Quinone-Fused Pyrazoles through 1,3-Dipolar Cycloadditions: Synthesis of Tricyclic Scaffolds and in vitro Cytotoxic Activity Evaluation on Glioblastoma Cancer Cells

Article abstract of DOI:10.1002/cmdc.201800251

A novel and straightforward synthesis of highly substituted isoquinoline-5,8-dione fused tricyclic pyrazoles is reported. The key step of the synthetic sequence is a regioselective, Ag₂CO₃ promoted, 1,3-dipolar cycloaddition of C-heteroaryl-N-aryl nitrilimines and substituted isoquinoline-5,8-diones. The broad functional group tolerability and mild reaction conditions were found to be suitable for the preparation of a small library of compounds. These scaffolds were designed to interact with multiple biological residues, and two of them, after brief synthetic elaborations, were analyzed by molecular docking studies as potential anticancer drugs. In vitro studies confirmed the potent anticancer effects, showing promising IC₅₀ values as low as 2.5 μm against three different glioblastoma cell lines. Their cytotoxic activity was finally positively correlated to their ability to inhibit PI3K/mTOR kinases, which are responsible for the regulation of diverse cellular processes in human cancer cells.

Full text of DOI:10.1002/cmdc.201800251

Accepted Article
Title: Quinone-fused pyrazoles through regio-selective 1,3-dipolar
cycloadditions: synthesis of tricyclic scaffolds and in vitro
cytotoxic activity evaluation on glioblastoma cancer cells
Authors: Mauro Comes Franchini, Giulio Bertuzzi, Mariafrancesca
Fochi, Erica Locatelli, Ilaria Monaco, Elena Strocchi, Paolo
Zani, Bianca Flavia Bonini, Pierpaolo Calandro, Mario
Chiariello, Simone Crotti, and Andrea Mazzanti
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
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800251
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800251
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10.1002/cmdc.201800251
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Quinone-fused pyrazoles through 1,3-dipolar cycloadditions:
synthesis of tricyclic scaffolds and in vitro cytotoxic activity
evaluation on glioblastoma cancer cells
Giulio Bertuzzi[a], Simone Crotti[a], Pierpaolo Calandro[b], Bianca Flavia Bonini[a], Ilaria Monaco[a], Erica
Locatelli[a], Mariafrancesca Fochi[a], Paolo Zani[a], Elena Strocchi[a], Andrea Mazzanti[a], Mario
Chiariello[b], Mauro Comes Franchini [a]*
[a]
Dipartimento di Chimica Industriale “Toso Montanari”
Università di Bologna
Viale Risorgimento 4, 40136 Bologna, Italy
mauro.comesfranchini@unibo.it:
[b]
Istituto di Fisiologia Clinica and Istituto Toscano Tumori, Core Research Laboratory,
Consiglio Nazionale delle Ricerche
Via Fiorentina 1, 53100, Siena, Italy.
Supporting information for this article is given via a link at the end of the document.
Abstract: A novel and straightforward synthesis of highly substituted
substrates represent a regiochemical challenge and undoubtedly
provide a much general platform for the synthesis of libraries of
isoquinoline-5,8-dione fused tricyclic pyrazoles is reported. The key
step of the synthetic sequence is a regioselective, Ag2CO3 promoted, compounds. Among them, isoquinolino-5,8-diones could be
1,3-dipolar cycloaddition of C-heteroaryl-N-aryl nitrilimines and
substituted isoquinoline-5,8-diones. The broad functional group
tolerability and the mild reaction conditions were suitable for the
preparation of a small library of compounds. These scaffolds were
employed in 1,3-DCs for the synthesis of different 5-membered
rings heterocycles. These represent
a
widely employed
substrate for regioselective amination reactions, [8] or Diels-Alder
cycloadditions,[9] targeting to biologically active products. On the
designed to interact with multiple biological residues and two of them, other hand, their 1,3-DC chemistry is much more limited: only
after brief synthetic elaborations, were analysed by molecular
docking studies in silico as potential anti-cancer drugs. In vitro
studies confirmed the potent anti-cancer capability, showing
interesting IC50 values down to 2.5 μM on three different
glioblastoma cell lines. Their cytotoxic activity was finally positively
correlated to their ability of inhibiting PI3K/mTOR kinases, which are
responsible for the regulation of diverse cellular processes in human
cancer cells.
one report disclosed the reaction between in situ generated
nitriloxides and 6-bromo-isoquinoline-5,8-diones, affording
isoxazole ring-fused products with good regioselectivity,
mediated by the presence of the bromide on the electrophilic
site.[10] Therefore, this class of substrates represents an
interesting building block for a 1,3-DCs aiming at the preparation
of tricyclic fused pyrazoles, both for the facility of preparation
and the interesting biological activities.
