New complex polycyclic compounds: Synthesis, antiproliferative activity and mechanism of action

Article abstract of DOI:10.1016/j.bioorg.2020.103989

Polycyclic or O-glycoconiugate polycyclic compounds 1a-g were previously tested for their in vitro antiproliferative activity. In this series of compounds, activity increases as log P decreases. Specifically, compounds 1d and 1g showed lower log P values together with the best antiproliferative profiles. With the aim of extending our understanding of the structure–activity relationship (SAR) of this class of compounds, we prepared new polycyclic derivatives 2a-c, which bear on each of the two phenyl rings hydrophilic substituents (OH, SO₂NH₂ or NHCOCH₃). These substituents are able to form hydrogen bonds and to decrease the partition coefficient value as compared with compound 1d. Compound 2a was slightly more active than 1d, while 2b and 2c had antiproliferative activity comparable to that of 1d. Finally, the role of the two phenyl groups of polycycle derivatives 1 was also investigated. The analog 3, which bears two methyls instead of the two phenyls had a lower log P value (2.94 ± 1.22) than all the other compounds, but it had negligible antiproliferative activity at 10 μM. The analysis of the most active derivative 2a revealed a significant antiproliferative activity against the triple-negative breast cancer cell line MDA-MB231. After a 24 h treatment, an autophagic process was activated, as demonstrated by an increase in monodansylcadaverine-positive cells as well as by the appearance of the autophagic markers Beclin and LC3II. Prolonging the treatment to 48 h, 2a caused cytotoxicity through the activation of caspase-dependent apoptosis.

Full text of DOI:10.1016/j.bioorg.2020.103989

BioorganicChemistry101(2020)103989
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New complex polycyclic compounds: Synthesis, antiproliferative activity
and mechanism of action
⁎
Giuseppe Daidonea,1, Antonella D'Anneob,1, Maria Valeria Raimondia, ,1, Demetrio Raffaa,
⁎
Ernest Hameld, Fabiana Plesciaa, , Marianna Lauricellac,2, Benedetta Maggioa,2
a Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), Medicinal Chemistry and Pharmaceutical Technologies Section –
University of Palermo, Via Archirafi 32, 90123 Palermo, Italy
b Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), Laboratory of Biochemistry, University of Palermo, Via del Vespro 129,
90127 Palermo, Italy
c Department of Biomedicine, Neurosciences and Advanced Diagnostics (BIND), Institute of Biochemistry, University of Palermo, Via del Vespro 129, 90127 Palermo, Italy
d Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer Research,
National Cancer Institute, National Institutes of Health, Frederick, MD 21702, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Polycyclic or O-glycoconiugate polycyclic compounds 1a-g were previously tested for their in vitro antiproliferative
activity. In this series of compounds, activity increases as log P decreases. Specifically, compounds 1d and 1g showed
lower log P values together with the best antiproliferative profiles. With the aim of extending our understanding of
the structure–activity relationship (SAR) of this class of compounds, we prepared new polycyclic derivatives 2a-c,
which bear on each of the two phenyl rings hydrophilic substituents (OH, SO2NH2 or NHCOCH3). These substituents
are able to form hydrogen bonds and to decrease the partition coefficient value as compared with compound 1d.
Compound 2a was slightly more active than 1d, while 2b and 2c had antiproliferative activity comparable to that of
1d. Finally, the role of the two phenyl groups of polycycle derivatives 1 was also investigated. The analog 3, which
O-glycoconjugate polycyclic compounds
Pyrazolo[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-
f]azocine
Antiproliferative activity
MDA-MB231 breast cancer cells
Autophagy
Apoptosis
bears two methyls instead of the two phenyls had a lower log P value (2.94
1.22) than all the other compounds,
but it had negligible antiproliferative activity at 10 µM. The analysis of the most active derivative 2a revealed a
significant antiproliferative activity against the triple-negative breast cancer cell line MDA-MB231. After a 24 h
treatment, an autophagic process was activated, as demonstrated by an increase in monodansylcadaverine-positive
cells as well as by the appearance of the autophagic markers Beclin and LC3II. Prolonging the treatment to 48 h, 2a
caused cytotoxicity through the activation of caspase-dependent apoptosis.
1. Introduction
clinically isolated cancer types (leukemia, lung, colon, melanoma,
renal, ovarian, brain, breast and prostate). The most active compounds
Previously we have reported the synthesis of the complex polycyclic
compounds 1a-h containing the 5,7:7,13-dimethanopyrazolo[3,4-b]
pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine system (Fig. 1). The structure
of these is characterized by a fused hexacyclic system that includes two
N-phenyl-substituted pyrazole rings (A and B in Fig. 1) that stabilize the
half-chair conformation of the adjacent two azepine rings (E and F in
Fig. 1). Finally, these compounds have two fused five-membered rings
(G and H in Fig. 1) [1–3].
were 1d and 1g, bearing on each of the two phenyl rings a methoxy or
glucose-containing moiety, respectively. Both of these moieties could be
involved in hydrogen bonds with a cell molecular target and produce
lower partition coefficient values than the inactive compounds 1b,c,e,
which all have very high log P values (Table 1).
Moreover, comparing 1d and 1g, the latter derivative was much
more active than the former. In fact, it showed antiproliferative ac-
tivity, expressed as a GI50 value (compound concentration that inhibits
50% net cell growth) in the range 0.29–5.43 µM against all the tumor
cell lines of the NCI panel, and the mean graph midpoint (MG_MID,
arithmetical mean value) calculated for LogGI50 (M) was −5.82 [3].
Compounds 1a-g were tested in vitro by the National Cancer
Institute (NCI, Frederick, MD, USA) for their antiproliferative activity
against a panel of 57 human cancer cell lines derived from nine
⁎ Corresponding authors.
E-mail addresses: mariavaleria.raimondi@unipa.it (M.V. Raimondi), fabiana.plescia@unipa.it (F. Plescia).
1 These authors contributed equally to this work.
2 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.103989
Received 18 February 2020; Received in revised form 25 April 2020; Accepted 29 May 2020
Availableonline01June2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Fig. 1. Compounds 1a-h and X-ray perspective of 1h.
Table 1
Calculated log P values of previously synthesized
compounds 1a-g.
Compound
log P
1a
1b
1c
1d
1e
1f
6.84
10.23
8.03
6.67
7.76
8.03
3.11
1.32
1.68
1.33
1.48
1.32
1.33
1.51
1g
For compound 1d, the GI50 values were instead in the 1.18–11.3 µM
range, and the mean graph midpoint value for LogGI50 (M) was −5.50
[2].
At this point, in order to obtain new polycyclic derivatives with
greater hydrophilic character than 1d, as well as to gain more insight
into the role of the glucose-containing moiety of 1g, we prepared new
polycyclic derivatives 2a,b,c, which bear on each of the phenyl rings
the hydrophilic groups OH, SO2NH2, or NHCOCH3 (Fig. 2).
Finally, we wanted to determine whether the two phenyl groups of
polycycle 1a could be replaced with methyl groups (compound 3,
Fig. 3) because this change decreased the partition coefficient value and
therefore might enhance antiproliferative activity.
Fig. 2. Structures of compounds 2a-c.
1) was obtained starting from the 3,N-dimethylpyrazole-5-amine deri-
vative 4, which was demethylated by reaction in 24% aqueous hydro-
bromic acid under microwave (MW) irradiation to give 5.
The latter compound was reacted with hexane-2,5-dione in 1,4-di-
oxane and in the presence of p-toluenesulfonic acid to afford 2a.
Compound 2b was obtained as described in Scheme 2.
2. Results and discussion
The pyrazole-5-amine derivative 8, bearing the 4-sulfamoylphenyl
moiety,
is
known.
It
was
obtained
from
4-hy-
2.1. Chemistry
drazinylbenzenesulfonamide 6 and 3-iminobutanenitrile 7 by mod-
ifying the reported procedure [4]. The 3-methyl-1-(4-sulfamoylphenyl)-
1H-pyrazole-5-amine 8 was formylated with 95% formic acid to give
The synthesis of polycyclic derivatives 2a-c and 3 (Figs. 2 and 3)
was performed as described in Schemes 1–3. The polycycle 2a (Scheme
2

