Combining X-ray crystallography and molecular modeling toward the optimization of pyrazolo[3,4-d ]pyrimidines as potent c-Src inhibitors active in vivo against neuroblastoma

Article abstract of DOI:10.1021/jm5013159

c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimi

Full text of DOI:10.1021/jm5013159

Article
pubs.acs.org/jmc
Combining X‑ray Crystallography and Molecular Modeling toward
the Optimization of Pyrazolo[3,4‑d]pyrimidines as Potent c‑Src
Inhibitors Active in Vivo against Neuroblastoma
Cristina Tintori,† Anna Lucia Fallacara,†,‡ Marco Radi,†,◆ Claudio Zamperini,† Elena Dreassi,†
Emmanuele Crespan,§ Giovanni Maga,§ Silvia Schenone,*,∥ Francesca Musumeci,∥ Chiara Brullo,∥
Andre Richters,⊥ Francesca Gasparrini,# Adriano Angelucci,∇ Claudio Festuccia,∇
́
Simona Delle Monache,∇ Daniel Rauh,⊥ and Maurizio Botta†,○
†Dipartimento di Biotecnologie, Chimica e Farmacia, Universita
‡Dipartimento di Chimica e Tecnologie Farmaceutiche, Sapienza Universita
§Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
∥Dipartimento di Farmacia, Universita
degli Studi di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
̀
degli Studi di Siena Via Aldo Moro 2, 53100 Siena, Italy
di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy
̀
̀
⊥Department of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund,
Germany
#Dipartimento di Medicina Molecolare, Sapienza Universita
̀
di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy
∇Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Universita
̀
degli Studi dell’Aquila, Via Vetoio, 67100 Coppito,
L’Aquila, Italy
○Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology,
Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United States
S
* Supporting Information
ABSTRACT: c-Src is a tyrosine kinase belonging to the Src-family
kinases. It is overexpressed and/or hyperactivated in a variety of
cancer cells, thus its inhibition has been predicted to have therapeutic
effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was
reported as a dual c-Src/Abl inhibitor. Herein we describe a
multidisciplinary drug discovery approach for the optimization of the
lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src
in complex with 3, Monte Carlo free energy perturbation calculations
were applied to guide the design of c-Src inhibitors with improved
activities. As a result, the introduction of a meta hydroxyl group on
the C4 anilino ring was computed to be particularly favorable. The
potency of the synthesized inhibitors was increased with respect to
the starting lead 3. The best identified compounds were also found
active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft
model using SH-SY5Y cells.
of NB consist of a multimodality approach, which includes
surgery, chemotherapy, radiotherapy, and biotherapy. Current
chemotherapeutic treatment for high-risk neuroblastoma uses
dose-intensive cycles of cisplatin and etoposide, alternating with
vincristine, doxorubicin, and cyclophosphamide. Furthermore,
isotretinoin could be used during the first remission. Despite
improvements in the overall cure rate of these patients, the
treatment strategies are still far from satisfaction especially
INTRODUCTION
■
Neuroblastoma (NB) is a rare cancer of the sympathetic
nervous system. It accounts for about 9% of malignancies in
patients younger than 15 years and for around 15% of all
pediatric oncology deaths.1 It is the most common extracranial
solid tumor in childhood and is a major cause of death from
neoplasia in infancy.2 The disease is remarkable for its broad
spectrum of clinical behavior. Although during the past few
decades a substantial improvement in the treatment of certain,
well-defined, subsets of patients has been observed, the
outcome of the disease for children with a high-risk clinical
phenotype has improved modestly, with long-term survival less
than 40%.3,4 The therapeutic options for the clinical managing
Special Issue: New Frontiers in Kinases
Received: August 28, 2014
© XXXX American Chemical Society
A
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because of the severe side effects.5,6 Accordingly, novel
therapeutic approaches are needed to improve the prognosis
of patients with NB. Since deregulation of tyrosine kinases
(TKs) has been associated with cancer, small molecule TK
inhibitors (TKIs) represent one of the largest drug family
currently targeted by pharmaceutical companies and academia
for the treatment of malignancies. Remarkably, TKIs, acting on
specific molecular targets, could be related with reduced toxic
side effects during antitumor treatments. For this reason, many
TKIs have been tested for their in vitro antiproliferative activity
and in vivo anticancer activity, and some of them have been
approved in clinical trials or are currently utilized in cancer
therapy.7,8 A subclass of nonreceptor TKs as target in the
treatment of human cancers is the Src-family tyrosine kinases
(SFKs). Among these, c-Src was found to stimulate cell
proliferation, migration, and invasion as well as angiogenesis.9
Hyperactivation of c-Src causes aberrant cell activity that
contributes to cancer development. High expression levels of c-
Src have been detected in several cancers and are generally
correlated to a poor prognosis with respect to overall survival.
Moreover, recent studies suggest that c-Src could be associated
with the development of acquired drug resistance.10 Accord-
ingly, many small molecule c-Src inhibitors have been
developed in the last 20 years belonging to different chemical
families such as purines,11,12 anilinoquinazolines,13 quinoline-
carbonitriles,14 benzotriazines,15 and thiazole-carboxamides.16
Remarkably, Src inhibitors showed antitumor effects in multiple
solid tumor types.17 Furthermore, it has been recently reported
that, among TKs, c-Src also plays a key role in the
differentiation, cell adhesion, and survival of NB cells, due to
its hyperactivation rather than overexpression.18 c-Src was also
hypothesized to have an oncogenic role in the progression of
aggressive NB forms.19 Addressing this kinase with small
molecule inhibitors and thereby inhibiting its catalytic activity
has recently been reported as an effective approach to the
treatment of NB. As a proof of concept, the well-known c-Src
inhibitor PP2 has recently been proved to inhibit cell survival/
proliferation and to reduce aggregation in NB cell lines20 while
the dual Src/Abl inhibitor Dasatinib has been proved to be
effective in reducing NB growth both in vitro and in vivo
(Figure 1).21 In recent years, our group conducted extensive
studies on a series of novel c-Src/Abl inhibitors endowed with a
pyrazolo[3,4-d]pyrimidine scaffold.22 Several members of this
family were found to induce apoptosis and to reduce cell
proliferation in different solid tumor cell lines (A431, 8701-BC,
SaOS-2, and PC3). A selected member of this family
(compound 1, Figure 1), characterized by a C6 methylthio
group on the pyrazolo[3,4-d]pyrimidine scaffold, displayed a
promising antiproliferative activity in SH-SY5Y cell cultures of
human neuroblastoma.23 Compound 1 has also been tested on
a panel of TKs showing an appreciable selectivity for Src.24
To get further insight into the potentiality of such
pyrazolo[3,4-d]pyrimidines for the treatment of NB, a small
collection of closely related analogues characterized by the
presence of a C6 methylthio group was synthesized to explore
the role of different functional groups in N1 and C4 for the
biological activity. Among the synthesized analogues, com-
pound 2 (Figure 1) was characterized by a potent inhibitory
activity against c-Src (Ki = 90 nM) and a considerable
antiproliferative effect on SH-SY5Y cells (IC50 = 80 nM).
However, despite its remarkable activities, this compound
suffers from a low water solubility (0.12 μg/mL) which
precludes oral administration.25 Accordingly, a series of more
Figure 1. Molecular structure of PP2, Dasatinib, and compounds 1, 2,
and 3.
soluble pyrazolo[3,4-d]pyrimidine derivatives has been ration-
ally designed and synthesized by our research group through
the introduction of polar groups in the solvent-exposed C6
position. This study led to the identification of the C4-anilino
derivative 3 (Figure 1), which showed a beneficial profile in
term of both biological activity and ADME properties, being
characterized by a high metabolic stability (95%), a good water
solubility (1.7 μg/mL), an efficient membrane permeability (10
× 10−6 cm/s), and a potent inhibitory activity against isolated c-
Src (Ki = 0.21 μM).19 Herein, taking into account these
preliminary data, we focus our effort on compound 3 with the
aim of developing second-generation inhibitors endowed with
improved affinity values toward c-Src and improved ADME
properties to be tested against neuroblastoma in vivo. To this
aim, a multidisciplinary approach combining X-ray crystallog-
raphy, structure-based drug design, synthesis, in vitro ADME
profiling, and in vitro/in vivo biological evaluation was applied.
Starting from the crystallographic complex of 3 and c-Src, an
efficient optimization of the pyrazolo[3,4-d]pyrimidine sub-
stituents has been guided by free energy perturbation (FEP)
calculations to direct the synthesis of c-Src inhibitors 5−29
(Table 1), many of which are endowed with nanomolar
potencies. A subset of compounds also showed a strong
antiproliferative activity against SHSY-5Y neuroblastoma cells
as well as optimal ADME characteristics. Accordingly, further
studies were conducted on compound 29, which is one of the
most promising derivatives, in order to test its efficacy against
neuroblastoma in vivo after oral administration in mice.
RESULTS AND DISCUSSION
■
X-ray Structure and Computational Studies. To gain a
deeper structural understanding of C6 substituted derivatives
binding mode, we determined the crystal structure of a complex
of the kinase domain of c-Src (aa 256−533) and the hit
compound 3 (see Experimental Section for detailed informa-
B
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Table 1. Enzymatic Activity, Cellular Activity, and ADME Properties of Compounds 3−29
a
b
Values are the mean of at least two experiments. Expressed as percentage of unmodified parent drug. *Compounds previously published,26,33,34
which have been retested according with the conditions described in Experimental Section. Empty cell means not determined.
tion and statistics). Diffraction data was collected to 2.1 Å
resolution, and subsequent data processing and refinement
exhibited two protein molecules within the crystallographic cell
unit which in this work will be referred to as chain A and chain
B. Comparative analysis of the empirically determined protein−
ligand structure and previous docking studies illustrated
coincident binding modes of 3 with respect to c-Src (Figure
2A).25 The C4 anilino substituent and the N1 side chain are
located within the hydrophobic regions I and II, respectively.
