 (2019) 367–379
Fig. 7. Compound 26c effects on H292
cell cycle distribution. Human lung
mucoepidermoid carcinoma H292 cells
were treated with 26c for the indicated
times, then the DNA content was eval-
uated by flow cytometry after propi-
dium iodide staining as reported in
Section 4.2.1. The percentage of cells in
the different cell cycle phases was cal-
culated by Expo 32 software.
consequent activation of caspase-8. The increase of TRAIL death re-
ceptors may be also a consequence of p53 activation, because it has
been reported that DR4 and DR5 are transcriptional targets of p53 [37].
Moreover, TRAIL-induced apoptotic signaling can also contribute,
through Bid activation, to down-regulate Bcl-2 levels [34]. A possible
schematic model of 26c cytotoxic effects in H292 is reported in Fig. 10.
Fig. 8. Western blotting of p53, acetylated p53 (Ac-p53) and Bcl-2 in 26c -treated H292 cells. All analyses were performed after 24 and 48 h of treatment with 26c
compound. The correct protein loading was ascertained by immunoblotting for γ-tubulin. Representative blots of three independent experiments and densitometric
analysis are shown. (*) p < 0.05 compared to the untreated sample.
374

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Bioorganic Chemistry 83 (2019) 367–379
Fig. 9. Western blotting analyses of death receptor-mediated pathway in 26c-treated H292 cells. DR4, DR5, c-FLIP and pro-caspase 8 analyses were performed after
4 and 48 h of treatment with 26c compound. The correct protein loading was ascertained by immunoblotting for γ-tubulin. Representative blots of three in-
dependent experiments and densitometric analysis are shown. (*) p < 0.05 compared to the untreated sample.
2
4
. Experimental
described by us [38]. Equimolar amounts of 4-nitrobenzoyl chloride 20
3 mmol, 0.56 g) and ethyl 5-amino-1-phenyl-1H-pyrazole-4-carbox-
ylate 21a or ethyl 5-amino-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylate
(
4
4
6
.1. Chemistry
2
1b or 1H-indazole-6amine 28, (3 mmol) are dissolved in 10 ml of
.1.1. General
acetonitrile and placed under reflux for 8 h. After this time, the solution
is evaporated under reduced pressure to give a solid which is crystal-
lized from ethanol.
Reaction progress was monitored by TLC on silica gel plates (Merck
0,
F
254
,
0.2 mm). Organic solutions were dried over Na
2
SO .
4
Evaporation refers to the removal of solvent on a rotary evaporator
under reduced pressure. All melting points were determined on a Büchi
4.1.3.1. Ethyl 5-(4-nitrobenzamido)-1-phenyl-1H-pyrazole-4-carboxylate
5
30 capillary melting point apparatus and are uncorrected. 1H NMR
(22a). 11% yield, mp 154–156 °C; 1H NMR (CDCl ) δ: 1.37 (t, 3H,
3
(
300 MHz) and 13C NMR (75 MHz) spectra were recorded with a
CH ), 4.34 (q, 2H, CH ), 7.36–8.31 (m, 10H, ArH and pyrazole-H), 9.46
3
2
Bruker AC-E spectrometer at r.t.; chemical shifts (δ) are expressed as
ppm values. Microanalyses data (C, H, N) were obtained by an
Elemental Vario EL III apparatus and were within ± 0.4% of the theo-
retical values. Yields refer to purified products and are not optimized.
The names of the compounds were obtained using Chem Draw Ultra
(s, 1H, NH). 13C NMR(δ) (CDCl ) 14.34, 60.84, 106.10, 123.09,
3
124.06, 128.42, 128.88, 129.27, 137.99, 139.46, 139.86, 140.53,
159.29, 163.08, 164.03. Anal. Calcd. for C
1
9 16 4 5
H N O : C, 60.00%; H,
4.24%; N, 17.73%; Found: C, 59.93%; H, 4.17%; N, 14.63.
1
2.0 software (CambridgeSoft).