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Fig. 3. Structure of compounds 1a and 3.
Scheme 1. Synthetic route to obtain compound 2a: i HBr 24%, MW: 150 °C, 80 W, 6 cycles of 10 min each; ii hexane-2,5-dione, p-toluenesulfonic acid, reflux.
derivative 9, which, in turn, was reduced with lithium aluminum hy-
dride to afford the 3,N-dimethyl-1H-pyrazole-5-amine derivative 10.
Finally, the latter compound was reacted with hexane-2,5-dione in 1,4-
dioxane, in the presence of p-toluenesulfonic acid, to give the polycyclic
derivative 2b.
2a,b,c and 3 were confirmed principally by NMR analysis. The 1H NMR
spectra of 2a,b,c and 3 are highly complex and very similar to that of
polycyclic compounds previously reported by us [1–3], and, as a con-
sequence, we assigned to them the same skeleton structure. In Fig. 1 is
shown the X-ray perspective of 1h that can be taken as the stereo-
chemical reference model for compounds 2 and 3. The 13C NMR (APT)
spectra of compounds 2 and 3 confirmed the assigned structures.
Compound 2c was prepared as described in Scheme 3, starting from
the pyrazole derivative 11, which was reduced by hydrogen and Pd/C
to give the amino derivative 12. The latter compound was treated with
LiAlH4 to afford compound 13. The amino group linked to phenyl ring
was protected by benzylchloroformiate to afford 14, which was reacted
with hexane-2,5-dione in 1,4-dioxane and in the presence of p-tolue-
nesulfonic acid to yield the intermediate 15. Compound 15 was de-
protected to give 16. Finally, 16 was acetylated by acetic anhydride to
afford the desired polycycle 2c.
2.2. Biological activity
The newly synthesized compounds 2a-c and 3 were evaluated by
the NCI against a panel of approximately 60 human cancer cell lines
according to the NCI standard protocol [5], as had been compounds 1a-
g previously [2,3].
Compound 3 was obtained as described in Scheme 5 by reacting
1,3,N-trimethyl-pyrazole-5-amine 17 with hexane-2,5-dione in dioxane
and in the presence of p-toluenesulfonic acid to afford 3.
A mean value was calculated for percent of cell growth inhibition at
a 10-5 M concentration, giving an average activity parameter for all the
cell lines. The mean growth percent for the new derivatives 2a-c and 3
are summarized in Table 2 along with the previously published [2,3]
data obtained with 1a-g for ease of comparison.
Despite some differences in the experimental procedures to obtain
the best yields of the synthesized products (Schemes 1–4), the reactions
proceeded with the same mechanism of action. In particular, the for-
mation of compounds 2a-c and 3 can be hypothesized as described in
scheme 5 for compound 2a, taken as an example. The reaction between
the N-methyl-1-phenyl-1H-pyrazol-5-amine 5 and the hexane-2,5-dione
18 in the presence of p-toluenesulfonic acid leads to the intermediate
19 containing a pyrazolo[3,4-b]azepine structure. The rearrangement
of one molecule of 19, by the action of a H+ ion, gives a new enamine
intermediate. This last reacts with another molecule of 19 to form a
methylene bridge between the two structures. A further rearrangement
of this adduct leads to the formation of 2a.
Our data (Table 1, Fig. 4) show that the best Mean Growth Percent
values were obtained by compounds with log P values falling in the
range 3.05–6.67. However, the greater hydrophilic character of 2a-c as
compared with 1d produced a slight increase in antiproliferative ac-
tivity only in the case of 2a, whereas for 2b and 2c their anticancer
activity was similar to that observed with 1d.
Compound 3, despite having the lowest log P value in this group of
compounds, was among the least active. Apparently, replacing the two
phenyl moieties with methyl groups led, perhaps, to the loss of some
fundamental interactions with the biological target(s). The polycycle 2b
bears two sulfonamido groups as substituents, and it has a log P value of
3.05, similar to the value of 3.11 of 1g. Despite these similar log P
The new compounds were identified on the basis of analytical as
well as spectroscopic data. In particular, the structures of compounds
3

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Scheme 2. Synthetic route to obtain compound 2b: i 50 °C, 10 min; 37% aqueous HCl, 15 min, reflux, NaOH; ii 95% formic acid, reflux; iii LiAlH4; iv hexane-2,5-
dione, p-toluenesulfonic acid, reflux.
values, 1g was significantly more active than 2b.
a 48 h treatment with 15 or 20 μM compound 2a. To determine whe-
ther the reduction in cell number caused by compound 2a on MDA-
MB231 cells was related to cell death induction, we evaluated by flow
cytometric analysis the effect of the compound on cell staining by
propidium iodide (PI), a fluorescent dye that binds to DNA by inter-
calating between base pairs.
These data thus support the hypothesis that the glucose moieties of
1g might play a role in the transport of 1g into the cancer cells. In
support of this idea, it is well documented that the majority of cancer
cells, to satisfy their high glucose requirement, increase their glucose
uptake by overexpressing a family of facilitative glucose transporters
(GLUTs) [6,7]. In this context, the glucose moieties of 1g might be an
effective substrate for these glucose transporters [3,8].
Since PI is a cell-impermeable dye that in isotonic conditions stains
only nuclei of cells that have lost plasma membrane integrity, it is
commonly used to detect dead cells. As shown in Fig. 5C, the percen-
tage of PI-positive cells increased from 2.9% in control cells to 50.8%
and 56.0% in MDA-MB231 cells treated with 15 or 20 μM compound
2a, respectively, for 48 h, thus inducing significant cell death. Further,
flow cytometric analyses were performed to investigate the distribution
of cells in the different phases of the cell cycle. These studies showed
that treatment of MDA-MB231 cells with compound 2a did not induce
significant changes in cell cycle distribution after a 24 h treatment, but
a 48 h treatment did increase the proportion of cells in the sub-G0/G1
region, characteristic of apoptotic cells with fragmented DNA (Fig. 6).
2.2.1. Compound 2a reduced viability of MDA-MB231 breast cancer cells
To gain insight into the mechanism of action of 2a, the compound
with the greatest antiproliferative activity, we evaluated its effect on
MDA-MB231 cells, a triple-negative breast cancer cell line. MDA-
MB231 cells were treated with various doses of compound 2a for dif-
ferent times, and cell viability was evaluated with the 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We
found that 2a strongly reduced MDA-MB231 cell viability in a time- and
dose-dependent manner (Fig. 5A). A modest reduction of viability was
observed in the first 24 h of treatment when a decrease of only 24% and
35% of control was observed with 15 and 20 μM compound 2a, re-
spectively. Prolonging the treatment time resulted in a further sig-
nificant reduction in the viability of the cells, so that at 48 h a reduction
of viable cells by 55% and 80% of control occurred with 15 and 20 μM
compound 2a, respectively.
2.2.2. Compound 2a induced autophagy in MDA-MB231 cells
The analysis of the effects exerted by compound 2a on MDA-MB231
cells showed that the compound had only a modest cytotoxic effect in
the first 24 h of treatment. This raised the possibility that a survival
mechanism was activated in a first phase of treatment.
The observation of MDA-MB231 cells under light microscopy
showed clear morphological changes in cells exposed to compound 2a.
In fact, as shown in Fig. 5B, control cells showed a clear epithelial-like
morphology, phenotypically appearing as spindle shaped cells. In con-
trast, there was a marked reduction in cell number and in cell size after
Autophagy is a self-degradative process that is activated in response
to many stresses, including nutrient deprivation, hypoxia and oxidative
stress [9,10]. Through the elimination of intracellular aggregates and
damaged organelles and the recycling of the breakdown products, au-
tophagy plays important roles in cell survival [11,12]. The process is
4