Furthermore, the X-ray structure confirmed the presence of two
predicted hydrogen bonds, involving the C4 amino group
which interacts with Thr338 side chain and the N2 of the
pyrazolopyrimidine scaffold taking contacts with the backbone
of Met341. Remarkably, the same binding orientation was
observed for compound 3 within the ATP binding pocket of
each chain (Figure 2B). However, despite many residues of the
activation loop were poorly defined (from 413 to 424 in chain
A and from 411 to 424 in chain B) significant differences
between the two chains were observed in the 3D rearrangement
of such flexible loop (aa 402−423) as well as in the position of
C
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Figure 2. (A) Inhibitor 3 in complex with wild-type c-Src (PDB code: 4O2P). The experimental electron density of 3 at 2.1 Å resolution is displayed
(2Fo − Fc map contoured at 1σ). The kinase domain is in the active DFG-in conformation and hydrogen-bond interactions of the inhibitor with
Thr338 (gatekeeper) and the backbone amide of Met341 are illustrated as red dotted lines. Hinge region (orange), helix C (turquoise), DFG-motif
(pink) and inhibitor 3 (yellow sticks). (B) Experimental electron density of chain A and B in complex with 3.
Figure 3. (A) Chains A (magenta) and B (aquamarine) of the crystal structure aligned each other. Differences between the two conformations were
observed at the level of activation loop, αC-helix and P-loop. (B) Chains A (magenta) and B (aquamarine) used for the MC/FEP calculations.
Missing residues in crystal structure were modeled.
D
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Table 2. MC/FEP Results for the Change in Free Energy of Binding upon Introduction of Chlorine, Bromine, Fluorine, and
Hydroxyl Substituents at the C4 Anilino Ring within Chain B
OH to H ΔΔGb
σ
Cl to H ΔΔGb
σ
Br to H ΔΔGb
σ
F to H ΔΔGb
σ
C2
C3
C4
C5
C6
5.11
4.89
0.10
0.11
0.13
0.12
0.09
4.8
0.11
0.09
0.09
0.21
0.07
1.28
−6.67
−9.33
−8.71
−5.43
0.11
0.11
0.10
0.27
0.11
1.96
0.25
0.05
0.03
0.05
0.08
0.05
−5.37
−6.73
−8.36
−1.05
1.49
−3
−3.66
0.71
−6.68
−5.08
a
ΔΔGb is the computed change in free energy of binding (kcal/mol) for introducing the substituents; σ is the computed uncertainty.
Table 3. MC/FEP Results for the Change in Free Energy of Binding upon Introduction of Chlorine, Bromine, Fluorine, and
Hydroxyl Substituents at the C4 Anilino Ring within Chain A
OH to H ΔΔGb
σ
Cl to H ΔΔGb
σ
Br to H ΔΔGb
σ
F to H ΔΔGb
σ
C2
C3
C4
C5
C6
−1.96
−9.41
−11.23
−7.51
0.06
0.08
0.10
0.08
0.17
0.13
−2.47
−2.70
−6.77
−12.14
−6.83
0.13
0.16
0.13
0.19
0.19
−3.47
−4.58
−12.79
−10.77
−11.03
0.14
0.18
0.20
0.15
0.16
0.49
−3.78
−1.55
−2.89
−6.48
0.05
0.06
0.06
0.10
0.07
a
ΔΔGb is the computed change in free energy of binding (kcal/mol) for introducing the substituents; σ is the computed uncertainty.
the αC-helix (aa 303−318) and in the glycine-rich loop
conformation (aa 273−281) (Figure 3A). In particular, in chain
A, the Glu310 side chain projects away from the ATP binding
site adopting a conformation similar to the closed and inhibited
one of c-Src phosphorylated on Tyr527 (PDB code: 2SRC).26
On the contrary, in chain B, Glu310 displays its side chain
turned toward the active site, forming a salt bridge with Lys295.
Moreover, in chain A, the solved amino acids of activation loop
(Phe405−Asp413) are arranged in a three-turn α helix in a
similar although not identical way as in 2SRC27 phosphorylated
c-Src. Vice versa, the determined activation loop of chain B
recalls the one solved in the 1Y5728 crystal structure. Another
significant difference between chains A and B resides in the
orientation of the DFG motif: in chain A, Glu404 projects its
side chain deeply into the ATP binding site, thereby reducing
the size of the hydrophobic pocket I which harbors the C4
substituent. Structural plasticity of c-Src in the presence of
small molecule inhibitors was recently described.29 To take into
account the conformational differences, both chains were used
in all the subsequent computational studies. A molecular
modeling protocol was first applied to fill the missing residues
(see Experimental Section for details), and the two refined
chains were aligned to each other (Figure 3B). Starting from
these completed structures, the optimization of 3 was pursued
using a computationally driven approach, primarily guided by
results of Monte Carlo free-energy perturbation (MC/FEP)
calculations.30 Notably, although the racemic mixture of 3 was
used for the preparation of the X-ray crystal structure, solely the
R-enantiomer was found to be able to bind within the kinase
active site in both the chains, pushing our studies toward
further investigation on the chiral center. No differences were
observed in the activities of the two enantiomers against c-Src
(see in vitro biological activity paragraph below, Table 1). Next,
we focused our attention on the C4 anilino ring with the aim of
optimizing the activity of 3 by increasing the affinity for the c-
Src kinase. MC/FEP halogen (chlorine, bromine, and fluorine)
and hydroxyl scans were performed to identify the most
promising sites and groups for substitutions of C4 anilino
hydrogens. In the present calculations, ortho positions 2,6 and
meta positions 3,5 are not equivalent as they do not
interconvert during the MC runs requiring separate simulations
for each conformer. According to the ring numbering in Table
2, replacement of hydrogen by OH was predicted to be
favorable (ΔΔGb represents the predicted change in free
energy of binding for the OH to H perturbation, thus a positive
value indicates that OH is preferred over H) by 5.11, 4.89, 1.49
kcal/mol at C2, C3, and C4, respectively, and unfavorable at C5
and C6 (ΔΔGb of −6.68 and −5.08 kcal/mol, respectively)
E
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Table 4. Halogen Bond Scanning for Chlorine and Bromine Atoms in Both Chain A and Chain B
chain A
Clσ to Cl (ΔΔGb)
chain B
Clσ to Cl (ΔΔGb)
Brσ to Br (ΔΔGb)
σ
σ
Brσ to Br (ΔΔGb)
σ
σ
C3
C5
−1.66
−0.89
0.10
0.06
−3.42
−0.79
0.06
0.05
−1.67
0.35
0.03
0.03
−0.89
1.14
0.03
0.05
a
ΔΔGb is the computed change in free energy of binding (kcal/mol) for introducing the substituents; σ is the computed uncertainty.
when initial complexes were built using chain B. Positive ΔΔGb
values were also found with the introduction of chlorine,
bromine, or fluorine at C2 (4.8, 1.28, and 1.96 kcal/mol,
respectively). On the contrary, in chain A, the entity of these
substituents resulted to be unfavorable with negative ΔΔGb
(Table 3). Taking into account the MC/FEP results, a focused
library of pyrazolo[3,4-d]pyrimidine derivatives bearing a m-
OH substituent at the C4 anilino ring was synthesized in order
to increase both water solubility and c-Src binding affinities of
compounds under study. Furthermore, analogues substituted
with bromine, chlorine, and fluorine in meta position were also
synthesized and tested in enzymatic assays to enlarge the
structure−activity relationships (Table 1) despite the pre-
diction of unfavorable outcomes. Concerning these last
substitutions, the possibility of halogen bonding between our
inhibitors and the ATP binding site was also investigated by
halogen bond scanning on both chain A and B considering m-
Br and m-Cl substituents. To this aim, the enhanced force field
OPLS/CM1Ax was employed in which the halogen represen-
tation has been modified to incorporate halogen bonding with
the addition of the partial positive charge which represents the
σ hole.31 FEP results then predicted the relative free energies of
binding from the modified halogen atom type to the classic one
(namely Brσ to Br and Clσ to Cl, Table 4). A marginal effect of
halogen bond interaction was found for chlorine and bromine
substituents at C5 position during chain B simulations (1.14
and 0.35 kcal/mol, respectively), while negative results were
obtained in case of using chain A. The calculated positive
contribution of halogen bonding was due to the interaction of
Cl or Br with the carbonyl backbone of Ile336, working as
Lewis base. However, this contribution has only limited effect
on the total free energy of binding calculated for the
introduction of bromine or chlorine at the meta position of
C4 anilino group, which still remains generally negative. In
summary, analysis of the MC/FEP results clearly highlighted
that c-Src binding affinity may be enhanced by introducing a
hydroxyl group at position 3 of C4 anilino ring, which allows
for the stabilization of the complex between the chain B
conformation of c-Src and the pyrazolo[3,4-d]pyrimidine
derivatives. The hydrogen-bond interaction between the 3-
OH of the ligand and the Glu310 side chain, usually involved in
the formation of a salt bridge with Lys295, undoubtedly gives
an important contribution to the binding affinity. This
interaction was not possible in chain A because of the different
orientation of the Glu310 side chain which points out from the
active site (Figure 4). On the other hand, the introduction of an
hydroxyl group or halogens at position 2 were also predicted as
favorable and will thus be subjected to our future studies.