4.1.3.2. Ethyl
5-(4-nitrobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-
carboxylate (22b). 43% yield, mp 139–140 °C; 1H NMR (DMSO) δ:
4
.1.2. General procedure for preparation of 2-nitrobenzoyl chlorides (25a-
1.20 (t, 3H, CH ), 4.21 (q, 2H, CH ), 7.42–8.47 (m, 9H, ArH and
3
2
c) [25]
pyrazole-H), 11.22 (s, 1H, NH). 13C NMR(δ) (DMSO) 15.47, 61.36,
111.42, 118.32, 124.79, 125.21, 130.60, 139.50, 140.12, 140.93,
142.92, 149.49, 150.92, 152.79, 162.84, 165.24. Anal. Calcd. for
The 3-phenylpropanoyl, 2-phenoxyacetyl and cinnamoyl chlorides
2
5a-c, were obtained by refluxing for 5 h the appropriate acid deriva-
tives 24a-c (0.01 mol) with thionyl chloride (7.25 ml). After evapora-
tion under reduced pressure, the crude liquid residue was used for
subsequent reactions without purification.
C H N O : C, 56.69%; H, 3.96%; N, 18.37%; Found: C, 56.60%; H,
1
8 15 5 5
3.90%; N, 18.44.
4
.1.4. Preparation of ethyl 5-(4-aminobenzamido)-1-phenyl-1H-pyrazole-
4
.1.3. Preparation of ethyl 5-(4-nitrobenzamido)-1-phenyl-1H-pyrazole-4-
4-carboxylate (23a) and ethyl 5-(4-aminobenzamido)-1-(pyridin-2-yl)-1H-
pyrazole-4-carboxylate (23b)
carboxylate (22a) and ethyl 5-(4-nitrobenzamido)-1-(pyridin-2-yl)-1H-
pyrazole-4-carboxylate (22b)
To a solution of 1.3 mmol of compound 22a,b in 75 ml of warm
ethanol, 100 mg of 10% Pd-C as catalyst was added. The mixture was
Compounds 22a,b were obtained following a method previously
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D. Raffa et al.
Bioorganic Chemistry 83 (2019) 367–379
Fig. 10. Schematic representation of 26c effect in H292 cells. 26c induces intrinsic apoptotic pathway by activating p53. Moreover, the compound is able to activate
TRAIL-inducing pathway by promoting activation of DR4 and DR5 death receptors, downregulation of c-FLIP
pathway may be responsible for Bcl-2 down-regulation and activation of executioner caspases.
L
and caspase-8 activation. Both intrinsic and extrinsic
left under hydrogenation in a Parr apparatus at 50 psi for 20 h. The
suspension was filtered and the filtrate was evaporated affording the
crude compound 23a,b which was crystallized from ethanol.
pyrazole-4-carboxylate 23a or ethyl 5-(4-aminobenzamido)-1-(pyridin-
2-yl)-1H-pyrazole-4-carboxylate 23b (1.425 mmol) and the appropriate
benzoyl chloride 25a-c (1.425 mmol) in acetonitrile (5 ml) was refluxed
for 8 h. The solvent was evaporated under reduced pressure and the
residue filtered and recrystallized from ethanol.
4
.1.4.1. Ethyl 5-(4-aminobenzamido)-1-phenyl-1H-pyrazole-4-carboxylate
(
23a). 83% yield, mp 105–106 °C; 1H NMR (CDCl
3
) δ: 1.34 (t, 3H,
); 6.59–7.67 (m, 10H, ArH and
) 14.36, 60.48,
CH
3
); 4.12 (s, 2H, NH
2
); 4.30 (q, 2H, CH
2
4.1.5.1. Ethyl
1-phenyl-5-(4-(3-phenylpropanamido)benzamido)-1H-
pyrazole-H); 9.20 (s, 1H, NH). 13C NMR(δ) (CDCl
3
pyrazole-4-carboxylate (26a). 52% yield, mp 200–202 °C; 1H NMR
(CDCl ) δ: 1.33 (t, 3H, CH ); 2.62 (t, 2H, CH ); 3.01 (t, 2H, CH );
1
1
04.93, 114.07, 121.75, 122.88, 127.84, 129.08, 129.80, 140.36,
3
3
2
2
40.49, 140.81, 150.91, 164.13, 164.59. Anal. Calcd. for
4.30 (q, 2H, CH ); 7.17–8.01 (m, 15H, ArH and pyrazole-H); 8.03 (s,
2
C
19
H
18
N
4
O
3
: C, 65.13%; H, 5.18%; N, 15.99%; Found: C, 64.73%; H,
1H, NH); 9.23 (s, 1H, NH). 13C NMR(δ) (CDCl ) 14.35, 31.34, 39.39,
3
4
.97%; N, 16.19.