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Scheme 3. Synthetic route to obtain compound 2c: i H2/Pd; ii LiAlH4; iii NaOH 4 M, benzylchloroformiate; iv p-toluenesulfonic acid, hexane-2,5-dione, reflux; v
EtOH/NaOH 6 M; vi acetic anhydride.
Scheme 4. Synthetic route to obtain compound 3: i p-toluenesulfonic acid, hexane-2,5-dione, reflux.
5

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Scheme 5. Hypothesized mechanism of reaction leading to polycycle 2a.
characterized by the formation of autophagic vacuoles (autophago-
somes) that sequestrate portions of cytoplasm and fuse themselves with
lysosomes, forming autolysosomes, to degrade the intra-autophago-
somal components by lysosomal hydrolases [13].
autophagy. MDC-positive fluorescent cells were observed after a 24 h
treatment with both 15 and 20 μM compound 2a, with little analogous
fluorescence in control cells.
Next, we analyzed the levels of the autophagic markers by Western
blotting (Fig. 7B). Beclin is a protein that plays an essential role during
the initiation of autophagy [14]. It is involved in autophagosome for-
mation, and its expression increases during autophagy [14]. The data
shown in Fig. 7B demonstrate that the Beclin protein level is sig-
nificantly higher (p < 0.001) after a 24 h treatment with either 15 or
20 μM compound 2a as compared to the untreated control.
Microtubule-associated protein light chain 3 (LC3) can be present in
The observation of cell morphology under light microscopy showed
the appearance of vacuoles in MDA-MB231 cells treated for 24 h with
compound 2a (Fig. 7A, upper panels). To verify the presence of au-
tophagic vacuoles, MDA-MB231 cells were stained with mono-
dansylcadaverine (MDC), a fluorescent marker for autophagic vacuoles.
Fig. 7A (lower panels) shows dot-like structures in the cytoplasm of
compound 2a-treated MDA-MB231 cells, suggesting the induction of
6

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Table 2
cycle that was observed in MDA-MB231 cells treated with compound 2a
Overview of the results of the in vitro single high dose (10-5 M) antitumor
for 48 h (Fig. 6).
screening for compounds 1-3.a
Compound
Caspases are cysteine aspartyl proteases involved in the induction
and execution of apoptotic cell death [17]. In healthy cells, these en-
zymes are present in inactive pro-caspase forms, which are activated
into an active form by cleavage of a pro-domain.
log P
No. Studiedb
% Mean Growthc
1a
1b
1c
1d
1e
1f
6.84
10.23
8.03
6.67
7.76
8.03
3.11
5.36
3.05
4.57
2.94
1.32
1.68
57
57
57
57
58
nt
57.21
105.75
104.41
34.55
92.11d
nt
The results shown in Fig. 8B demonstrate that, when MDA-MB231
cells were treated for 48 h with 15 or 20 µM compound 2a, the intensity
of the pro-caspase-9 band significantly (p < 0.001) decreased, sug-
gesting the activation of the protease. Moreover, the decrease of pro-
caspase-9 content was not observed after a 24 h treatment (Fig. 8B),
thus confirming that the activation of apoptosis occurred only after a
prolonged treatment time.
1.33
1.48
1.32
1.33
1.51
1.48
1.48
1.48
1.22
1g
2a
2b
2c
3
56
55
55
55
56
0.74
29.90
36.87
36.34
93.65
3. Conclusions
a
Data obtained from the NCI’s in vitro disease-oriented human tumor cell
screen.
We synthesized novel polycyclic derivatives 2a-c bearing on each of
the two phenyl rings the hydrophilic groups OH, SO2NH2, or
NHCOCH3. These groups are more able than the OCH3 group of 1d to
produce hydrogen bonds and to decrease the partition coefficient value
of the compounds. The greater hydrophilic character of 2a-c as com-
pared with 1d produced only a slight increase in antiproliferative ac-
tivity and only in the case of 2a (the activities of 2b and 2c were almost
identical to that of 1d). We also synthesized compound 3, eliminating
the two phenyl rings to produce an even more hydrophilic compound,
but it was almost inactive at 10 µM. We conclude that replacing the
phenyl moieties with methyl groups probably results in loss of inter-
actions with the biological target.
b
c
Refers to the number of cell lines.
Arithmetical mean value of growth percent (%MG) for all tested cancer cell
lines.
d
nt: not tested.
Although derivative 2b had a log P value (3.05) very similar to that
of the glycoconjugate 1g, the latter compound had a much higher an-
tiproliferative effect than 2b at 10 µM. This different activity supports
the hypothesis that the glucose moiety of 1g might be a substrate for
GLUTs.
Finally, compound 2a, the most active among the newly synthesized
agents, was selected for further biological studies. Analysis of the effects
of compound 2a on triple negative MDA-MB231 breast cancer cells
showed that 2a activated within 24 h an autophagic response, de-
monstrated by the appearance of MDC-positive cells and the increase of
the autophagic markers Beclin and LC3II. Prolonging the treatment to
48 h, compound 2a induced an apoptotic cell death, as demonstrated by
the activation of caspase-9.
Fig. 4. Mean Growth Percent values versus log P for compounds 1a-e,g, 2a-c
and 3.
the cells in two different forms: LC3I, a soluble cytosolic form, and
LC3II, a form conjugated with phosphatidylethanolamine and stably
associated with autophagosome structure [15]. During the induction of
autophagy, LC3I is cleaved to LC3II by the cysteine protease Atg4 and is
recruited to autophagosomal membranes. As shown in Fig. 7B, Western
blotting analysis demonstrated that treatment with compound 2a fa-
vored the accumulation of LC3II (p < 0.001) with a concomitant re-
duction of LC3I.
4. Experimental
4.1. Chemistry
4.1.1. General
Reaction progress was monitored by TLC on silica gel plates (Merck
60, F254, 0.2 mm). Organic solutions were dried over Na2SO4.
Evaporation refers to the removal of solvent on a rotary evaporator
under reduced pressure. All melting points were determined on a Büchi
530 capillary melting point apparatus and are uncorrected. IR spectra
were recorded with a Perkin Elmer Spectrum RXI FT-IR System spec-
trophotometer, with compound as a solid in a KBr disc or nujol. 1H
NMR (300 MHz) and APT (75 MHz) spectra were recorded with a
Bruker AC-E spectrometer at r.t.; chemical shifts (δ) are expressed as
ppm values. Microanalytic data (C, H, N) were obtained with an
Elemental Vario EL III apparatus and were within 0.4% of the theo-
retical values. Yield refers to purified products and is not optimized.
Log P values were calculated with the ACD/ChemSketch 14.01 2012
freeware version. The names of the compounds were obtained using
ChemDraw Ultra 12.0 software (CambridgeSoft).
2.2.3. Compound 2a induced apoptosis in MDA-MB231 cells
The autophagic process is activated in response to cellular stress
and, by improving the cell environment, it can represent a pro-survival
mechanism. However, if the stress signal is very intense and persistent,
the autophagic process cannot ensure cellular survival. If this is the
case, the process can switch to apoptosis to eliminate damaged cells
[12,16].
To investigate the activation of apoptosis in our experimental con-
dition, cells were examined for the characteristic morphological fea-
tures of apoptotic cell death by fluorescence microscopy. Using Hoechst
33342 to stain the nuclei, it was observed that, in comparison to normal
cells, treatment with 15 or 20 μM compound 2a induced, in a high
percentage of MDA-MB231 cells, chromatin condensation and nuclear
fragmentation, which are typical morphological changes occurring in
apoptotic cell death (Fig. 8A). These results are in accordance with the
increase in the proportion of cells in the subG0/G1 phase of the cell
4.1.2. Procedure to prepare 4-[3-methyl-5-(methylamino)-1H-pyrazol-1-
yl]phenol 5
To a solution of 1-(4-methoxyphenyl)-N,3-dimethyl-1H-pyrazol-5-
7