Figure 4. Hydrogen-bond between the 3-OH of the ligand and the
Glu310 of chain B (aquamarine) is highlighted with red dashed line.
The orientation of Glu310 side-chain in chain A (magenta) is also
visualized.
Chemistry. The synthesis of compounds 5−18 is reported
in Scheme 1. Alkylation of the thiocarbonyl group of derivatives
30a−b25 with iodomethane, iodoethane, or 4-(2-chloroethyl)-
morpholine afforded the 6-alkylthio derivatives 31a−d, which
were in turn treated with the Vilsmeier complex (POCl3/DMF,
1:1) in CH2Cl2 at reflux for 6−8 h to obtain compounds 32a−d
bearing a chlorine atom in C4. Finally, reaction of 32a−d with
the suitable anilines in absolute ethanol at reflux for 3−5 h gave
desired compounds 5−18 in good yields.
The synthesis of compounds 19−29 was performed starting
from intermediates 33a−e already reported by us (Scheme
2).22,26,32 The suitable 4-chloro derivatives 33a−e were reacted
with the proper aniline in absolute ethanol at reflux for 3−5 h
to give desired compounds 19−29 with good yields.
In Vitro Biological Activity. All synthesized compounds,
including reference compound 3, were initially tested in a cell-
free assay (see Experimental Section) to evaluate their affinity
toward isolated c-Src (Table 1). Compounds 4, 5, 8, and 27
already published by us26,33,34 have been also inserted in Table
1 for comparison purpose. First, to characterize the biological
profile of each enantiomer, the racemic mixture of 3 was
resolved by semipreparative HPLC (see Supporting Informa-
tion, Figure S1−S2), thus obtaining each single optically pure
entity (3-R and 3-S) with high enantiomeric excess (>98%).
Compounds 3-R and 3-S were then submitted to biological
F
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a
Scheme 1. Preparation of Derivatives 5−18
a
Reagents and conditions: (i) Method A, CH3I, anhyd THF, reflux, 12 h (for 31a); method B, 4-(2-chloroethyl)morpholine, NaOH, EtOH, anhyd
DMF, reflux, 6 h (for 31b and 31d); method C, C2H5I, K2CO3, anhyd DMF, rt, 20 h (for 31c). (ii) POCl3/DMF, CH2Cl2, reflux, 6−8 h. (iii)
R2NH2, EtOH, reflux, 3−5 h.
a
Scheme 2. Preparation of Derivatives 19−29
a
Reagents and conditions: (i) R2NH2, EtOH, reflux, 3−5 h.
evaluation in a cell-free assay. Comparable Ki values were found
for the two enantiomers (Ki values of 0.20 and 0.23 μM for 3-R
and 3-S, respectively). Accordingly, the remaining synthesized
compounds were tested as racemic mixture. Still focusing on
the chiral center, the chlorine substitution determined an
improvement of the activity against c-Src (compare 4 with 9
and 17). On the other hand, as it can be appreciated from
Table 1, the rationally designed derivatives 10, 18, 19, 24, and
28 showed potent in vitro inhibitory effect against c-Src with Ki
values in the low nanomolar range (40, 70, 30, 10, and 7 nM,
respectively). These potencies were most likely evoked due to
the contribution of the hydroxyl group in meta position of the
anilino ring, as hypothesized by our molecular modeling
calculations and further confirmed by the observed structure−
activity relationships. With the simple addition of a m-OH
substituent on the C4 anilino ring, potent agents were
identified with 2−30-fold increased activities (compare 8 with
5, 18 with 15, 24 with 21, and 28 with 25 in Table 1). On the
contrary, no clear trends were observed with the introduction
of fluorine, chlorine, or bromine at the same position. However,
compounds with remarkable activities were identified also in
these series such as 13, 26, and 29, showing Ki values of around
100 nM. Biological results on halogen derivatives were in
contrast to the expectation from the computed ΔΔG values. A
feasible explanation for this discrepancy may be due to the fact
that ligand-induced conformational changes are much more
pronounced than those explored by Monte Carlo because of
the lack of sampling of the protein backbone whose atoms are
frozen during the simulations. The most active c-Src inhibitors
10, 18, 19, 24, and 28 were also tested against Abl. These
compounds maintained the dual Src/Abl inhibitory profile of
lead structures 3 and 4 but showed Ki values of 1 order of
magnitude higher than for Src, possessing a significant
selectivity for c-Src over Abl. Selected c-Src inhibitors were
then evaluated for their ability to inhibit the proliferation of
neuroblastoma SH-SY5Y cells by spheroid formation assay.
Cells were treated for 72 h with increasing concentrations of
the inhibitors (0.01−50 μM), and IC50 values were calculated
considering the mean area of spheroids respect to control
(Table 1). The strongest effect on SH-SY5Y was obtained by
19 and 29 that showed IC50 values of 0.12 and 0.34 μM,
respectively. The growth rate of spheroids in the presence of 29
and representative images are shown in Supporting Information
(Figure S3).
It is noteworthy, from the structure−activity relationships
(SAR) analysis, two moieties reported in literature as
toxicophoric groups, the phenol and the alkyl halide, emerged
as crucial for the activity. To evaluate the nonspecific cytotoxic
effect of 19 and 29, we treated in vitro normal embryonic
fibroblasts Wi38 with increasing doses of the drugs. 19 and 29
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affected Wi38 cell viability starting from concentration greater
than 100 μM.
Biological effect of 29 was also evaluated through analysis of
cell cycle (Figure 5). SH-SY5Y cells were treated with
drug candidate selection. Accordingly, in vitro ADME studies
were conducted on the most potent c-Src inhibitors reported
herein in order to early assess their absorption/stability. In
particular, aqueous solubility, parallel artificial membrane
permeability (PAMPA), and human liver microsomes (HLM)
stability were evaluated for the most active c-Src inhibitors
(Table 1). Overall, optimal ADME properties were measured
for the entity of studied compounds. Indeed, the metabolic
stability was found to be higher than 90% and membrane
permeability values ranged from 1.72 to 9.33 × 10−6 cm/s,
highlighting a sufficient ability to cross the cell membranes.
Furthermore, water solubility of some compounds was
increased by about 2- to greater than 57-fold compared to
that of reference 3, with the more soluble compound 10
showing a solubility value of 97 μg/mL.
In Vivo Studies. Among the most promising c-Src
inhibitors, compound 29 was selected for the in vivo studies
because it showed an appropriate balance of different ADME
properties, remarkable activity in the cell-free assay, and
promising submicromolar potency against SH-SY5Y neuro-
blastoma cells. The anticancer activity of 29 was tested in vivo
using a xenograft mouse model. Mice inoculated with SH-SY5Y
neuroblastoma cells were treated daily with 50 mg/kg 29
starting from the appearance of a visible tumor mass, and the
tumor volume was evaluated at regular intervals. 29 caused a
significant reduction in tumor volume after 60 days of oral
treatment with a reduction of more than 50% in mean tumor
volume compared to placebo treated mice (Figure 6A).
Remarkably, mice did not show signs of distress or weight
loss. In vivo Dasatinib treatment (50 mg/kg) determined a very
similar inhibitory trend, but the appearance of palpable tumor
masses was earlier in Dasatinib group with respect to mice
treated with 29. It is notable that the tumor associated
angiogenesis at the end point was significantly compromised in
mice treated with 29 (data not shown). Thus, a three-
dimensional in vitro sprouting assay was performed to analyze
the effect of 29 on angiogenic response of endothelial cells.
Spheroids from endothelial HBMECs were seeded on Matrigel.
Twenty-four hours after the beginning of the experiment, we
observed a significant reduction of angiogenesis as demon-
strated by the reduction of the number of sprouts derived from
spheroid treated with the compound, at 0.5 and 1 μM
concentrations, compared with untreated cells (Figure 6B).
Figure 5. Analysis of the cell cycle distribution of SH-SY5Y cells after
treatment with increasing concentrations of 29 and in comparison with
0.1 μM Dasatinib. SH-SY5Y cells status was investigated by
cytofluorimetry after propidium iodide staining, and results were
expressed as percentage of cells in each phase of cell cycle respect to
total viable cells. Apoptosis was evaluated by calculating the number of
hypodiploid cells and was expressed as percentage of apoptotic cells
respect to total cells (viable and dead cells). Results are the mean of
three different experiments. *p < 0.01 respect the value of the
untreated cells.
increasing concentration of 29 (0.1−10 μM), and the
percentage of cells in each phase of cell cycle was evaluated
by cytofluorimetric analysis of DNA content. 29 determined a
significant and dose-dependent accumulation of cells in the G1
phase of cell cycle starting from 0.1 μM. In parallel we observed
a progressive accumulation of hypodiploid cells, indicating the
presence of apoptotic cells. The comparison with the reference
compound Dasatinib at 0.1 μM demonstrated that the
biological effect by 29 was not significantly different with
respect to cytotoxic effect by Dasatinib. The treatment with 10
μM 29 induced the apoptosis in about 50% of treated cells.
ADME Studies. It is well-known that kinase inhibitors are
generally affected by solubility issues because of their lipophilic
nature. Therefore, the early evaluation of ADME properties in
this field represents, more than ever, a key step to guide the
Figure 6. Evaluation of antitumoral effect of 29 and of reference compound Dasatinib. (A) Inhibition of tumor growth by 50 mg/kg 29 or 50 mg/kg
Dasatinib treatment in a rodent model of neuroblastoma. Tumor xenografts were monitored measuring the diameters of tumor mass. (B)
Antiangiogenic effect of 29 evaluated by sprouting assay with endothelial cells. Histogram shows the mean number of sprouts per spheroid for each
experimental condition (CTR = untreated cells). Representative images are shown on the right. *p < 0.01 according Student-t test.