60.59, 105.46, 119.31, 122.96, 126.50, 127.74, 128.08, 128.35,
1
28.70, 128.86, 129.15, 140.08, 140.13, 140.35, 140.57, 141.97,
4
.1.4.2. Ethyl
5-(4-aminobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-
163.94, 164.31, 170.70. Anal. Calcd. for C28
H
26
N
4
O : C, 69.70%; H,
4
carboxylate (23b). 49% yield, mp 185–187 °C; 1H NMR (DMSO) δ:
5.43%; N, 11.61%; Found: C, 69.66%; H, 5.62%; N, 11.81.
1
9
1
1
.17 (t, 3H, CH
3
), 4.17 (q, 2H, CH
2
), 5.93 (s, 2H, NH ), 6.59–8.49 (m,
2
H, ArH and pyrazole-H); 10.58 (s, 1H, NH). 13C NMR(δ) (DMSO)
5.49, 61.08, 109.83, 114.05, 118.15, 120.56, 124.41, 131.00, 140.90,
41.22, 142.73, 149.29, 153.25, 154.22, 163.26, 165.59. Anal. Calcd.
4
.1.5.2. Ethyl
pyrazole-4-carboxylate (26b). 56% yield, mp 182–184 °C; 1H NMR
CDCl ) δ: 1.34 (t, 3H, CH ); 4.31 (q, 2H, CH ); 4.61 (s, 2H, OCH );
.97–8.02 (m, 15H, ArH and pyrazole-H); 8.49 (s, 1H, NH), 9.31 (s, 1H,
NH). 13C NMR(δ) (CDCl ) 14.37, 60.63, 67.55, 76.83, 105.44, 114.84,
19.66, 122.69, 122.98, 128.11, 128.52, 129.04, 129.18, 129.99,
140.08, 140.58, 140.89, 156.79, 163.97, 164.17, 166.66. Anal. Calcd.
for C27 : C, 66.93%; H, 4.99%; N, 11.56%; Found: C, 66.93%;
H, 5.23%; N, 11.81.
5-(4-(2-phenoxyacetamido)benzamido)-1-phenyl-1H-
(
3
3
2
2
for C18
H
17
N
5
O : C, 61.53%; H, 4.88%; N, 19.93%; Found: C, 61.55%;
3
6
H, 4.90%; N, 19.77.
3
1
4
.1.5. General procedure [38] for preparation of compounds (26a-c) and
27a-c)
An equimolar amount of ethyl 5-(4-aminobenzamido)-1-phenyl-1H-
(
H
24
N O
4 5
376

D. Raffa et al.
Bioorganic Chemistry 83 (2019) 367–379
4
.1.5.3. Ethyl
carboxylate (26c). 24% yield, mp 203–205 °C; 1H NMR (DMSO) δ:
.39 (t, 3H, CH ); 4.17 (q, 2H, CH ); 6.86 (d, 1H, J = 15.9 Hz, olefinic-
5-(4-cinnamamidobenzamido)-1-phenyl-1H-pyrazole-4-
Anal. Calcd. for C14
H
12
N
4
O: C, 66.65%; H, 4.79%; N, 22.21%; Found:
C, 67.01%; H, 4.90%; N, 22.13.
1
3
2
H); 7.43–7.92 (m, 15H, ArH and olefinic-H); 8.18 (s, 1H, pyrazole-H);
4.1.8. General procedure [39] for preparation of compounds (31a,b)
To a stirred cold (ice bath, 0–5 °C) solution of N-(1H-indazol-6-yl)-4-
aminobenzamide 30 (400 mg, 1.6 mmol) in pyridine (1.3 ml), the ap-
propriate benzoyl chloride 25a-c (1.6 mmol) was added dropwise.
Stirring was continued for 24 h then the white slurry was poured into
crushed ice. The solid that separated out was filtered off and re-
crystallized to give pure 31a,b.