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Fig. 5. Effects of compound 2a on viability of MDA-MB231 breast cancer cells. (A) Cells (8 × 103) were treated with various doses of compound 2a (5–25 µM) for 24 or
48 h. Cell viability was assessed by the MTT assay. Values are the means of three independent experiments
S.D. (*) p < 0.05, (**) p < 0.01 compared to the
control. (B) Effects of compound 2a on the morphology of MDA-MB231 cells after treatment for 48 h with compound 2a. (C) Flow cytometric analysis of dead cells
using PI staining after a 48 h incubation with compound 2a. Data are representative of three independent experiments
S.D.
amine 4 (0.150 g, 0.69 mmol) in toluene (4 mL), a 24% HBr aqueous
solution (4 mL) was added. The reaction mixture was irradiated with a
MW power of 80 W at 150 °C for 10 min, and the MW cycle was re-
peated six times. The toluene was removed, and the solution was ba-
sified to pH = 8.5 with 40% aqueous NaOH. The white solid that
precipitated was removed by filtration and crystallized from ethyl
acetate to give 5 (0.073 g).
4.1.3. Procedure
to
prepare
4,4′-{1,4,5,8,11,13a-hexamethyl-12-
methylene-4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo
[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine-3,9-diyl}diphenol 2a
A solution of 5 (0.175 g, 0.86 mmol), 2,5-hexanedione (0.102 mL,
0.86 mmol) and p-toluenesulfonic acid monohydrate (20% mol/mol of
5) in anhydrous dioxane was refluxed for 120 h. After the first 24 h,
four portions of 2,5-hexanedione and p-toluenesulfonic acid mono-
hydrate (50% of the above amounts) were added at intervals of 24 h.
Then, the solvent was removed under reduced pressure, and the oily
residue was purified by flash chromatography [18] on silica gel
(0.063–0.2 mm) (200 g): external diameter of the column 4.5 cm, ethyl
acetate/cyclohexane (1:1) (1.3 L) as eluent. Fractions 21–25 (each
25 mL) were collected and evaporated to afford crude 2a (0.011g) as a
white solid, which was crystallized from ethanol.
4.1.2.1. 4-[3-methyl-5-(methylamino)-1H-pyrazol-1-yl]phenol (5). 52%
yield; mp 227–228 °C; IR (KBr) (cm−1): 3482 (OH); 3350 (NH); 1H
NMR (DMSO‑d6) (δ) 2.09 (3H, s, CH3); 2.63 (3H, s, NCH3); 5.14 (1H, s,
NH, exchangeable with D2O); 5.28 (1H, s, pyrazole H-4); 6.84, 7.26
(4H, dd, C6H4); 9.59 (1H, s, OH, exchangeable with D2O); 13C NMR
(CDCl3) (δ) 14.32 (CH3); 32.41 (NCH3); 86.92 (pyrazole CH); 115.90
(2xCH); 125.51 (2xCH); 131.26 (C); 147.45 (C); 150.56 (C); 156.28 (C).
Anal Calcd for C11H13N3O: C, 65.01; H, 6.45; N, 20.68. Found: 65.15; H,
6.52; N, 20.62.
4.1.3.1. 4,4′-{1,4,5,8,11,13a-hexamethyl-12-methylene-
4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo[3,4-b]
pyrazolo[3′,4′:2,3]azepino[4,5f]azocine-3,9-diyl}diphenol
(2a). 42%
yield; mp 298 °C; IR (KBr) (cm−1): 3450–3156 (broad band, OH); 1H
8