H
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refinement, and Ramachandran statistics are provided in Supporting
Information (Table S1).
CONCLUSIONS
■
In summary, for the first time an X-ray crystal structure of c-Src
in complex with one of our patented prazolo[3,4-d]pyrimidines
was obtained in good resolution. The solved structure
confirmed the binding mode previously predicted by us for
C6-substituted derivatives within the ATP binding site of c-Src.
Starting from this crystal structure, a computationally driven
hit-to-lead optimization primarily guided by results of free-
energy perturbations was successfully conducted, leading to the
identification of novel derivatives with improved potencies
against c-Src. A hydroxyl group at the meta position of the C4
aniline ring was identified as the key structural feature for the
inhibitory activity toward c-Src. The m-OH group was
predicted to be efficient in establishing hydrogen-bond
interaction with the conserved residue Glu310. This polar
interaction was already proposed as crucial for the development
of potent c-Src inhibitors.35 In this regard, our results are also in
line with, and reinforce, this finding. It is noteworthy that the
introduction of the hydroxyl group further led to a beneficial
effect in terms of water solubility which was increased by 10 or
more fold with respect to the starting hit. Furthermore, on the
basis of the knowledge that the tyrosine kinase c-Src is
overexpressed or hyperactivated in many solid cancers,
including NB, the antitumor activity of the optimized
pyrazolo[3,4-d]pyrimidines reported herein was assessed by
measuring their effect on SH-SY5Y NB cell proliferation. The
most promising agents inhibited cell viability with IC50 values in
the submicromolar range. Next, to test the efficacy in vivo, SH-
SY5Y cells were subcutaneously injected in nude mice and drug
treatment conducted until day 60. Tumor growth was
significantly inhibited by compound 29 at the dose of 50
mg/kg. Subsequent observations on excised tumor masses and
in vitro assays suggest that c-Src inhibitor was active on both
cancer cells and tumor-associated endothelial cells inhibiting
their migratory capacity and angiogenesis. Overall, these results
illustrated how the combination of structural analysis, computa-
tional studies, organic chemistry, and biological experiments in
vitro and in vivo can be effectively utilized to develop
anticancer agents active against neuroblastoma.
Computer Modeling. Loops Modeling Protocol. The FASTA
sequence of c-Src was used as query, the coordinates of the two chains
of our crystal structure (c-Src in complex with 3) were in turn
employed as templates and the missing residues were built by using
the program Prime.46 For each chain, the serial loop sampling
approach was applied by choosing “Extended” as level of accuracy
(recommended for loop length between 6 and 11 residues) and the
lowest energy conformation was saved for the next analysis. Similarly,
Prime was used to fill the A-loop of the chain B by the building of the
Cys277 missing residue and to construct the amino acids 300 and 301
absent in the chain A.47 The maximum number of structures to return
was set to 10. An energy cutoff of 10 kcal/mol was applied. Loop
conformations were clustered, and representatives of each cluster were
selected. The best scoring loop structure was finally selected.
Monte Carlo/Free Energy Perturbation. MC/FEP calculations
were performed with the MCPRO program and following standard
protocols.48,49 Z-Matrix for the c-Src−ligand complexes were obtained
with the molecular growing program BOMB48 starting from the pose
of 3 within our crystal structure (PDB code: 4O2P). The models
included the 160 amino acid residues nearest to the ligand. Short
conjugate-gradient minimizations were carried out on the initial
structures for all complexes to relieve any unfavorable contacts.
Coordinates for the free ligands were obtained by extraction from the
complexes. Next, 1500 steps of conformational search analysis was
carried out on the ligands using BOSS50 program with the OPLS/
CM1A and OPLS/CM1Ax force fields and GB/SA hydration. The
resultant conformer with the lowest energy was used for FEP
calculation. The unbound ligands and complexes were solvated with
TIP4P water spheres (“caps”) with a 25 Å radius. The water molecules
in too close contact with solute atoms were removed. A few remote
side chains were neutralized in order to maintain overall charged
neutrality for each system. The ligand and the protein side chains
within 10 Å of any ligand were sampled during the MC simulations.
The only constraints were the bond lengths in side chains, and all
backbone atoms were frozen after a short conjugate-gradient
minimization. The energetics for the systems were evaluated with
the OPLS-AA and OPLS-AAx force fields for the protein and OPLS/
CM1A and OPLS/CM1Ax force fields for ligands.31 The CM1A
atomic charges were scaled by 1.14 for neutral molecules. Differences
in free energies of binding were determined from the usual
thermodynamic cycle that requires conversion of one ligand to
another both free in water and bound to the protein. The FEP
calculations utilized 11 windows of simple overlap sampling. For the
unbound ligand, each window consisted of 40 M configurations of
equilibration and 60 M configurations for averaging. For the bound
calculations, each window covered 20 M configurations of solvent only
equilibration, 40 M configurations of full equilibration, and 50 M
configurations of averaging. In the case of halogen bond scanning, the
number of configurations was increased to 60 M of equilibration and
80 M of averaging. All MC simulations were run at 298 K.
EXPERIMENTAL SECTION
■
Crystallization and Structure Determination of c-Src-3.
Inhibitor 3 was cocrystallized with c-Src using conditions similar to
those previously reported by Michalczyk et al.36 Briefly, final
concentrations of 540 μM inhibitor (100 mM stock in DMSO) and
180 μM wild-type c-Src (stored in 50 mM Tris pH 8.0, 100 mM NaCl,
1 mM DTT, 5% glycerol (v/v)) were preincubated for 1 h on ice to
form the enzyme−inhibitor complex prior to crystallization. Crystals
were grown using the hanging drop method at 20 °C after mixing 1 μL
of protein−inhibitor solution with 1 μL of reservoir solution (0−30
mM NaCl, pH 7.0, 9−20% ethylene glycol). All crystals were frozen
with further addition of 30% (v/v) glycerol. Diffraction data of the c-
Src−3 complex crystals were collected at the PX10SA beamline of the
Swiss Light Source (PSI, Villingen, Switzerland) to a resolution of 2.1
Å, using wavelengths close to 1 Å. The data set was processed with
XDS2 and scaled using XSCALE.37,38
Structure Determination and Refinement of c-Src-3. The c-
Src−inhibitor complex structure was solved by molecular replacement
with PHASER39 using the published c-Src structure 2OIQ40 as
template. The two c-Src molecules in the asymmetric unit were
manually modified using the program COOT.41 The model was first
refined with CNS42 using simulated annealing to remove model bias.
The final refinement was performed with REFMAC5.43 Inhibitor
topology files were generated using the Dundee PRODRG2 server.44
Refined structures were validated with PROCHECK.45 Detailed data,
Chemistry. Starting materials were purchased from Aldrich-Italia
(Milan, Italy). Melting points were determined with a Buchi 530
̈
apparatus and are uncorrected. IR spectra were measured in KBr or
1
CHCl3 with a PerkinElmer 398 spectrophotometer. H NMR spectra
were recorded at 400 MHz in CDCl3 or [D6]DMSO on a Bruker
Avance DPX400 spectrometer. Chemical shifts are reported as δ
1
(ppm) relative to TMS as the internal standard, J in Hz. H patterns
are described using the following abbreviations: s = singlet, d =
doublet, t = triplet, q = quartet, quint = quintet, sx = sextet, sept =
septet, m = multiplet, and br s = broad singlet. TLC was carried out
using Merck TLC plates silica gel 60 F254. Chromatographic
purifications were performed on columns packed with Merck 60 silica
gel, 230−400 mesh, for flash technique. Elemental analyses
(Supporting Information, Table S2) were determined with an
elemental analyzer EA 1110 (Fison-Instruments, Milan, Italy), and
the purity of all synthesized compounds was >95%. Analyses for C, H,
N, and S were within 0.3% of the theoretical value. Mass spectra
(MS) data were obtained using an Agilent 1100 LC/MSD VL system
I
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(G1946C) with a 0.4 mL/min flow rate using a binary solvent system
of 95:5 methanol/water. UV detection was monitored at 254 nm. MS
were acquired in positive and negative modes, scanning over the mass
range 50−1500. The following ion source parameters were used:
drying gas flow, 9 mL/min; nebulizer pressure, 40 psig; drying gas
temperature, 350 °C.
4.66 (m, 2H, CH2N), 7.12−7.31 (m, 5H Ar), 8.01 (s, 1H, H-3). MS:
m/z [M + 1]+ 334. Anal. (C16H17N4ClS) C, H, N, S.
4-Chloro-6-[(2-morpholin-4-ylethyl)thio]-1-(2-phenylpropyl)-1H-
pyrazolo[3,4-d]pyrimidine (32d). Yellow oil (288 mg, 69%). 1H NMR
(CDCl3): δ 1.22 (d, J = 6.8 Hz, 3H, CH3), 2.50−2.66, 2.73−2.82,
3.26−3.41 and 3.45−3.58 (4m, 8H, 2CH2N morph + SCH2CH2),
3.68−3.85 (m, 5H, 2CH2O morph + CHCH3), 4.40−4.56 (m, 2H,
CH2N pyraz), 7.07−7.30 (m, 5H Ar), 7.96 (s, 1H, H-3). MS: m/z [M
+ 1]+ 419. Anal. (C20H24N5OClS) C, H, N, S.