1
6
1
1
0.39 (s, 1H, NH); 10.55 (s, 1H, NH). 13C NMR(δ) (CDCl ) 14.56,
3
0.24, 110.16, 119.04, 122.30, 124.20, 127.76, 128.31, 128.89,
29.34, 129.53, 129.73, 130.48, 135.01, 138.38, 139.20, 141.45,
41.76, 143.32, 162.03, 164.42, 166.43. Anal. Calcd. for
C
28
H
24
N
4
O : C, 69.99%; H, 5.03%; N, 11.66%; Found: C, 69.82%; H,
4
5
.43%; N, 12.06.
4
.1.5.4. Ethyl 5-(4-(3-phenylpropanamido)benzamido)-1-(pyridin-2-yl)-
4.1.8.1. N-(1H-Indazol-6-yl)-4-(3-phenylpropanamido)benzamide
1H-pyrazole-4-carboxylate (27a). 73% yield, mp 137–138 °C; 1H NMR
(31a). 63% yield, mp > 250 °C (ethyl acetate); 1H NMR (DMSO) δ:
(
CDCl
3
) δ: 1.28 (t, 3H, CH
3
); 2.68 (t, 2H, CH
2
); 3.02 (t, 2H, CH
2
); 4.29
2.68 (t, 2H, CH
2
); 2.93 (t, 2H, CH ); 7.19–8.26 (m, 13H, ArH and
2
(
q, 2H, CH
2
); 7.18–8.02 (m, 13H, ArH and pyrazole-H); 8.24 (s, 1H,
) 14.31,
indazole-H); 10.22 (s, H, NH); 10.23 (s, H, NH); 12.95 (s, 1H, indazole-
NH). 13C NMR(δ) (DMSO) 31.15, 38.51, 100.38, 115.55, 118.63,
119.77, 120.82, 126.50, 128.71, 128.80, 129.11, 129.63, 133.76,
137.92, 140.75, 141.55, 142.63, 165.89, 171.60. Anal. Calcd. for
NH); 8.38 (d, 1H, ArH); 11.32 (s, 1H). 13C NMR(δ) (CDCl
3
3
1
1
1.35, 39.25, 60.70, 107.43, 116.72, 119.33, 122.49, 126.35, 127.71,
28.39, 128.61, 128.94, 140.26, 140.58, 140.75, 142.26, 142.34,
45.88, 152.20, 162.96, 163.44, 171.14. Anal. Calcd. for
C
23
H
20
N
4
O : C, 71.86%; H, 5.24%; N, 14.57%; Found: C, 72.02%; H,
2
C
27
H
25
N
5
O
4
: C, 67.07%; H, 5.21%; N, 14.48%;. Found: C, 67.30%; H,
5.18%; N, 14.57.
4
.82%; N, 14.43.
4
.1.8.2. N-(1H-Indazol-6-yl)-4-(2-phenoxyacetamido)benzamide
4
.1.5.5. Ethyl
5-(4-(2-phenoxyacetamido)benzamido)-1-(pyridin-2-yl)-
(31b). 95% yield, mp > 250 °C; 1H NMR (CDCl ) δ: 4.76 (s, 2H,
OCH ); 7.02–8.27 (m, 13H, ArH); 10.29 (s, 1H, NH), 10.43 (s, 1H, NH),
3
1
H-pyrazole-4-carboxylate (27b). 48% yield, mp 173–174 °C; 1H NMR
2
(
CDCl
3
) δ: 1.29 (t, 3H, CH
3
); 4.31 (q, 2H, CH
2
); 4.63 (s, 2H, OCH
2
);
12.97 (s, 1H, indazole-NH). 13C NMR(δ) (CDCl ) 68.39, 101.32,
3
6
1
1
1
1
1
.98–8.39 (m, 14H, ArH and pyrazole-H); 8.58 (s, 1H, NH), 11.51 (s,
116.01, 116.45, 120.21, 120.68, 121.77, 122.60, 130.04, 130.92,
H, NH). 13C NMR(δ) (CDCl ) 14.34, 60.49, 67.54, 107.51, 114.81,
3
131.12, 134.66, 138.82, 141.65, 142.75, 159.13, 166.49, 168.46.