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Fig. 6. Effects of compound 2a on cell cycle distribution of MDA-MB231 breast cancer cells. Cells (1.5 × 104) were treated with 15 or 20 µM compound 2a for the
indicated times. Then, cells were centrifuged and resuspended in a hypotonic solution containing Nonidet-P-40 and PI. Cell cycle analysis by quantitation of DNA
content was performed by flow cytometry using a Beckman Coulter Epics XL flow cytometer. At least 10,000 events were acquired for each condition. Histograms of
the gated subG0/G1, G0/G1 and G2/M cells were analyzed by Expo32 software. Data are representative of three independent experiments.
NMR (DMSO‑d6) (δ) 1.16–2.67 (26H, a set of signals, 6xCH3, 3xCH2,
4.1.5.1. N-[3-methyl-1-(4-sulfamoylphenyl)-1H-pyrazol-5-yl]formamide
(9). 22% yield; mp 200–202 °C. IR (cm−1): 3342 (NH), 3253 (NH2),
1703 (CO); 1H NMR δ: 2.22 (3H, s, CH3); 6.38 (1H, s, pyrazole H-4);
7.56 (2H, br s, NH2, exchangeable with D2O); 7.71–7.92 (4H, dd,
C6H4,); 8.20 (1H, s, CHO) 10.37 (1H, s, NH, exchangeable with D2O).
Anal Calcd for C11H12N4O3S: C, 47.14; H, 4.32; N, 19.99. Found: C
46.80; H, 3.95; N, 20.26.
2xCH); 4.93, 4.98 (2H, br dd, methylene CH2); 6.77–7.40 (8H, a set of
signals, 2xC6H4); 9.59, 9.63 (2H, s, 2xOH, exchangeable with D2O). 13
C
NMR (DMSO‑d6) (δ) 15.17 (CH3); 16.69 (CH3); 23.85 (CH3); 25.16
(CH3); 34.41 (CH3); 35.80 (CH2); 36.02 (CH3); 44.83 (CH2); 46.49 (C);
55.91 (CH2); 61.61 (CH); 66.33 (CH); 77.32 (C); 110.16 (C); 111.78
(CH2, C); 115.25 (4xCH); 124.75 (2xCH); 124.99 (2xCH); 131.38 (C);
132.55 (C); 142.09 (C); 144.56 (C); 144.76 (C); 145.03 (C); 147.00 (C);
155.57 (C); 155.73 (C). Anal Calcd for C34H38N6O2: C, 72.57; H, 6.81;
N, 14.94. Found: C 72.49; H, 6.85; N, 15.07.
4.1.6. Procedure to prepare 4-[3-methyl-5-(methylamino)-1H-pyrazol-1-
yl]benzenesulfonamide 10
To
a suspension of lithium aluminum hydride (0.420 g,
4.1.4. Procedure to prepare 4-(5-amino-3-methyl-1H-pyrazol-1-yl)
benzenesulfonamide 8
11.07 mmol) in anhydrous tetrahydrofuran (15 mL), a suspension of 9
(1.000 g, 3.50 mmol) in anhydrous tetrahydrofuran (20 mL) was added
with stirring. The mixture was refluxed for 4 h, then the excess lithium
aluminum hydride was destroyed with methanol and water. The solvent
was then removed at reduced pressure, and the solid residue was ex-
tracted 5 times with ethyl acetate (5x40 mL). The organic extract was
evaporated under reduced pressure to give a residue that was crystal-
lized from ethanol to give 10 (0.420 g).
4-Hydrazinylbenzenesulfonamide 6 (5.000 g, 26.73 mmol) was so-
lubilized in a mixture of H2O (67.5 mL) and 37% HCl (2.8 mL), and the
reaction was heated for 10 min at 50 °C. Then a solution of diacetoni-
trile 7 (1.800 g) in ethanol (22.5 mL) was slowly added. The reaction
mixture was kept at 50 °C for another 10 min. After this time, 13.5 mL
of 37% HCl was added, and the mixture was refluxed for 15 min, then
cooled and basified to pH 8. A precipitate formed and was recovered by
filtration (3.507 g). This was used for subsequent reactions without
purification. Product 8 is known. Melting point, IR and 1H NMR spectra
were in agreement with those reported in the literature [6].
4.1.6.1. 4-[3-methyl-5-(methylamino)-1H-pyrazol-1-yl]
benzenesulfonamide (10). 45% yield; mp 224–30 °C. IR (cm−1) 3373
(NH), 3281 (NH2); 1H NMR δ 2.11 (3H, s, CH3); 2.65 (3H, d, CH3); 5.38
(1H, s, pyrazole H-4); 5.74 (1H, q, NH, exchangeable with D2O); 7.41
(2H, s, NH2, exchangeable with D2O); 7.73–7.88 (4H, dd, C6H4). Anal
Calcd for C11H14N4O2S: C, 49.61; H, 5.30; N, 21.04. Found: C 49.75; H,
5.40; N, 21.16.
4.1.5. Procedure to prepare N-[3-methyl-1-(4-sulfamoylphenyl)-1H-
pyrazol-5-yl] formamide 9
A mixture of 8 (2.830 g, 11.22 mmol) and 95% formic acid
(12.7 mL) was refluxed with magnetic stirring for 2 h. After that time,
the reaction mixture was neutralized with 40% aqueous NaOH. The
precipitated solid was recovered by filtration, air-dried and crystallized
from ethanol to give pure 9 (0.692 g).
9

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Fig. 7. Compound 2a treatment induced an autophagic flux in MDA-MB231 breast cancer cells. (A) Cells (1.5 × 104) were treated with 15 or 20 µM compound 2a for
24 h. The generation of the autophagic process was studied using MDC, a fluorescent dye that specifically labels the lysosomal/autophagic vacuoles. Fluorescence
images were taken using a Leica DC300F microscope using a DAPI filter. At least three independent experiments were performed. A representative image
(400 × original magnification) of the effects of 2a treatment is shown. (B) Western blot analysis of autophagic markers Beclin and LC3 II in MDA-MB231 cells
incubated in the presence or absence of compound 2a (15 or 20 µM) for 24 h. All data were normalized to the housekeeping control represented by γ-tubulin.
Densitometry analyses of each band, performed by ImageJ, were from three independent experiments and are shown in the center and right-hand panels (*)
p < 0.05 compared to the untreated sample.
4.1.7. Procedure
to
prepare
4,4′-{1,4,5,8,11,13a-hexamethyl-12-
set of signals, 8H, 2 × C6H4,). 13C NMR (DMSO‑d6) (δ): 15.26 (CH3);
16.75 (CH3); 23.64 (CH3); 24.96 (CH3); 35.23 (CH3); 36.58 (CH3);
44.50 (CH2); 46.55 (2xCH2); 61.24 (CH); 61.61 (CH); 66.84 (2xC);
77.37 (2xC); 111.59 (CH2); 112.89 (2xC); 122.16 (2xCH); 122.64
(2xCH); 126.67 (2xCH); 126.81 (2xCH); 140.90 (C); 141.28 (C);
142.09 (C); 143.03 (C); 145.39 (C); 146.99 (C); 147.27 (C); 147.55
(C). Anal Calcd for C34H40N8O4S2: C, 59.28; H, 5.85; N, 16.27. Found: C
58.97; H, 5.54; N, 16.52.
methylene-4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo
[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine-3,9-diyl}
dibenzenesulfonamide 2b
To a suspension of 10 (0.590 g, 2.21 mmol) in anhydrous dioxane
(12 mL), 2,5-hexanedione (0.26 mL, 2.21 mmol) and p-toluenesulfonic
acid monohydrate (20% mol/mol of 10) were added. The mixture was
refluxed for 160 h. After the first 24 h of reflux, five portions of 2,5-
hexanedione and p-toluenesulfonic acid monohydrate (50% of the
above amounts) were added at intervals of 24 h. Then, the solvent was
removed under reduced pressure and the residue treated with ethanol
(4 mL). From the solution, left at room temperature for 24 h, a solid
formed, and this was recovered by filtration and crystallized from DMF/
H2O (0.244 g).
4.1.8. Procedure to prepare N-[1-(4-aminophenyl)-3-methyl-1H-pyrazol-5-
yl]formamide 12
To a solution of N-[3-methyl-1-(4-nitrophenyl)-1H-pyrazol-5-yl]
formamide 11 (0.200 g, 0.81 mmol) in hot methanol (20 mL), 10% Pd/
C (0.020 g) as a catalyst was added. The mixture was left under hy-
drogenation in a Parr apparatus at 50 psi for 20 h. After this time, the
suspension was filtered, and the filtrate was evaporated to afford a solid
that was crystallized to give 12 (0.128 g).
4.1.7.1. 4,4′-{1,4,5,8,11,13a-hexamethyl-12-methylene-
4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo[3,4-b]
pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine-3,9-diyl}dibenzenesulfonamide
(2b). 16% yield; mp: 312 °C. IR (cm−1): 3286 (NH2); 1H NMR
(DMSO‑d6) (δ): 1.19–2.89 (multiple signals, 26H, CH3, CH2, CH); 5.06
(d, 2H, CH2); 7.43 (s, 4H, exchangeable with D2O, 2xNH2); 7.71–7.95 (a
4.1.8.1. N-[1-(4-aminophenyl)-3-methyl-1H-pyrazol-5-yl]formamide
(12). 64% yield; mp 201–202 °C (ethanol); IR (KBr) (cm−1
)
3391–3315 (multiple bands, NH2 and NH), 1684 (CO); 1H NMR
10