Isoquinoline-5,8-diones displayed, indeed, a wide range of
biological activities such as antibacterial, antifungal, antimalarial
and antitumor agents.[11,12] This moiety is present as an
important core in a number of cytotoxic agents, such as naturally
occurring Caulibugulones of type I (Figure 1), which exhibited
IC50 values of 0.03–1.67 µM against murine tumor cells.[13]
Moreover, Pixantrone (II), an aza-anthraquinone commercialized
under the trade name Pixuvri, is an antineoplastic drug, that has
completed phase III clinical trials and stands in the family of
antitumor antibiotics for the treatment of different type of
cancers.[14] On the other hand, a large number of commercially
available drugs containing pyrazoles can be found; among
1,3-Dipolar cycloadditions (1,3-DCs) of nitrilimines, reactive
species generated in situ upon basic treatment of hydrazonoyl
halides, with various activated π-systems represent a powerful
and general tool for the synthesis of substituted pyrazoles.[1]
Both electron-rich and electron-poor double and triple bonds
have been widely employed by us as dipolarophiles in the
regioselective formation of various pyrazoles.[2] Among these,
cyclic systems such as cycloalkenones,[3] α,β-unsaturated
lactones, thiolactones and lactams have gained much attention
for the preparation of fused structures.[4] Quinones as well would
represent a privileged platform for the synthesis of ring-fused
pyrazoles, given their wide applications in [4+2] or [3+2]
cycloaddition reactions.[5] In 1991, Argyropoulos and Terzis
reported a pioneering work in which various 1,4-benzoquinones
were reacted with some nitrilimines, proving the process to be
feasible.[6] However, the difficulties encountered in the
chemoselectivity and the low isolated yields prevented it to
represent a useful preparative procedure. To our knowledge, no
other report of 1,3-DCs between quinones and nitrilimines has
been published. On the other hand, fused quinones would
represent an easy alternative to simple 1,4-benzoquinones to
introduce molecular diversity and avoid site-selectivity issues.
Naphthoquinone-fused pyrazoles were indeed prepared by
others,
they
exhibit
antispasmodic,
anti-inflammatory,
antibacterial, antihyperglicemic, antidepressive and antitumor
activities.[1] For example, Axitinib (III), an anti-angiogenic drug
approved for renal cancer, has been recently discovered to be
effective as an inhibitor for ABL1 gatekeeper mutant drug-
resistant leukaemia patients.[15] In order to supply phase II
clinical trials, Pfizer has developed a scalable process for the
preparation of indazol-4-one IV (disclosed by Huang in 2009), an
Hsp90 inhibitor exhibiting low nanomolar antiproliferative
properties across multiple cancer cell lines.[16]
hydrazones
or
diazomethane
reaction
with
simple
naphthoquinones.[7] However, unsymmetrically substituted
1
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10.1002/cmdc.201800251
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COMMUNICATION
enaminoesters 3a-c in the abovementioned isoquinolino-5,8-
diones formation reaction (scheme 2, top and middle). Synthesis
of 1a-c[20] and 3a-c[21] was straightforward, following known
literature procedures. Thus, we prepared six new isoquinolino-
5,8-diones 2a-c and 4a-c in good yields (71-90%), proving that
such unprecedented substitutions, (phenyl, 3-pyridyl and 2-furyl)
were well tolerated in the protocol disclosed by Valderrama,
originally limited to ketones and esters as EWGs at the C-4
position, and methyl as R substituent. [19]
For the synthesis of hydrazonoyl chlorides 7 and 8, we
employed a methodology already reported by us.[2] Coupling of
picolinic acid with suitable phenylhydrazines was undertaken by
DCC-NHS activation: a milder method, more suitable for the
presence of the basic pyridine moiety.[22] Chlorination of
hydrazides 5 and 6 under Appel conditions gave the respective
hydrazonoyl chlorides
respectively, representing the first example of C-heteroaryl
7 and 8 in 80% and 62% yield
Figure 1. Biologically active structures I-IV
substitution of these substrates (Scheme 2, bottom).