15.82, 119.70, 122.02, 122.62, 129.10, 129.28, 129.97, 139.54,
Anal. Calcd. for C22
H
18
N
4
O : C, 68.38%; H, 4.70%; N, 14.50%;
3
39.79, 140.77, 141.96, 146.89, 153.38, 156.82, 162.91, 162.98,
Found: C, 68.73%; H, 4.47%; N, 14.17.
66.68. Anal. Calcd. for C26
H
23
N
5
O : C, 64.32%; H, 4.78%; N,
5
4.43%; Found: C, 64.65%; H, 4.68%; N, 14.57.
4.1.9. Procedure for preparation of 4-nitro-N-(1-(4-nitrobenzoyl)-1H-
indazol-3-yl)benzamide (34)
4
4
1
.1.5.6. Ethyl 5-(4-cinnamamidobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-
Attempt to obtain the N-(1H-indazol-3-yl)-4-nitrobenzamide 33
(Scheme 3) with the same method [38] used for compound 29 failed.
Immediately a precipitate is formed which appears to be the 4-nitro-N-
(1-(4-nitrobenzoyl)-1H-indazol-3-yl)benzamide 34.
-carboxylate (27c). 15% yield, mp 204–206 °C; 1H NMR (DMSO) δ:
.20 (t, 3H, CH
3
); 4.21 (q, 2H, CH ); 6.90 (d, 1H, J = 15.6 Hz, olefinic-
2
H); 7.45–8.50 (m, 15H, ArH, pyrazole-H and olefinic-H); 10.59 (s, 1H,
NH); 10.87 (s, 1H, NH). 13C NMR(δ) (DMSO) 14.60, 60.29, 109.80,
1
1
1
6
17.40, 119.12, 122.23, 123.71, 128.02, 128.32, 129.38, 129.52,
4.1.9.1. 4-Nitro-N-(1-(4-nitrobenzoyl)-1H-indazol-3-yl)benzamide
(34). 73% yield, mp 222–225 °C; 1H NMR (DMSO) δ: 7.47–8.49 (m,
12H, ArH); 11.67 (s, 1H, NH). 13C NMR(δ) (DMSO) 115.80, 121.28,
123.46, 123.77, 124.02, 125.47, 130.25, 130.96, 132.09, 139.17,
30.46, 135.03, 139.59, 139.99, 141.43, 141.94, 143.32, 148.54,
52.14, 162.15, 164.43, 164.76. Anal. Calcd. for C27
H
23
N
5
O : C,
4
7.35%; H, 4.81%; N, 14.54%; Found: C, 67.42%; H, 4.62%; N, 14.83.
1
39.60, 140.71, 146.83, 149.55, 150.05, 165.23, 166.30. Anal. Calcd.
4.1.6. Preparation [39] of N-(1H-indazol-6-yl)-4-nitrobenzamide (29)
for C21
H
13
N
5
O : C, 58.47%; H, 3.04%; N, 16.24%; Found: C, 58.32%;
6
To a stirred cold (ice bath, 0–5 °C) solution of 1H-indazole-6-amine
H, 2.90%; N, 16.35.
2
8 (611 mg, 5.4 mmol) in pyridine (16 ml), 4-nitro-benzoyl chloride 20
(
1 g, 5.4 mmol) was added dropwise. Stirring was continued for 24 h
4.2. Biology
then the white slurry was poured into crushed ice. The solid that se-
parated out was filtered off and recrystallized from ethanol to give pure
4.2.1. Materials and methods
2
9.
4.2.1.1. Cell culture and treatment conditions. The human lung
mucoepidermoid carcinoma H292 cells (American Type Culture
Collection, ATCC) and human dermal fibroblasts HDFα (gently
provided by Dr Marta Di Carlo) were cultured in RPMI-1640 medium
(Sigma Aldrich, Milan, Italy) supplemented with 10% heat-inactivated
Fetal Bovine Serum, 100 U/mL streptomycin, and 100 U/mL penicillin
4
.1.6.1. N-(1H-Indazol-6-yl)-4-nitrobenzamide
(29). 58%
yield,
: C,
mp > 250 °C; 1H NMR (CDCl
3
) δ: 8.39–7.39 (m, 8H, ArH); 10.71 (s,
1
H, NH); 13.03 (s, 1H, indazole-H). Anal. Calcd. for C14
H
10
N
4
O
3
5
9.57%; H, 3.57%; N, 19.85%; Found: C, 59.82%; H, 3.85%; N, 19.83.