G. Daidone, et al.
BioorganicChemistry101(2020)103989
Fig. 8. Prolonged treatment with compound 2a induced DNA fragmentation and activation of caspase-9. (A) Analysis of DNA condensation and fragmentation was
evaluated by Hoechst 33342 staining after a 48 h treatment. Images (200x magnification) were acquired with a Leica fluorescent microscope using a DAPI filter. (B)
Western blotting analysis of pro-caspase 9. Immunoblot was quantified by densitometry analysis and normalized against γ-tubulin. Results are from three in-
dependent experiments. (*) p < 0.05 compared to the untreated sample.
(DMSO‑d6) (δ) 2.16 (3H, s, CH3); 3.58 (2H, br s, NH2, exchangeable
with D2O); 6.32 (1H, s, pyrazole H-4); 6.62–7.06 (4H, dd, C6H4); 8.14
(1H, d, CHO), 10.02 (1H, br s, NH, exchangeable with D2O). Anal Calc
for C11H12N4O: C, 61.10; H, 5.59; N, 25.91. Found: C 61.19; H, 5.65; N,
25.86.
4.1.10. Procedure to prepare benzyl {4-[3-methyl-5-(methylamino)-1H-
pyrazol-1-yl]phenyl}carbamate 14
To a magnetically stirred cold solution (T = 0–5 °C) of 13 (0.270 g,
1.34 mmol) in a 2 M sodium hydroxide water/dioxane (1:1) (v/v) so-
lution (2 mL), benzyl chloroformate (0.12 mL, 1.47 mmol) was added
dropwise. Stirring was continued overnight at room temperature. Then
the mixture was diluted with cold water and stirred until the formation
of a solid. The product was removed by filtration and purified by flash
chromatography on silica gel (0.063–0.2 mm) (150 g): diameter of
column bed 3.5 cm, ethyl acetate/cyclohexane (1:1) (1.3 L) as eluent.
Fractions 27–33 (each 20 mL) were collected and evaporated to afford
14 as a white solid, which was crystallized from ethyl acetate (0.340 g).
4.1.9. Procedure to prepare of 1-(4-aminophenyl)-N,3-dimethyl-1H-
pyrazol-5-amine 13
To a suspension of LiAlH4 (0.110 g, 11 mmol) in anhydrous tetra-
hydrofuran (6.5 mL), compound 12 (0.200 g, 3.6 mmol) in anhydrous
tetrahydrofuran (3.5 mL) was added slowly with vigorous stirring. The
mixture was refluxed for 30 min. After this time, the excess LiAlH4 was
destroyed with ethanol and water, the organic solvent was evaporated
under reduced pressure, and the aqueous phase was extracted with
ethyl acetate (5 × 5 mL). Then, the solvent was removed under reduced
pressure, and the oily residue was purified by flash chromatography
[18] on silica gel (0.063–0.2 mm) (150 g): diameter of column bed
3.5 cm, ethyl acetate (1.3 L) as eluent. Fractions 29–39 (each 20 mL)
were collected and evaporated, affording 13 (0.100 g) as a pure oil that
solidified with ethanol.
4.1.10.1. Benzyl {4-[3-methyl-5-(methylamino)-1H-pyrazol-1-yl]phenyl}
carbamate (14). 75% yield; mp 72–74 °C; MS 337 (MH+); IR (KBr)
(cm−1) 3383–3235 (multiple bands, 2xNH), 1684 (CO); 1H NMR
(DMSO‑d6) (δ) 2.08 (3H, s, CH3); 2.61 (3H, d, NCH3, J = 5.10 Hz);
5.16 (2H, s, CH2); 5.28 (1H, s, pyrazole H-4); 5.32 (1H, apparent d, NH,
exchangeable with D2O); 7.38–7.55 (9H, a set of signals, C6H4 and
C6H5); 9.90 (1H, br s, NH, exchangeable with D2O). Anal Calcd for
C
19H20N4O2: C, 67.84; H, 5.99; N, 16.66. Found: C 67.91; H, 6.05; N,
16.67.
4.1.9.1. 1-(4-aminophenyl)-N,3-dimethyl-1H-pyrazol-5-amine (13). 44%
yield; mp 106–108 °C (ethanol); IR (KBr) (cm−1) 3383–3235 (multiple
bands, NH2 and NH); 1H NMR (DMSO‑d6) (δ) 2.05 (3H, s, CH3); 2.59
(3H, d, NCH3, J = 5.10 Hz); 4.98 (1H, apparent d, NH, exchangeable
with D2O); 5.20 (2H, br s, NH2, exchangeable with D2O); 5.21 (1H, s,
pyrazole H-4); 6.57–7.06 (4H, a set of signals, C6H4). Anal Calcd for
4.1.11. Procedure to prepare benzyl {3-[9-(4-{[(benzyloxy)carbonyl]
amino}phenyl)-1,4,5,8,11,13a-hexamethyl-12-methylene-
5,6,6a,8,9,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo[3,4-b]
pyrazolo[3′,4′:2,3]azepino[4,5-f]azocin-3(4H)-yl]phenyl}carbamate 15
A solution of equimolar amounts of 14 (0.290 g, 0.86 mmol), 2,5-
hexanedione (0.102 mL, 0. 86 mmol), and p-toluenesulfonic acid
monohydrate (20% mol/mol of 14) in anhydrous dioxane was refluxed
for 120 h. After the first 24 h, four portions of 2,5-hexanedione and p-
toluenesulfonic acid monohydrate (50% of the above amounts) were
C
11H14N4: C, 65.32; H, 6.98; N, 27.70. Found: C 65.41; H, 7.05; N,
27.77.
11