Compounds 4, 5, 8, and 27 and intermediates 30a,b, 31a, 32a, and
33a−e were already reported by us.21,23,25,30,32
General Procedure for the Synthesis of Compounds 31b and
31d. A mixture of 1-substituted 6-thioxo-1,5,6,7-tetrahydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one 30a,b (1 mmol), 4-(2-chloroethyl)-
morpholine (224 mg, 1.5 mmol), NaOH (40 mg, 1 mmol) in
anhydrous DMF (1 mL), and absolute ethanol (3 mL) was stirred at
reflux for 6 h. After cooling to room temperature, the solvent was
evaporated under reduced pressure and the mixture was poured into
cold water (20 mL). The obtained solid was filtered, washed with
water, and recrystallized from absolute ethanol.
General Procedure for the Synthesis of Compounds 6, 7, 9, 11−
13, 15−17, 21−23, 25, 26, 29. The suitable aniline (2 mmol) was
added to a solution of the 4-chloro derivative 32a−d and 33a−e (1
mmol) in absolute ethanol (5 mL), and the mixture was refluxed for
3−5 h. After cooling to room temperature, the obtained solid was
filtered, washed with water, and recrystallized from absolute ethanol.
N-(3-Fluorophenyl)-6-(methylthio)-1-(2-phenylethyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6). White solid (220 mg, 58%);
mp 133−135 °C. 1H NMR (CDCl3): δ 2.59 (s, 3H, SCH3), 3.18 (t, J
= 7.6 Hz, 2H, CH2Ar), 4.57 (t, J = 7.6 Hz, 2H, CH2N), 6.92−6.95 and
7.15−7.37 and 7.47−7.50 (3m, 10H, 9 Ar + H-3). IR (cm−1): 3100−
2500 (NH). MS: m/z [M + 1]+ 381. Anal. (C20H18N5FS) C, H, N, S.
N-(3-Bromophenyl)-6-(methylthio)-1-(2-phenylethyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (7). White solid (273 mg, 62%);
6-[(2-Morpholin-4-ylethyl)thio]-1-(2-phenylethyl)-1,5-dihydro-
4H-pyrazolo[3,4-d]pyrimidin-4-one (31b). White solid (197 mg,
1
51%); mp 197−198 °C. H NMR (CDCl3): δ 2.55−2.70 (m, 4H,
2CH2N morph), 2.80−2.84 (m, 2H, CH2N), 3.17−3.24 (m, 4H,
CH2S + CH2Ar), 3.80−3.85 (m, 4H, 2CH2O morph), 4.50 (t, J = 7.6
Hz, 2H, CH2N pyraz), 7.10−7.26 (m, 5H Ar), 8.02 (s, 1H, H-3). IR
(cm−1): 3500−2800 (NH), 1667 (CO). MS: m/z [M + 1]+ 386. Anal.
(C19H23N5O2S) C, H, N, S.
1
mp 233−234 °C. H NMR (CDCl3 + [D6]DMSO): δ 2.50 (s, 3H,
SCH3), 3.12 (t, J = 7.6 Hz, 2H, CH2Ar), 4.97 (t, J = 7.6 Hz, 2H,
CH2N), 7.04−7.19 and 7.59−7.61 (2m, 9H Ar), 8.00 (s, 1H, H-3). IR
(cm−1): 2929 (NH). MS: m/z [M + 1]+ 441. Anal. (C20H18N5BrS) C,
H, N, S.
6-[(2-Morpholin-4-ylethyl)thio]-1-(2-phenylpropyl)-1,5-dihydro-
4H-pyrazolo[3,4-d]pyrimidin-4-one (31d). White solid (200 mg,
50%); mp 128−130 °C. 1H NMR (CDCl3): δ 1.24 (d, J = 6.8 Hz, 3H,
CH3), 2.60−2.70 (m, 4H, 2CH2N morph), 2.80−2.90 and 3.15−3.25
(2m, 4H, SCH2CH2), 3.45−3.52 (m, 1H, CHCH3), 3.80−4.00 (m,
4H, 2CH2O morph), 4.38−4.40 (m, 2H, CH2N pyraz), 7.18−7.28 (m,
5H Ar), 7.99 (s, 1H, H-3). IR (cm−1): 3450−2900 (NH), 1678 (CO).
MS: m/z [M + 1]+ 400. Anal. (C20H25N5O2S) C, H, N, S.
N-(3-Chlorophenyl)-6-[(2-morpholin-4-ylethyl)thio]-1-(2-phenyl-
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (9). White solid (233
mg, 47%); mp 235−237 °C. 1H NMR (CDCl3): δ 2.88−2.95 (m, 4H,
2CH2N morph), 3.15 (t, J = 7.0 Hz, 2H, CH2Ar), 3.22−3.30 (m, 2H,
CH2N), 3.69−3.74 (m, 2H, SCH2), 4.00−4.49 (m, 4H, 2CH2O
morph), 4.64 (t, J = 7.0 Hz, 2H, CH2N pyraz), 7.16−7.38 (m, 9H Ar),
7.51 (s, 1H, H-3). IR (cm−1): 3300−3100 (NH). MS: m/z [M + 1]+
496. Anal. (C25H27N6OClS) C, H, N, S.
6-(Ethylthio)-N-(3-fluorophenyl)-1-(2-phenylpropyl)-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine (11). White solid (208 mg, 51%); mp 133−
135 °C. 1H NMR (CDCl3): δ 1.25 (d, J = 6.8 Hz, 3H, CH3CH), 1.50
(t, J = 7.5 Hz, 3H, SCH2CH3), 3.20 (q, J = 7.5 Hz, 2H, SCH2), 3.50−
3.55 (m, 1H, CHCH3), 4.39−4.51 (m, 2H, CH2N), 6.91−6.95 and
7.08−7.49 (2m, 10H, 9 Ar + H-3). IR (cm−1): 3290 (NH). MS: m/z
[M + 1]+ 409. Anal. (C22H22N5FS) C, H, N, S.
Synthesis of 6-(Ethylthio)-1-(2-phenylpropyl)-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one 31c. A mixture of 1-(2-phenyl-
propyl)-6-thioxo-1,5,6,7-tetrahydro-4H-pyrazolo[3,4-d]pyrimidin-4-
one 30b (286 mg, 1 mmol), iodoethane (172 mg, 1.1 mmol), and
K2CO3 (138 mg, 1 mmol) in anhydrous DMF (2 mL) was stirred at
room temperature for 20 h. The mixture was poured into cold water
(50 mL). The obtained solid was filtered, washed with water, and
recrystallized from absolute ethanol. White solid (192 mg, 61%); mp
1
155−156 °C. H NMR ([D6]DMSO): δ 1.22 (d, J = 7.0 Hz, 3H,
CH3CH), 1.36 (t, J = 7.2, 3H, SCH2CH3), 3.17 (q, J = 7.2 Hz, 2H,
SCH2), 3.40−3.50 (m, 1H, CHCH3), 4.26−4.50 (m, 2H, CH2N),
7.07−7.30 (m, 5H Ar), 7.94 (s, 1H, H-3), 12.10 (br s, 1H, NH
disappears with D2O). IR cm−1: 3110−2800 (NH), 1681 (CO). MS:
m/z [M + 1]+ 315. Anal. (C16H18N4OS) C, H, N, S.
N-(3-Chlorophenyl)-6-(ethylthio)-1-(2-phenylpropyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (12). White solid (136 mg, 32%);
1
mp 69−70 °C. H NMR ([D6]DMSO): δ 1.22 (d, J = 7.0 Hz, 3H,
CH3CH), 1.36 (t, J = 7.4 Hz, 3H, SCH2CH3), 3.18 (q, J = 7.4 Hz, 2H,
SCH2), 3.95−4.00 (m, 1H, CHCH3), 4.29−4.45 (m, 2H, CH2N),
7.11−8.15 (m, 10H, 9 Ar + H-3), 10.13 (br s, 1H, NH disappears with
D2O). IR (cm−1): 3592 (NH). MS: m/z [M + 1]+ 425. Anal.
(C22H22N5ClS) C, H, N, S.
General Procedure for the Synthesis of Compounds 32b−d. The
Vilsmeier complex, previously prepared from POCl3 (0.74 mL, 8
mmol) and anhydrous DMF (590 mg, 8 mmol) was added to a
suspension of 31b−d (1 mmol) in CH2Cl2 (10 mL). The mixture was
refluxed for 6−8 h. For compounds 32b and 32d, the solution was
washed with a 4N NaOH solution (2 × 10 mL) and water (2 × 10
mL), dried (MgSO4), filtered, and concentrated under reduced
pressure. For compound 32c, the solution was washed with water (2
× 10 mL), dried (MgSO4), filtered, and concentrated under reduced
pressure. The crude oil was purified by column chromatography
(Florisil, 100−200 mesh), using diethyl ether as the eluent, to afford
the pure product.
4-Chloro-6-[(2-morpholin-4-ylethyl)thio]-1-(2-phenylethyl)-1H-
pyrazolo[3,4-d]pyrimidine (32b). Yellow oil (323 mg, 80%). 1H NMR
(CDCl3): δ 2.51−2.90 (m, 6H, 2CH2N morph + CH2N), 3.22 (t, J =
7.2 Hz, 2H, CH2Ar), 3.30−3.40 (m, 2H, SCH2), 3.68−3.88 (m, 4H,
2CH2O morph), 4.62 (t, J = 7.2 Hz, 2H, CH2N pyraz), 7.09−7.26 (m,
5H Ar), 8.01 (s, 1H, H-3). MS: m/z [M + 1]+ 405. Anal.
(C19H22N5OClS) C, H, N, S.