All these features considered, as part of our on-going effort
directed towards the preparation of cytotoxic pyrazoles,[17] we
decided to develop
a
new synthetic protocol for the
straightforward preparation of some class of isoquinolino-5,8-
dione-fused pyrazoles, characterized by a tricyclic core (Scheme
1). A flat polycyclic structure containing the quinone moiety
fused with N-heterocycles has indeed been found responsible
for the potent antitumor activity displayed by compounds such
as mytomicin C.[12] Moreover, in order to decorate the structure
with some different biologically appealing moieties, we took the
opportunity to introduce some halogens as substituents on one
of the aryl rings, as this structure itself is frequently recurring in
approved drugs and bioactive molecules.[18] Variation of the
isoquinolino-5,8-dione
was
undertaken
with
different
(hetero)aromatic cycles and EWG groups. Finally, to explore the
first C-heteroaryl substituted nitrilimine in such 1,3-DCs and to
introduce an H-bond acceptor moiety, a 2-pyridyl ring was
chosen as the privileged substituent on the pyrazole structure.
Scheme 2. Preparation of isoquinolino-5,8-diones 2 and 4 and hydrazonoyl
chlorides 7 and 8. Reaction conditions: a) NH4OAc, MeNO2, reflux, 18h. b)
MeONH2 hydrochloride, TEA, DMF, 0 °C, 15 min; then t-BuOK, DMF, 0 °C to
RT, 1 h. c) Ag2O (3 equiv), MgSO4, DCM, RT, 18h. d) (MeO)2CO, NaH, PhMe,
reflux, 3h. e) NH4OAc, MeOH, reflux, 18h. f) DCC, NHS, THF, 0 °C to RT, 18 h.
g) CCl4, PPh3 CH3CN, 50 °C to 30 °C, 18 h. [TEA, triethylamine; DMF, N,N’-
dimethylformamide;
dicyclohexylcarbodiimide;
tetrahydrofurane.]
DCM,
dichloromethane;
DCC,
N,N’-
THF,
NHS,
N-hydroxysuccinimide;
Scheme 1. Retrosynthetic analysis towards tricyclic pyrazoles
With the dipolarophiles and the precursors of the 1,3-dipoles in
hand, we moved to the cycloaddition-oxidation reaction taking 7
and 2a as model reactive partners; the first step renders, in fact,
two regioisomeric pyrazolines that, upon oxidative treatment,
afford the corresponding regioisomeric pyrazoles. While the
second step was rather trivial and always accomplished by
treatment of the crude first-step mixture with cerium(IV)
ammonium nitrate (CAN) in a THF/H2O mixture at 0 °C, the
For the synthesis of isoquinolino-5,8-diones 2 and 4 we sought
to employ a literature procedure by Valderrama et al.,[19]
involving a cyclocondensation-oxidation reaction between 2,5-
dihydroxybenzaldehyde and electron-poor enamines, promoted
by silver oxide. In order to synthesize a small library of
substituted compounds, we decided to engage three different
(hetero)aryl substituted β-amino nitroolefins 1a-c and β-
2
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COMMUNICATION
Table 1. Scope of the 1,3-DC reaction followed by oxidation ]a]
.
Entry [
Dipolarophile
Precursor of 1,3-
dipole
Major Product
Minor Product
Yield (%)[b]
Ratio[c]
1[d]
2[d]
3[d]
4[e]
5[e]
6[e]
7[d]
8[d]
9[d]
2a
2b
2c
4a
4b
4c
2a
2b
2c
7
7
7
7
7
7
8
8
8
9a
10a
10b
10c
12a
12b
12c
14a
14b
14c
67
55
72
54
45
61[f]
58
70
61
80:20
83:17
86:14
86:14
86:14
75:25
85:15
83:17
83:17
9b
9c
11a
11b
11c
13a
13b
13c
[a] Reaction conditions: 2 (or 4) (1 mmol), 7 (or 8) (1.2 mmol, 0.2 equiv added hourly), Ag2CO3 (2.0 mmol), 1,4-dioxane (5.0 mL, 0.2 M), 50 °C or reflux, 6h; then
the mixture is filtered over a Celite® pad, evaporated in vacuo and treated with: CAN (2.5 mmol), THF/H2O (4:3, 21 mL), 0 °C, 2h. [b] Isolated yield of the major
regioisomer (minor regioisomer impurity below 10%) after column chromatography. [c] Determined on the crude mixture by 1H-NMR spectroscopy. [d] Reaction
run at 50 °C. [e] Reaction run at 100 °C. [f] Yield of the inseparable mixture of regioisomers.