G. Daidone, et al.
BioorganicChemistry101(2020)103989
added at intervals of 24 h. Then, the solvent was removed under re-
duced pressure, and the oily residue was purified by flash chromato-
graphy [18] on silica gel (0.063–0.2 mm) (200 g): diameter of column
bed 4.5 cm, ethyl acetate/cyclohexane (1:1) (1.3 L) as eluent. Fractions
13–16 (each 25 mL) were collected and evaporated to afford a crude
product that was crystallized from ethanol to give 15 as a white solid
(0.150 g).
35.14 (CH3); 36.63 (CH3); 36.94 (CH2); 39.32 (CH2); 45.50 (CH2);
47.20 (C); 62.07 (3xCH); 67.11 (C); 111.31 (C); 112.67 (C); 113.16
(CH2); 120.07 (2xCH); 120.29 (2xCH); 124.10 (2xCH); 124.51 (2xCH);
135.50 (C); 135.87 (C); 136.86 (C); 137.14 (C); 141.89 (C); 146.20 (C);
146.86 (C); 147.13 (C); 147.83 (C); 169.24 (CO); 174.91 (CO). Anal
Calcd for C38H44N8O2: C, 70.78; H, 6.88; N, 17.38. Found: C 70.85; H,
6.93; N, 17.45.
4.1.11.1. Benzyl {3-[9-(4-{[(benzyloxy)carbonyl]amino}phenyl)-1,4,5,8,
11,13a-hexamethyl-12-methylene-5,6,6a,8,9,12,13,13a-octahydro-
5,7:7,13-dimethanopyrazolo[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-f]
azocin-3(4H)-yl]phenyl}carbamate (15). 50% yield; mp 206–208 °C; IR
(KBr) (cm−1) 3324 (broad band, 2xNH), 1735 (2xCO); 1H NMR (CDCl3)
(δ) 1.29–2.85 (26H, a set of signals, 6xCH3, 3xCH2, 2xCH); 5.01, 5.04
(2H, br dd, methylene CH2); 5.22 (4H, s, 2× benzyl CH2); 6.90 (2H, br
s, 2xNH, exchangeable with D2O); 7.34–7.63 (18H, a set of signals,
2xC6H4 and 2xC6H5). Anal Calcd for C50H52N8O4: C, 72.44; H, 6.32; N,
13.52. Found: C 72.49; H, 6.35; N, 13.57.
4.1.14. Procedure
to
prepare
1,3,4,5,8,9,11,13a-octamethyl-12-
methylene-3,4,5,6,6a,8,9,12,13,13a-decahydro-5,7:7,13-
dimethanopyrazolo[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine 3
To a solution of N,1,3-trimethyl-1H-pyrazol-5-amine 17 (0.950 g,
7.60 mmol) in anhydrous dioxane (19 mL), 2,5-hexanedione (0.89 mL,
7.60 mmol) and p-toluenesulfonic acid monohydrate (20% mol/mol of
17) were added. The mixture was refluxed for 160 h. After the first
24 h, five portions of 2,5-hexanedione and p-toluenesulfonic acid
monohydrate (50% of the above amounts) were added at intervals of
24 h. Then, the solvent was removed under reduced pressure, and the
residue was chromatographed following the flash chromatography
procedure [18] on silica gel (0.040–0.063 mm) (250 g): diameter of bed
column 5.4 cm, ethyl acetate/cyclohexane (6:4 v/v) as eluent. Fractions
42–51 (each 50 mL) were collected and evaporated to afford a product
that was crystallized from ethyl acetate to give 3 (0.556 g) as a colorless
solid.
4.1.12. Procedure to prepare 4,4′-{1,4,5,8,11,13a-hexamethyl-12-
methylene-4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo
[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine-3,9-diyl}dianiline 16
To a solution of 15 (0.090 g, 0.11 mmol) in ethanol (3 mL), 6 M
NaOH (0.9 mL) was added. After 30 min of reflux, the organic solvent
was removed under reduced pressure to afford a solid product that was
recovered by filtration. The solid was purified by flash chromatography
[18] on silica gel (0.063–0.2 mm) (100 g): diameter of column bed
2.8 cm, ethyl acetate (1 L) as eluent. Fractions 30–54 (each 10 mL) were
collected and evaporated affording a crude product as a white solid that
was crystallized from ethanol to give pure 16 (0.038 g).
4.1.14.1. 1,3,4,5,8,9,11,13a-octamethyl-12-methylene-
3,4,5,6,6a,8,9,12,13,13a-decahydro-5,7:7,13-dimethanopyrazolo[3,4-b]
pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine
(3). 18%
yield;
mp:
212–214 °C; 1H NMR (CDCl3) (δ): 1.14 (s, 3H, CH3); 1.37 (s, 3H,
CH3); 1.57–2.80 (multiple signals, 17H, 3xCH3, 3xCH2, 2xCH); 2.80 (s,
3H, CH3); 3.65 (s, 6H, 2xCH3); 4.92 (br signal, 2H, CH2). 13C NMR
(CDCl3) (δ): 15.22 (CH3); 16.41 (CH3); 24.02 (CH3); 27.01 (CH3); 34.98
(CH3); 35.49 (CH3); 35.96 (CH2); 36.61 (CH3); 37.30 (CH3); 38.87
(CH2); 45.15 (CH2); 47.13 (C); 61.81 (CH); 62.08 (CH); 67.09 (C);
78.12 (C); 109.80 (C); 111.56 (CH2); 112.42 (C); 142.43 (C); 144.56
(C); 145.22 (C); 147.24 (C); 148.05 (C). Anal Calcd for C24H34N6: C,
70.90; H, 8.43; N, 20.67. Found: C 70.67; H, 8.64; N, 20.52.
4.1.12.1. 4,4′-{1,4,5,8,11,13a-hexamethyl-12-methylene-
4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo[3,4-b]
pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine-3,9-diyl}dianiline
(16). 62%
yield; mp 310–312 °C; IR (KBr) (cm−1): 3437–3346 (multiple bands,
2xNH2); 1H NMR (CDCl3) (δ) 1.15–2.68 (26H, a set of signals, 6xCH3,
3xCH2 and 2xCH); 3.43 (4H, br s, 2xNH2, exchangeable with D2O);
4.91, 4.96 (2H, br dd, methylene CH2); 6.64–7.56 (8H, a set of signals,
2xC6H4); 13C NMR (CDCl3) (δ) 15.53 (CH3); 16.74 (CH3); 24.29 (CH3);
25.65 (CH3); 34.85 (CH3); 36.45 (CH3); 45.41 (CH2); 47.05 (2XCH2);
62.01 (CH); 62.20 (CH); 66.95 (2XC); 77.82 (2XC); 111.08 (C); 112.47
(CH2); 115.02 (2xCH); 115.15 (2xCH); 125.12 (2xCH); 125.51 (2xCH);
129.89 (C); 130.56 (C); 142.45 (C); 145.01 (C); 145.55 (C); 145.75 (C);
145.93 (C); 146.26 (C); 147.66 (C). Anal Calcd for C34H40N8: C, 72.83;
H, 7.19; N, 19.98. Found: C 72.85; H, 7.18; N, 19.97.
4.2. Biology
4.2.1. Cell cultures
Triple negative breast cancer MDA-MB231 cells, obtained from
Istituto Scientifico Tumori (Genoa, Italy), were grown as monolayers in
culture medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum (FCS), 2 mM glutamine and 1% non-essential amino acids at
37 °C in a humidified atmosphere containing 5% CO2 as previously
described [19]. After plating on 96- or 6-well plates, cells were allowed
to adhere overnight in culture medium before treatment with com-
pounds. All reagents used for cell cultures were purchased from Euro-
clone (Pero, Italy).
4.1.13. Procedure to prepare N,N'-{[1,4,5,8,11,13a-hexamethyl-12-
methylene-4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo
[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine-3,9-diyl]bis(4,1-
phenylene)}diacetamide 2c
To a suspension of 16 (0.050 g, 0.09 mmol) in anhydrous di-
chloromethane (1.5 mL), acetic anhydride (0.019 mL) was added. After
30 min of reflux, the organic solvent was removed under reduced
pressure, and the crude white solid that formed was recovered by fil-
tration and washed with water. The solid was crystallized from ethanol
to give 2c (0.039 g).
4.2.2. Cell viability assay
To evaluate the effect of compound 2a on cell viability, the MTT
colorimetric assay was used as previously described [20]. MDA-MB231
cells (8 × 103/200 µL/well) were plated in 96-well plates and treated
with various concentrations of 2a for different times. Then, 20 μL MTT
(11 mg/mL) was added, and the cells were incubated at 37 °C for 2 h.
Finally, after removing the medium, 100 µL of lysis buffer (20% sodium
dodecyl sulfate in 50% N,N-dimethylformamide) was added. At the end,
the absorbance of the formazan was measured directly at 490 nm with
630 nm as a reference wavelength using an automatic ELISA plate
reader (OPSYS MR, Dynex Technologies, Chantilly, VA). Cell viability
was expressed as the percentage of the absorbance value of 2a-treated
cells compared with untreated samples used as control. Stock solutions
of compound 2a were prepared in dimethyl sulfoxide (DMSO) and di-
luted to final concentrations in DMEM. The concentrations of DMSO
4.1.13.1. N,N'-{[1,4,5,8,11,13a-hexamethyl-12-methylene-
4,5,6,6a,8,12,13,13a-octahydro-5,7:7,13-dimethanopyrazolo[3,4-b]
pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine-3,9-diyl]bis(4,1-phenylene)}
diacetamide (2c). 68% yield; mp 300–301 °C; IR (KBr) (cm−1) (CO);
(multiple bands, 2xNH); 1H NMR (CDCl3) (δ) 1.28–2.81 (32H, a set of
signals, 8xCH3, 3xCH2 and 2xCH); 5.01 (2H, br dd, methylene CH2);
7.32–7.55 (8H, a set of signals, 2xC6H4); 8.69 (1H, s, NH, exchangeable
with D2O); 8.84 (1H, s, NH exchangeable with D2O); 13C NMR (CDCl3)
(δ) 15.38 (CH3); 16.68 (CH3); 20.93 (CH3); 24.23 (2xCH3); 25.83 (CH3);
12