(
Life Technology, Milan, Italy) in a humidified atmosphere with 5%
4
.1.7. Preparation of N-(1H-indazol-6-yl)-4-aminobenzamide (30)
CO
2
, as previously reported [31]. The cells were grown as monolayers
2
Compound 30 was obtained with the same method used for the
attached to 75 cm culture flasks and cultured at 37 °C in a 5% CO
2
compounds 23a,b solubilizing the nitro compound 29 (880 mg,
humidified incubator.
3
.12 mmol) in warm ethanol (mL 120) adding 88 mg of 10% Pd-C as
Synthesized compounds 26a-c, 27a-c and 31a,b were prepared as a
20 mM stock solution in dimethyl sulfoxide (DMSO), stored at 20 °C and
freshly dissolved immediately before use. The maximum final con-
centration of DMSO in the medium was less than 0.01%. Working di-
lutions were made in sterile medium and added to the complete cell
culture medium at the appropriate concentrations. Twenty-four hours
after seeding, when the cells reached 70% confluency, the cultures were
treated with a range of concentrations of compounds. Cells were
catalyst.
4
.1.7.1. N-(1H-Indazol-6-yl)-4-aminobenzamide
(30). 42%
yield,
mp > 250 °C; 1H NMR (DMSO) δ: 5.82 (s, 2H, NH
2
); 6.64–7.98 (m,
7
H, ArH); 8.30 (s, 1H, indazole-H); 9.95 (s, 1H, NH), 12.95 (s, 1H,
indazole NH). 13C NMR(δ) (DMSO) 99.88, 112.87, 115.46, 119.28,
20.51, 121.44, 129.72, 133.52, 138.29, 140.73, 152.46), 165.91.
1
377

D. Raffa et al.
Bioorganic Chemistry 83 (2019) 367–379
routinely photographed before and after incubation with the com-
pounds to record morphological changes occurring in the cells, using an
inverted light microscope equipped with phase contrast rings (LEICA
DM-IRB, Leica Microsystem, Milan, Italy).
peroxidase-labeled antibodies were purchased from Amersham Life
Science Inc. Immunoreactive signals were detected using enhanced
chemiluminescence (ECL) reagents (Bio-Rad). The correct protein
loading was confirmed by stripping the immunoblot and reprobing with
primary antibody for γ-tubulin (diluted 1:5000; Sigma).
4
.2.1.2. MTT assay. The viability of both lung mucoepidermoid
carcinoma H292 cells and human dermal fibroblasts HDFα treated
4.2.1.6. Statistical analysis. The statistical analysis of data was
performed using the GraphPad Prism 5 software package (San Diego,
CA). The data obtained were compared by the One-way ANOVA
analysis of variance using Bonferroni post-hoc multiple comparisons.
The data are expressed as means ± SD. The statistical significance
threshold was fixed at p < 0.05.
with compounds 26a-c, 27a-c and 31a,b was measured using 3-(4,5-
dimethylthiazol-2-yl)-2,3-diphenyltetrazolium
bromide
(MTT)
3
colorimetric assay. Briefly, 5 × 10 cells were plated in 200 µl of
complete medium per well in 96-well plates and treated with various
concentrations of compounds. Then, cell viability analysis by MTT was
performed as described [40]. MTT is reduced to purple formazan in the
mitochondria of living cells. The absorbance of the formazan was
measured at 570 nm, with 630 nm as a reference wavelength by
scanning the relative cell viability with an ELISA plate reader (OPSYS
MR, Dynex Technologies).
Acknowledgement
Financial support from “Fondo di Finanziamento della Ricerca di
Ateneo, University of Palermo” is gratefully acknowledged.
Cell viability was expressed as the percentage of the OD value of
inhibitor-treated cells compared with untreated samples used as con-
trol. Each experiment was performed in triplicate.
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