N-(3-Bromophenyl)-6-(ethylthio)-1-(2-phenylpropyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (13). White solid (192 mg, 41%);
mp 83−84 °C. 1H NMR (CDCl3): δ 1.23 (d, J = 7.2 Hz, 3H,
CH3CH), 1.45 (t, J = 7.6 Hz, 3H, SCH2CH3), 3.19 (q, J = 7.6 Hz, 2H,
SCH2), 3.49−3.55 (m, 1H, CHCH3), 4.39−4.50 (m, 2H, CH2N),
7.16−7.46 (m, 9H Ar), 7.84 (s, 1H, H-3). IR cm−1: 3300−3000 (NH).
MS: m/z [M + 1]+ 469. Anal. (C22H22N5BrS) C, H, N, S.
6-[(2-Morpholin-4-ylethyl)thio]-N-phenyl-1-(2-phenylpropyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (15). White solid (332 mg, 70%);
mp 212−213 °C. 1H NMR (CDCl3): δ 1.14 (d, J = 6.8 Hz, 3H, CH3),
2.80−3.00 (m, 2H, SCH2), 3.20−3.45, 3.46−3.60, 3.72−3.85 and
4.02−4.15 (4m, 11H, 4CH2 morph + CH2N + CHCH3), 4.30−4.44
(m, 2H, CH2N pyraz), 6.70−6.81 and 7.07−7.35 (2m, 10H Ar), 7.49
(s, 1H, H-3). IR (cm−1): 3500−2800 (NH). MS: m/z [M + 1]+ 476.
Anal. (C26H30N6OS) C, H, N, S.
N-(3-Fluorophenyl)-6-[(2-morpholin-4-ylethyl)thio]-1-(2-phenyl-
propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (16). White solid (182
mg, 37%); mp 236−237 °C. 1H NMR (CDCl3): δ 1.24 (d, J = 7.0 Hz,
3H, CH3), 2.08−2.60, 2.78−3.17, 3.27−3.74 and 3.97−4.38 (4m, 13H,
SCH2 + 4CH2 morph + CH2N + CHCH3), 4.40−4.50 (m, 2H, CH2N
4-Chloro-6-(ethylthio)-1-(2-phenylpropyl)-1H-pyrazolo[3,4-d]-
1
pyrimidine (32c). Yellow oil (300 mg, 90%). H NMR (CDCl3): δ
1.31 (d, J = 7.2 Hz, 3H, CH3CH), 1.47 (t, J = 7.4 Hz, 3H, SCH2CH3),
3.21 (q, J = 7.4 Hz, 2H, SCH2), 3.48−3.63 (m, 1H, CHCH3), 4.42−
J
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pyraz), 5.90−6.40 and 7.03−7.50 (2m, 10H, 9 Ar + H-3), 9.33 (br s,
1H, NH, disappears with D2O). IR (cm−1): 3450−3100 (NH). MS:
m/z [M + 1]+ 494. Anal. (C26H29N6OFS) C, H, N, S.
3-{[6-[(2-Morpholin-4-ylethyl)thio]-1-(2-phenylethyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-yl]amino}phenol Hydrochloride (10).
Pale-yellow solid (261 mg, 51%); mp 261−262 °C. 1H NMR
(CDCl3): δ 3.05−3.58 (m, 10H, CH2Ar + 2CH2N morph + SCH2 +
CH2N), 3.77−3.95 (m, 4H, 2CH2O morph), 4.57 (t, J = 7.0 Hz, 2H,
CH2N pyraz), 6.52−6.63 and 7.08−7.32 (2m, 10H, 9 Ar + H-3), 8.22
(br s, 1H, NH disappears with D2O), 9.61 (br s, 1H disappears with
D2O), 10.18 (br s, 1H disappears with D2O). IR (cm−1): 3500−3100
(NH + OH). MS: m/z [M + 1]+ 478. Anal. (C25H29N6O2ClS) C, H,
N, S.
N-(3-Chlorophenyl)-6-[(2-morpholin-4-ylethyl)thio]-1-(2-phenyl-
propyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (17). Pale-yellow solid
(265 mg, 52%); mp 247−249 °C. 1H NMR ([D6]DMSO): δ 1.23 (d, J
= 7.0 Hz, 3H, CH3), 2.52−2.67, 2.74−2.81, 3.24−3.40 and 3.43−3.59
(4m, 8H, 2CH2N morph + SCH2CH2), 3.65−3.80 (m, 4H, 2CH2O
morph), 3.85−3.90 (m, 1H, CHCH3), 4.40−4.50 (m, 2H, CH2N
pyraz), 7.20−7.40 (m, 9H Ar), 7.97 (s, 1H, H-3), 10.40 (br s, 1H, NH
disappears with D2O). IR (cm−1): 3450−3100 (NH). MS: m/z [M +
1]+ 510. Anal. (C26H29N6OClS) C, H, N, S.
1-(2-Chloro-2-phenylethyl)-6-(isopropylthio)-N-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (21). Pale-yellow solid (263 mg,
62%); mp 152−153 °C. 1H NMR (CDCl3): δ 1.33 (d, J = 6.0 Hz, 3H,
CH3), 1.38 (d, J = 6.0 Hz, 3H, CH3), 4.00 (sept, J = 6.0 Hz, 1H,
SCH), 4.70−4.99 (m, 2H, CH2N), 5.45−5.55 (m, 1H, CHCl), 7.00−
7.51 (m, 11H, 10 Ar + H-3). IR (cm−1): 3200−2800 (NH). MS: m/z
[M + 1]+ 425. Anal. (C22H22N5ClS) C, H, N, S.
1-(2-Chloro-2-phenylethyl)-N-(3-fluorophenyl)-6-(isopropylthio)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine (22). White solid (208 mg,
47%); mp 217−218 °C. 1H NMR (CDCl3): δ 1.31 (d, J = 6.0 Hz, 3H,
CH3), 1.34 (d, J = 6.0 Hz, 3H, CH3), 4.05 (sept, J = 6.0 Hz, 1H,
SCH), 4.67−4.87 (m, 2H, CH2N), 5.25−5.32 (m, 1H, CHCl), 6.88−
7.51 (m, 10H, 9 Ar + H-3). IR (cm−1): 3000−2500 (NH). MS: m/z
[M + 1]+ 443. Anal. (C22H21N5ClFS) C, H, N, S.
N-(3-Bromophenyl)-1-(2-chloro-2-phenylethyl)-6-(isopropylthio)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine (23). Yellow solid (276 mg,
55%); mp 233−234 °C. 1H NMR (CDCl3): δ 1.28 (d, J = 6.0 Hz, 3H,
CH3), 1.31 (d, J = 6.0 Hz, 3H, CH3), 4.10 (sept, J= 6.0 Hz, 1H, SCH),
4.78−4.91 (m, 2H, CH2N), 5.35−5.47 (m, 1H, CHCl), 6.76−7.51 (m,
10H, 9 Ar + H-3). IR (cm−1): 3000−2500 (NH). MS: m/z [M + 1]+
504. Anal. (C22H21N5BrClS) C, H, N, S.
3-{[6-(Ethylthio)-1-(2-phenylpropyl)-1H-pyrazolo[3,4-d]-
pyrimidin-4-yl]amino}phenol (14). Pale-yellow solid (296 mg, 73%);
1
mp 184−186 °C. H NMR (CDCl3): δ 1.22 (d, J = 6.8 Hz, 3H,
CH3CH), 1.44 (t, J = 7.6 Hz, 3H, SCH2CH3), 3.19 (q, J = 7.6 Hz, 2H,
SCH2), 3.46−3.52 (m, 1H, CHCH3), 4.36−4.89 (m, 2H, CH2N),
6.82−6.84, 6.93−6.95, 6.99−7.05 and 7.16−7.24 (4m, 10H, 9 Ar + H-
3). IR (cm−1): 3200−2900 (NH + OH). MS: m/z [M + 1]+ 407. Anal.
(C22H23N5OS) C, H, N, S.
3-{[6-[(2-Morpholin-4-ylethyl)thio]-1-(2-phenylpropyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-yl]amino}phenol Hydrochloride (18).
Pale-yellow solid (248 mg, 47%); mp 177−178 °C. 1H NMR
(CDCl3): δ 1.16−1.33 (m, 3H, CH3), 2.60−2.80, 2.88−3.03 and
3.30−3.64 (3m, 9H, 2CH2N morph + CHCH3 + CH2CH2S), 3.78−
3.97 (m, 4H, 2CH2O morph), 4.38−4.55 (m, 2H, CH2N pyraz),
6.54−6.74 and 7.09−7.33 (2m, 9H Ar), 7.58 (br s, 1H, disappears with
D2O), 7.92 (s, 1H, H-3), 8.18 (br s, 1H, disappears with D2O). IR
(cm−1): 3500−3100 (NH + OH). MS: m/z [M + 1]+ 492. Anal.
(C26H31N6O2ClS) C, H, N, S.
3-{[1-(2-Chloro-2-phenylethyl)-6-(methylthio)-1H-pyrazolo[3,4-
d]pyrimidin-4-yl]amino}phenol (19). Light-brown solid (132 mg,
32%); mp 82−83 °C. 1H NMR (CDCl3): δ 2.60 (s, 3H, CH3), 4.72−
4.77 and 4.84−4.90 (2m, 2H, CH2N) 5.47−5.51 (m, 1H, CHCl),
6.78−6.80, 6.94−7.00 and 7.20−7.38 (3m, 11H, 9Ar + H-3 + 1H, 1H
disappears with D2O). IR (cm−1): 3300−2910 (NH + OH). MS: m/z
[M + 1]+ 413. Anal. (C20H18N5OClS) C, H, N, S.