G. Daidone, et al.
BioorganicChemistry101(2020)103989
used as vehicle never exceeded 0.04% and did not exert toxic effects on
MDA-MD231 cells in comparison with the control. Each experiment
was performed in triplicate.
compared to control (untreated) cells. The data are expressed as
means
SD. The statistical significance threshold was fixed at
p < 0.05.
Cell morphology images were acquired using an inverted Leica
microscope equipped with a DC 300F camera (Leica Microsystems,
Wetzlar, Germany).
Declaration of Competing Interest
In order to determine changes in nuclear morphology, MDA-MB231
cells were stained with Hoechst 33342, as previously described [21]. In
particular, cells (7 × 103/well) were stained with Hoechst 33342
(2.5 µg/mL in medium) for 30 min, washed with phosphate-buffered
saline (PBS), resuspended in culture medium and treated for various
times with compound 2a. A DAPI filter was used to examine cell
morphology, and images were taken with a Leica DC 300F microscope
(Leica Microsystems, Wetzlar, Germany) and acquired with Leica Q
Fluoro Software.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by Ministero dell’Istruzione dell’Università
e della Ricerca (MIUR) of the Italian Government [grant numbers
PJ_RIC_FFABR_2017_001683 and PJ_RIC_FFABR_2017_160599]. The
authors wish to thank the Developmental Therapeutics Program of the
National Cancer Institute of the United States of America for performing
the antiproliferative screening of compounds in the 60 cell line assay.
This research was supported in part by the Developmental Therapeutics
Program in the Division of Cancer Treatment and Diagnosis of the
National Cancer Institute, which includes federal funds under Contract
No. HHSN261200800001E. The content of this publication does not
necessarily reflect the views or policies of the Department of Health and
Human Services, nor does mention of trade names, commercial pro-
ducts, or organizations imply endorsement by the U.S. Government.
Cell death was assessed by staining the cells with PI, a membrane
impermeable dye generally excluded from viable cells. Cells were har-
vested, washed with PBS and incubated for 10 min at 4 °C in a solution
of PBS containing 2 μg/mL PI. After staining, red fluorescence was
measured using the FL3 channel using a 620-nm BP filter with a Coulter
Epics XL flow cytometer (Beckman Coulter).
4.2.3. Flow cytometric analysis of cell cycle distribution
Cell cycle analysis was performed as previously described [21,22].
Cells were harvested by trypsinization and resuspended in a hypotonic
solution containing 50 µg/mL PI, 0.1% sodium citrate, 0.01% Nonidet
P-40 and 10 mg/mL RNase A. The cell cycle phase distribution was
evaluated by an Epics XL flow cytometer (Beckman Coulter) using
Expo32 software.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103989.
4.2.4. Western blot analysis
After treatment with 2a, MDA-MB231 cells were lysed as described
[23], and protein concentration was determined with the Bradford
Protein Assay (Bio-Rad Laboratories S.r.L., Segrate, Milan, Italy). 40 µg
of protein/sample were then separated by sodium dodecyl sulfate–po-
lyacrylamide gel electrophoresis, after which the proteins were trans-
ferred to nitrocellulose membranes. Next, the nitrocellulose membranes
were blocked with 1% milk-TBST (Tris-buffered saline, 0.1% Tween 20)
and incubated overnight with 1 mg/mL of primary antibodies directed
against Beclin (Santa Cruz Biotechnology, St. Cruz, CA), LC3 (Novus
Biologicals, Littleton, CO), caspase-9 (Cell Signalling, Beverly, MA) or γ-
tubulin (Sigma Aldrich, Milan, Italy). Membranes were then incubated
with horseradish peroxidase-conjugated secondary antibody (1:5000)
(Pierce, Thermo Fisher Scientific, UK), and the signals were detected
using enhanced chemiluminescence (ECL) reagents (Cyanagen, Bo-
logna, Italy). The signal obtained was visualized and photographed
with Chemi Doc XRS (BioRad, Hercules, CA). The intensity of the bands
was quantitated using Quantity One software (BioRad, Hercules, CA).
Expression of proteins was normalized to γ-tubulin and compared to the
appropriate controls. All the blots shown are representative of at least
three separate experiments.
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