cycloaddition step required some optimization (see Supporting
Information for an extensive reaction scheme and optimization
table). When the reaction was run in 1,4-dioxane at 50 °C,
Ag2CO3 was employed as the base and the total amount of
hydrazonoyl chloride 7 was added portionwise, we obtained an
optimal satisfactory yield of 67% and a good regioisomeric ratio
(80:20 in favour of 9a, Table 1, entry 1). The structure of major
regioisomer 13b was assesed by means of single crystal X-ray
analysis and extended for analogy to all compounds 9, 11 and
13 (see Supporting Information for more details).
With the optimal conditions in hand, we subjected dipolarophiles
2 and 4 to the cycloaddition-oxidation protocol with precursor 7,
obtaining fused pyrazoles 9a-c and 11a-c as major regioisomers
Scheme 3. Preparation of targets 15a and 15b. Reaction conditions: a)
(10a-c and 12a-c respective minor isomers), with overall useful
synthetic yields and moderate to good regioselectivities, proving
the disclosed protocol to be of some robustness. Moreover,
isoquinolino-5,8-diones 2a-c were also reacted with hydrazonoyl
halide 8, allowing the preparation of a library of nine novel,
tricyclic-fused, fully substituted pyrazoles (Table 1).
Finally, in order to obtain the desired targets 15a and 15b,
products 13a and 13b were hydrogenated with ammonium
formate and catalytic Pd/C in MeOH at 50 °C (Scheme 3).
HCOONH4, Pd/C, MeOH, 50 °C, 18h.
We then underwent an “in silico” study to predict the potential
affinity of the novel tricyclic fused pyrazole derivatives 15a and
15b to the catalytic active site of PI3K and mTOR kinases,
involved in the crucial pathway to many antitumor processes
(cell growth, proliferation, survival and apoptosis).[23] These
targets have been chosen on the basis of the mode of action of
3
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10.1002/cmdc.201800251
ChemMedChem
COMMUNICATION
the below described antitumor drugs, pertaining to the category
of pyrazole derivatives. Docking simulations were performed to
characterize the binding pose of our tricyclic pyrazoles derived
inhibitor candidates to the pocket site of the above indicated
PI3K and mTOR. Our tricyclic fused pyrazole derivatives were
found to have the minimum requirements for tight binding at the
pocket active site of both the PI3K and mTOR domains. The
selected target proteins for the docking evaluation of the
proposed compounds were retrieved from the Protein Data Bank
(http:/www.rcsb.org/pdb/) and were: PI3K (PDB: 4HVB) and
mTOR (PDB: 4JT6). Binding modes and binding affinities of the
evaluated compounds within the catalytic pocket site of the
selected macromolecules were calculated using the Autogrid 4.0
and Autodock 4.2 programs. [24]
We analyzed the location and orientation of the evaluated
compounds and the interactions into the binding site of the
selected PI3K and mTOR above indicated. The predicted
binding free energy (BE, that includes intermolecular energy and
torsional free energy) was used as the criterion for ranking. The
conformation with the lowest ranking docked binding energy
(BE) was considered to be the best docking result.
Figure 2. Low energy conformations of molecules 15a and 15b into the PI3K
and mTOR binding sites. Top: best docking binding poses of compound 15a
(blue) and 15b (red) are shown into the pocket binding site of PI3K (on the left)
and mTOR (on the right) and - in the bottom part - the amino acid residues of
the PI3K (on the left) and mTOR (on the right) binding sites within 5 A° from
the compound 15a (blue) and 15b (red) are displayed.
Satisfactory docking results with good affinity parameters were
observed for both compounds (15a and 15b) either for the PI3K
and mTOR; virtual constants of inhibition (Ki) at micromolar
concentration were contextually calculated (1.65 μM and 1.21
μM for 15a and 15b with PI3K; 73.27 μM and 43.55 μM for 15a
and 15b with mTOR). Both compounds were docked and nicely
fitted into the active pocket site of the examined PI3K and
mTOR kinases, as indicated from binding free energy and from
analysis of ligand binding pose inside the active binding sites.