3-{[6-(Butylthio)-1-(2-chloro-2-phenylethyl)-1H-pyrazolo[3,4-d]-
pyrimidin-4-yl]amino}phenol (20). Light-brown solid (436 mg, 96%);
mp 105−106 °C. 1H NMR (CDCl3): δ 0.99 (t, J = 7.2 Hz, 3H, CH3),
1.52 (sx, J = 7.2 Hz, 2H, CH2CH3), 1.79 (quint, J = 7.2 Hz, 2H,
CH2CH2CH3), 3.14−3.24 (m, 2H, SCH2), 4.68−4.73 and 4.83−4.89
(2m, 2H, CH2N), 5.29 (br s, 1H, disappears with D2O), 5.46−5.50
(m, 1H, CHCl), 6.78−6.80, 6.94−6.99 and 7.18−7.38 (3m, 11H, 9 Ar
+ H-3 + 1H, 1H disappears with D2O). IR (cm−1): 3400−3200 (NH +
OH). MS: m/z [M + 1]+ 455. Anal. (C23H24N5ClOS) C, H, N, S.
3-{[1-(2-Chloro-2-phenylethyl)-6-(isopropylthio)-1H-pyrazolo-
[3,4-d]pyrimidin-4-yl]amino}phenol (24). Light-brown solid (339 mg,
77%); mp 198−199 °C. 1H NMR (CDCl3): δ 1.46 (d, J = 6.0 Hz, 3H,
CH3), 1.49 (d, J = 6.0 Hz, 3H, CH3), 3.97−4.07 (m, 1H, SCH), 4.70−
4.75 and 4.83−4.88 (2m, 2H, CH2N) 5.48−5.51 (m, 1H, CHCl),
6.78−6.80, 6.94−7.02 and 7.21−7.41 (3m, 10H, 9 Ar + H-3). IR
(cm−1): 3300−3100 (NH + OH). MS: m/z [M + 1]+ 441. Anal.
(C22H22N5ClOS) C, H, N, S
1-(2-Chloro-2-phenylethyl)-6-(cyclopentylthio)-N-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (25). White solid (369 mg, 82%);
1
mp 125−126 °C. H NMR (CDCl3): δ 1.52−1.78 (m, 8H, 4CH2
cyclopent), 3.95−4.12 (m, 1H, SCH), 4.50−4.62 and 4.70−4.85 (2m,
2H, CH2N), 5.22−5.40 (m, 1H, CHCl), 7.10−7.50 (m, 11H, 10 Ar +
H-3). IR (cm−1): 3363 (NH). MS: m/z [M + 1]+ 451. Anal.
(C24H24N5ClS) C, H, N, S.
1-(2-Chloro-2-phenylethyl)-6-(cyclopentylthio)-N-(3-fluorophen-
yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (26). White solid (206 mg,
1
44%); mp 226−227 °C. H NMR (CDCl3): δ 1.78−1.84 and 2.12−
2.43 (2m, 8H, 4CH2 cyclopent), 3.98−4.17 (m, 1H, SCH), 4.54−4.68
and 4.73−4.89 (2m, 2H, CH2N), 5.26−5.44 (m, 1H, CHCl), 5.54 (br
s, 1H, NH disappears with D2O), 6.93−7.53 (m, 10H, 9 Ar + H-3). IR
(cm−1): 2835 (NH). MS: m/z [M + 1]+ 469. Anal. (C24H23N5ClFS)
C, H, N, S.
N-(3-Bromophenyl)-1-(2-chloro-2-phenylethyl)-6-[(2-morpholin-
4-ylethyl)thio]-1H-pyrazolo[3,4-d]pyrimidin-4-amine (29). White
1
solid (350 mg, 61%); mp 232−233 °C. H NMR (CDCl3): δ 2.90−
3.99 (m, 12H, 4CH2 morph + CH2N + CH2S), 4.63−4.85 and 5.04−
5.21 (2m, 2H, CH2N pyraz), 5.55−5.70 (m, 1H, CHCl), 7.03−8.52
(m, 10H, 9 Ar + H-3), 11.33 (br s, 1H, NH disappears with D2O). IR
3-{[1-(2-Chloro-2-phenylethyl)-6-(cyclopentylthio)-1H-pyrazolo-
[3,4-d]pyrimidin-4-yl]amino}phenol (28). White solid (359 mg,
1
(cm−1): 3450 (NH). MS: m/z [M
+
1]+ 575. Anal.
77%); mp 160−161 °C. H NMR (CDCl3): δ 1.62−2.24 (m, 8H,
4CH2 cyclopent), 4.03−4.22 (m, 1H, SCH), 4.75−4.95 (m, 2H,
CH2N), 5.45−5.56 (m, 1H, CHCl), 6.80−7.60 (m, 9H Ar), 8.06 (s,
1H H-3), 10.20 (br s, 1H, disappears with D2O). IR (cm−1): 3500−
3000 (NH + OH). MS: m/z [M + 1]+ 467. Anal. (C24H24N5OClS) C,
H, N, S.
(C25H26N6OBrClS) C, H, N, S.
General Procedure for the Synthesis of Compounds 10, 14, 18,
19, 20, 24, and 28. The 3-aminophenol (545 mg, 5 mmol) was added
to a solution of the suitable 4-chloro derivative 32a−d and 33a−d (1
mmol) in absolute ethanol (10 mL), and the mixture was refluxed for
3−5 h. After cooling to room temperature, the solvent was evaporated
under reduced pressure and the crude was solved in ethyl acetate (10
mL), washed with 0.1 N HCl solution (2 × 10 mL), 1 N NaOH
solution (10 mL) and brine (2 × 10 mL), dried (MgSO4), filtered, and
concentrated under reduced pressure to give a brown oil which
crystallized at 4 °C by adding a 1:1 mixture of diethyl ether/petroleum
ether (bp 40−60 °C) (compounds 10, 14, 18, 19, 20, and 24) or
cycloexane (28). If necessary, the solid obtained was purified by
Silicagel chromatography column using CH2Cl2 as the eluent.
ADME Assays. Chemicals. All solvents and reagents were from
Sigma-Aldrich Srl (Milan, Italy), and Brain Polar Lipid Extract
(Porcine) was from Avanti Polar Lipids, Inc. (Alabama, USA).
Dodecane was purchased from Fluka (Milan, Italy). Pooled male
donors 20 mg mL−1 HLM were from BD Gentest-Biosciences (San
Jose, California). Milli-Q quality water (Millipore, Milford, MA, USA)
was used. Hydrophobic filter plates (MultiScreen-IP, clear plates, 0.45
μm diameter pore size), 96-well microplates, and 96-well UV-
transparent microplates were obtained from Millipore (Bedford, MA,
USA).
Compounds 10 and 18 were obtained as hydrochloride salts.
K
dx.doi.org/10.1021/jm5013159 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Parallel Artificial Membrane Permeability Assay (PAMPA). Donor
solution (0.5 mM) was prepared by diluting 1 mM dimethyl sulfoxide
(DMSO) compound stock solution using phosphate buffer (pH 7.4,
0.025 M). Filters were coated with 5 μL of a 1% (w/v) dodecane
solution of phosphatidylcholine or 4 μL of brain polar lipid solution
(20 mg mL−1 16% CHCl3, 84% dodecane), prepared from CHCl3
solution 10% w/v, for intestinal permeability and BBB permeability,
respectively. Donor solution (150 μL) was added to each well of the
filter plate. To each well of the acceptor plate were added 300 μL of
solution (50% DMSO in phosphate buffer). All compounds were
tested in three different plates on different days. The sandwich was
incubated for 5 h at room temperature under gentle shaking. After the
incubation time, the plates were separated and samples were taken
from both receiver and donor sides and analyzed using LC with UV
detection at 280 nm.
LC analysis were performed with a Varian Prostar HPLC system
(Varian Analytical Instruments, USA) equipped with a binary pump
with a manual injection valve and model Prostar 325 UV−vis detector.
Chromatographic separation were conducted using a Polaris C18-A
column (150−4.6 mm, 5 μm particle size) at a flow rate of 0.8 mL
min−1 with a mobile phase composed of 60% ACN/40% H2O−formic
acid 0.1%.
Quantification of the single compound was made by comparison
with apposite calibration curves realized with standard solutions in
methanol.
Microsomal Stability Assay. Each compound in DMSO solution
was incubated at 37 °C for 60 min in 125 mM phosphate buffer (pH
7.4) and 5 μL of human liver microsomal protein (0.2 mg mL−1), in
the presence of a NADPH-generating system at a final volume of 0.5
mL (compound final concentration, 50 μM); DMSO did not exceed
2% (final solution). The reaction was stopped by cooling on ice and
adding 1.0 mL of acetonitrile. The reaction mixtures were then
centrifuged, and the parent drug and metabolites were subsequently
determined by LC−UV−MS.
Chromatographic analysis was performed with an Agilent 1100 LC/
MSD VL system (G1946C) (Agilent Technologies, Palo Alto, CA)
constituted by a vacuum solvent degassing unit, a binary high-pressure
gradient pump, an 1100 series UV detector, and an 1100 MSD model
VL benchtop mass spectrometer.
Chromatographic separation was obtained using a Varian Polaris
C18-A column (150−4.6 mm, 5 μm particle size) and gradient elution:
eluent A being ACN and eluent B consisting of an aqueous solution of
formic acid (0.1%). The analysis started with 2% of eluent A, which
was rapidly increased up to 70% in 12 min, then slowly increased up to
98% in 20 min. The flow rate was 0.8 mL min−1 and injection volume
was 20 μL.