Better docking results were observed for the examined PI3K
than with mTOR, for both compounds; the best poses into active
binding sites of compounds 15a and 15b exhibit slight better
values of binding free energies to PI3K with an estimated
binding free energy of -7.89 kcal/mol and -8.07 kcal/mol,
respectively. Both compounds showed lower, although still
satisfying results for the examined mTOR kinase (-5.64 kcal/mol
and -5.89 kcal/mol for 15a and 15b respectively).
The binding conformation to PI3K showed a superimposable
pose for both 15a and 15b, where the molecules of the tricyclic
fused pyrazoles are entirely inserted and retained inside the
active site. Differently, the binding conformations to mTOR, are
characterized by the tricyclic fused pyrazole of both molecules
entering the active site and pyridine/phenyl groups retained
along the rime region of the pocket (with superimposable
conformation).
Additional polar interactions between the pyridine or the
benzene group of 15a and 15b respectively and ASP841,
between the sulphanyl of MET953 and the pyrazole ring,
between the carboxylate of VAL882 and the difluorobenzene
moiety, stabilize the molecules inside the active site. The
examined compounds revealed a common binding mode in the
hydrophobic cleft, through a complementary apolar interaction
surface with the hydrophobic chain of ILE831.
The best poses of both molecules, obtained after docking to
PI3K and mTOR PDB structures, suggest that these are typical
of aspecific kinase inhibitors. In all the cases, the synthesized
molecules fitted and entered into the pocket binding site with
good binding affinity. These docking results on the two above
described structures encouraged us to continue the investigation
by testing the synthesized compounds as PI3K/mTOR inhibitors
in vitro.
The PI3K/AKT/mTOR pathway regulates diverse cellular
processes, including cell growth, proliferation, survival, and
metabolism [23] and, indeed, it is activated in a large percentage
of human cancers through a variety of mechanisms including
Ras mutation, loss of PTEN, activation of growth factor
receptors such as EGFR, and mutations in PIK3CA.[25]
On the basis of our results demonstrating promising molecular
docking of compounds 15a and 15b to PI3K and mTOR proteins,
we next studied the ability of the drugs of affecting viability of
glioblastoma (GBM) cells. Indeed, amplification of the EGFR
gene, mutation and deletion of the PTEN gene, and mutations of
the PIK3CA gene are among the most frequent alterations
observed in primary GBM.[26] Specifically, we choose a panel of
three cell lines, U251, DBTRG, and U87MG and demonstrated,
in all of them, that the most active compound, in terms of
cytotoxicity, was 15b, with an IC50 value of ~2.5 μM after 72h,
while the other compound (15a) resulted less active with IC50
values higher than 10 μM (Figure 3). While these differences in
the cytotoxicity of the two compounds against GBM cell lines
may be surprising, these data suggest that, although predicted
common binding modes by docking analysis, their ability of
effectively interfering with kinases activity may differ, explaining
observed biological consequences.
Only compound 15a is stabilized inside the catalytic site of the
selected PI3K by two hydrogen bonds between the amino group
of ASP (ASP964) and the amino group of the tricyclic pyrazole
and between the amino group of ALA (ALA885) and the 3F of
fluorobenzene moiety. At distances within 5 Å, a possible
different set of polar interactions are observed between the
pyridine moiety (connected to pyrazole) of both 15a and 15b,
and the polar residues THR887 and LYS890.
4
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10.1002/cmdc.201800251
ChemMedChem
COMMUNICATION
6h
2.5 µM 11.64 µM
2.93 µM
23.85 µM
A
B
120
A
C
120
100
80
60
40
20
0
6h
CTR 15a 15b
100
CTR 15a 15b
WB
WB
-P-4EBP1
15a
15b
an>-P-4EBP1
-P-4EBP1
80
an>-P-4EBP1
15a
15b
WB
60
40
WB
-4EBP1
-Erk2
an>-4EBP1
-4EBP1
-Erk2
an>-4EBP1
WB
WB
20
an>-Erk2
U251
an>-Erk2
P-4EBP1:4EBP1 1.00 0.79 0.54
DBTRG
0
P-4EBP1:4EBP1 1,00 0.90 0.59
-1
0
1
2
3
4
-1
0
1
2
3
log[], µM
log[], µM
U251 cells
DBTRG cells
2.40 µM
32.60 µM
C
120
100
80
60
40
20
0
6h
6h
B
an>-P-p70
D
an>-P-p70
CTR 15a 15b
CTR 15a 15b
WB
WB
15a
15b
-P-p70
-p70
-P-p70
-p70
WB
an>-p70
WB
WB
an>-p70
WB
-Erk2
-Erk2
U87
an>-Erk2
an>-Erk2
P-p70:p70
P-p70:p70 1.00 0.77 0.44
1,00 1,15 0.65
-1
0
1
log[], µM
2
3
4
U251 cells
DBTRG cells
Figure 3. Cytotoxicity evaluation in different GBM cell lines. IC50 evaluation,
after 72h treatment of U251, DBTRG, and U87MG cells, of compounds 15a
and 15b, at different concentrations, obtained by cell counting through a Z2
coulter counter. The results shown are averages of triplicate samples from a
typical experiment.
Figure 4. Inhibition of PI3K/mTOR signalling in U251 and DBTRG cells. Cells
were incubated 6 h with vehicle (CTR), and compounds 15a and 15b (10 µM).
Corresponding lysates were next immunoblotted with (A and C) anti-phospho-
4EBP1 and “total” 4EBP1 antibodies, and (B and D) anti-phospho-p70 and
“total” p70 antibodies. Equal loading was confirmed by immunoblot with anti-
Erk2 antibody. Below each experiment, phospho-4EBP1/4EBP1 or phospho-
p70/p70 ratios of intensitometric signals are shown. Anti-Erk2 levels are also
shown to confirm equal loading of samples.
Therefore, we next sought to investigate if the cytotoxic activity
of these compounds on GBM cells was indeed correlated to their
ability of inhibiting PI3K/mTOR kinases. Therefore, upon
treatment with 15a and 15b, we evaluated phosphorylation of
4EBP1, a well-established substrate of mTOR[27] and of p70 S6
Kinase, which acts predominantly downstream of PI3K,[28] in our
GBM cells panel. Interestingly, 15b was the most effective
compound at inhibiting PI3K/mTOR biochemical activities in both
U251 and DBTRG cells (Figure 4 and Figures S2A and S2B,
see Supporting Information), confirming the consistent higher
efficiency of this compound in cytotoxicity studies (see above).
Ultimately, the two compounds showed very limited activity on
PI3K/mTOR in U87MG cells (Figures S2C and S2D).
diverse cellular processes in human cancer cells. Further in vivo
experiments using these molecules are still underway and will
be released in due course.
Experimental Section
Synthetic procedures, reaction optimization parameters, spectral
characterizations and additional biological essays are provided in the
Supporting Information. The following preparation (and characterization)
of compound 15b through cycloaddition-oxidation followed by nitro group
reduction may be taken as a representative example.
In conclusion, products characterized by high molecular
complexity and great functional group diversity were easily
obtained. The previously described reaction sequence was
shown to be a convenient synthetic protocol to afford libraries of
substituted compounds, bearing a free amine, an isoquinolino-
In an oven dried Schlenk tube equipped with a magnetic stirring bar and
under nitrogen atmosphere, isoquinoline-5,8-dione 2b (281.2 mg, 1
mmol) and Ag2CO3 (441 mg, 1.6 mmol) were suspended in dry dioxane
(5 mL) and heated to 50 °C. Then, hydrazonoyl chloride 8 (53.5 mg, 0.2
mmol) was added and the resulting mixture was stirred for 1 h. Then,
another portion (53.5 mg, 0.2 mmol) of 8 was added hourly until 1.2
equivalents (320.6 mg, 1.2 mmol) were reached after 5 hours. The
5,8-dione-fused pyrazole,
a terminal pyridine, a halogen
substituted aryl ring and a different (hetero)aryl moiety. These
scaffolds were designed to interact with multiple biological
residues and two of them, after brief synthetic elaborations, were
analysed by molecular docking studies in silico as potential anti-
cancer drugs. Further in vitro studies confirmed the potent anti-
cancer capability, showing interesting IC50 values down to 2.5
μM on three different glioblastoma cell lines. Their cytotoxic
activity was finally positively correlated to their ability of inhibiting
PI3K/mTOR kinases, which are responsible for the regulation of
reaction was then stirred for
1 additional hour, cooled to room
temperature, filtered over a Celite® pad and washed repeatedly with
DCM. The filtrate was evaporated in vacuo and suspended in 21 mL of a
4:3 THF/H2O mixture and cooled to 0 °C. Cerium (IV) ammonium nitrate
(1.37 g, 2.5 mmol) was added in portions and the resulting slurry was
stirred at 0 °C for 2 h. Hereafter, the THF was evaporated in vacuo,
EtOAc (20 mL) was added and the phases were separated. The organic
phase was washed repeatedly with water and once with brine (10 mL),
dried over MgSO4, filtered and evaporated in vacuo. The crude product
was analyzed by means of 1H NMR spectroscopy to calculate the
regioisomeric ratio 13b/14b of the reaction, which was found to be 83:17,
and finally purified by column chromatography on silica gel (n-
hexane/EtOAc 1:1) to afford pure compound 13b in 70% (357 mg) as a
dark yellow solid, which was completely charcterized (see Supporting
Information).
In a Schlenk tube equipped with a magneting stirring bar and under
nitrogen atmosphere, compound 13b (50,8 mg, 0.1 mmol) and
5
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ammonium formate (63 mg, 1 mmol) were dissolved in degassed MeOH
(1 mL) and heated to 50 °C. Then, 10% wt. Pd/C (3.2 mg, 3 mol%) was
added and the reaction mixture was stirred at 50 °C overnight under
nitrogen. Hereafter, DCM (5 mL) was added, the resulting suspension
was filtered over a Celite® pad and washed repeatedly with DCM. The
solvent was evaporated in vacuo and the crude product was purified by
column chromatography on silica gel (EtOAc/MeOH 10:1) to afford 43 mg
of pure product 15b as an intense cherry-red powder in 90% yield. 1H
NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 2.3, 0.9 Hz, 1H, pyridine-H),
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Valença, T. V. Baiju, F. G. Brito, M. H. Araujo, C. Pessoa, B. C.
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Almeida, S. T. Sivanandan, C. A. de Simone, L. M. Brito, B. C.
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8.83 (ddd,
J = 4.8, 1.8, 0.9 Hz, 1H, pyridine-H), 8.77 (s, 1H,
isoquinoline-H), 8.76 – 8.75 (m, 1H, pyridine-H), 8.22 (dt, J = 7.9, 1.1
Hz, 1H, pyridine-H), 8.04 (ddd, J = 7.9, 2.3, 1.7 Hz, 1H, pyridine-H),
7.90 (td, J = 7.8, 1.8 Hz, 1H, pyridine-H), 7.68 (tdd, J = 8.2, 5.7, 0.9 Hz,
1H, ArF-H), 7.50 (ddd, J = 7.8, 4.9, 0.9 Hz, 1H, pyridine-H), 7.43 (ddd, J
= 7.6, 4.9, 1.2 Hz, 1H, pyridine-H), 7.12 – 6.99 (m, 2H, ArF-H)
overlapped with 7.00 (bs, 2H, NH2) ppm; 13C NMR (101 MHz, cdcl3) δ
182.8, 174.8, 163.5 (dd, J = 253.6, 11.2 Hz), 157.4 (dd, J = 256.1, 12.8
Hz), 153.0, 151.8, 150.8, 149.9, 149.4, 149.3, 142.6, 139.3, 136.5, 136.4,
136.3, 132.7, 129.3 (d, J = 10.2 Hz), 125.2, 125.1, 124.3, 124.0, 123.5
(dd J = 12.6, 4.1 Hz), 120.9, 116.9, 111.9 (dd, J = 22.9, 3.8 Hz), 105.0
(dd, J = 26.7, 23.1 Hz) ppm; 19F NMR (282 MHz, CDCl3) δ -105.33 (qd, J
= 8.3, 5.6 Hz, 1F), -116.97 (q, J = 8.8 Hz, 1F) ppm; ESIMS m/z = 481 [M
+ H+], 503 [M + Na+].
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Acknowledgements
University of Bologna is gratefully acknowledged.
Keywords: Pyrazole, Quinone, 1,3-Dipolar Cycloaddition,
Anticancer Drugs, Molecular Docking
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