The Agilent 1100 series mass spectra detection (MSD) single-
quadrupole instrument was equipped with the orthogonal spray API-
ES (Agilent Technologies, Palo Alto, CA). Nitrogen was used as
nebulizing and drying gas. The pressure of the nebulizing gas, the flow
of the drying gas, the capillary voltage, the fragmentor voltage, and the
vaporization temperature were set at 40 psi, 9 L/min, 3000 V, 70 V,
and 350 °C, respectively. UV detection was monitored at 280 nm. The
LC−ESI−MS determination was performed by operating the MSD in
the positive ion mode. Spectra were acquired over the scan range m/z
100−1500 using a step size of 0.1 μ. The percentage of not
metabolized compound was calculated by comparison with reference
solutions.
Permeability (Papp) for PAMPA was calculated according to the
following equation, obtained from Wohnsland and Faller51 and Sugano
et al.52 equation with some modification in order to obtain
permeability values in cm s−1,
VDVA
(VD + VA)At
P
=
− ln(1 − r)
app
where VA is the volume in the acceptor well, VD is the volume in the
donor well (cm3), A is the “effective area” of the membrane (cm2), t is
the incubation time (s), and r the ratio between drug concentration in
the acceptor and equilibrium concentration of the drug in the total
volume (VD + VA). Drug concentration was estimated by using the
peak area integration.
Membrane retentions (%) were calculated according to the
following equation:
Biological Studies. Enzymatic Assay on Isolated Src. Recombi-
nant human Src was purchased from Upstate (Lake Placid, NY).
Activity was measured in a filter-binding assay using a commercial kit
(Src Assay Kit, Upstate), according to the manufacturer’s protocol,
using 150 μM of the specific Src peptide substrate (KVEKIGEG-
TYGVVYK) and in the presence of 0.125 pMol of Src and 10 μM of
[γ-32P]-ATP. The apparent affinity (Km) values of the Src preparation
used for its peptide and ATP substrates were determined separately
and found to be 30 μM and 5 μM, respectively.
Enzymatic Assay on Isolated Abl. Recombinant human Abl was
purchased from Upstate. Activity was measured in a filter binding assay
using an Abl specific peptide substrate (Abtide, Upstate). Reaction
conditions were: 10 μM [γ-32P]-ATP, 50 μM peptide, 0.022 μM c-Abl.
The apparent affinity (Km) values of the Abl preparation used for its
peptide and ATP substrates were determined separately and found to
be 1.5 and 10 μM, respectively.
[r − (D + A)] × 100
%MR =
Eq
where r is the ratio between drug concentration in the acceptor and
equilibrium concentration, D, A, and Eq represented drug concen-
tration in the donor, acceptor, and equilibrium solution, respectively.
Water Solubility Assay. Each solid compound (1 mg) was added to
1 mL of water. The samples were shaked in a shaker bath at room
temperature for 24−36 h. The suspensions were filtered through a
0.45 μm nylon filter (Acrodisc), and the solubilized compound
determined by LC−MS−MS assay. For each compound, the
determination was performed in triplicate.
For the quantification was used an LC−MS system consisted of a
Varian apparatus (Varian Inc.) including a vacuum solvent degassing
unit, two pumps (212-LC), a triple quadrupole MSD (model 320-LC)
mass spectrometer with ES interface, and Varian MS Workstation
System Control version 6.9 software. Chromatographic separation was
obtained using a Pursuit C18 column (50 mm × 2.0 mm) (Varian)
with 3 μm particle size and gradient elution: eluent A being ACN and
eluent B consisting of an aqueous solution of formic acid (0.1%). The
analysis started with 0% of eluent A, which was linearly increased up to
70% in 10 min, then slowly increased up to 98% up to 15 min. The
flow rate was 0.2 mL min−1, and injection volume was 5 μL. The
instrument operated in positive mode and parameters were: detector
1850 V, drying gas pressure 25.0 psi, desolvation temperature 300.0
°C, nebulizing gas 40.0 psi, needle 5000 V, and shield 600 V. Nitrogen
was used as nebulizer gas and drying gas. Collision induced
dissociation was performed using argon as the collision gas at a
pressure of 1.8 mTorr in the collision cell. The transitions as well as
the capillary voltage and the collision energy used for each compound
are summarized in Supporting Information, Table S3.
Cell Cycle and Apoptosis Analysis. Cells were seeded in 60 mm
Petri dishes at a density of 3 × 105. After treatment and subsequent
incubation for 24 h at 37 °C and 5% CO2 in humidified atmosphere,
harvested cells were washed and fixed overnight with 70% ethanol.
Then ethanol was removed by centrifugation and the cells
resuspended in PBS and stained with 50 μg/mL propidium iodide
(PI) at 4 °C for 30 min in the dark. Stained cells were analyzed by Tali
image based cytometer (Life Technologies, Carlsbad, CA, USA)
counting 20 fields for sample and exported fcs raw. Apoptosis led to
the fragmentation of DNA and formation of apoptotic bodies with a
progressive loss of DNA content from cells in the G1 phase. For this
reason, the fluorescence signals lower but adjacent to the G1 phase
signals (hypodiploid peak) are considered an effective estimation for
apoptotic cells (percentage respect to total events). Data were
elaborated by Flowing software (v. 2.5.0, by Perttu Terho, University
of Turku, Finland).
Spheroid Growth Assay. The in vitro antitumoral action of
inhibitors was evaluated by neuroblastoma spheroid assay. The SH-
L
dx.doi.org/10.1021/jm5013159 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
SY5Y cell line was utilized as cell model of human neuroblastoma.
Cells were purchased from American Type Culture Collection
(ATCC, Manassas, VA, USA) and were cultured in DMEM medium
supplemented with 10% fetal bovine serum. IBIDI angiogenesis
microslides (IBIDI GmbH) were coated with growth factor reduced
Matrigel (BD, Bioscience) and allowed to polymerize for 30 min. SH-
SY5Y cells were seeded in a 96-multiwell plate in the presence or not
(CTR) of inhibitors. Starting from 24 h after the seeding, in basal
condition, cellular aggregates with spheroidal appearance (diameter
>100 μm) were visible. The size of cellular spheres, in terms of area
and diameter, was determined using an Image Pro-plus v 4.5 analysis
system considering five random fields/treatment (400× magnifica-
tion). IC50 (drug concentration that determined the 50% of growth
inhibition) was calculated by Grafit v4.0 (Erithacus Software Limited)
software using the best fitting sigmoid curve. For cytotoxicity studies,
the Wi38 human diploid cell line was derived from normal embryonic
lung tissue and was purchased from ATCC.
Animals and Experimental in Vivo Model. Male CD1 nude mice
(Charles River, Milan, Italy) were maintained under the guidelines
established by our Institution (University of L’Aquila, Medical School
and Science and Technology School Board Regulations, complying
with the Italian government regulation no. 116, January 27, 1992, for
the use of laboratory animals). Before any invasive manipulation, mice
were anesthetized with a mixture of ketamine (25 mg/mL)/xylazine (5
mg/mL). Tumor grafts were obtained by injecting sc 1 ×106 SH-SY5Y
cells in 100 μL of 12 mg/mL Matrigel (Becton Dickinson, Franklin
Lakes, NJ, USA). Tumor growth was monitored daily by measuring
the average tumor diameter. The tumor volume was expressed in mm3
according to the formula 4/3πr3. For in vivo administration, 29 was
prepared as suspension in 0.5% methylcellulose solution. Each mouse
received daily oral administration of methylcellulose vehicle or of 50
mg/kg 29.
Cytofluorimetric Analysis. Analysis of DNA content was performed
for the evaluation of the cell cycle; 0.5 × 106 SH-SY5Y cells were
plated in 100 mm dishes and treated the next day with 29 for 72 h.
Then cells were detached with trypsin, fixed in cold 70% ethanol at 4
°C for 2 h, resuspended in 500 μL of staining solution (40 μg/mL
propidium iodide and 500 μg/mL RNase A in PBS; all from Sigma)
for 30 min at 37 °C, and analyzed by flow cytometry. A FACSCalibur
(BD Biosciences) flow cytometer was used, and the analysis was
performed with FlowJo software (BD Biosciences).
Sprouting Assay. The brain microvascular endothelial cell line
hBMEC was purchased from ScienCell Research Laboratory
(Carlsbad, CA, USA). HBMEC cells were suspended in culture
medium containing 20% methylcellulose, seeded at a density of 1000
cells/well, into nonadhesive 96-well plate and cultured at 37 °C (5%
CO2, 100% humidity). Under these conditions, suspended endothelial
cells (EC) form spontaneously within 4 h a single cell aggregate
known as a spheroid. Spheroids were harvested within 24 h and used
for in-gel sprouting angiogenesis experiments. Briefly, spheroids were
seeded in microslides coated with Matrigel and images were observed
after 24 h, captured by an inverted microscope and analyzed with the
NIH ImageJ analysis system. For statistical analysis, number of sprouts
per spheroid, with a minimum of 10 spheroids for each treatment, was
considered.
Present Address
◆For M.R.: Dipartimento di Farmacia, University of Parma,
Parco Area delle Scienze 27/A, Parma 43124, Italy.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to Lead Discovery Siena (LDS) and MIUR
(Ministero dell’Istruzione dell’Universita
■
̀
e della Ricerca; Art.11
of D.M. n.593). Cost Action CM1106 “Chemical Approaches
to Targeting Drug Resistance in Cancer Stem Cells” is also
acknowledged. S.S. gratefully acknowledges the National
Interest Research Project PRIN_2010_5YY2HL. This work
was partially supported by the Italian Association of Cancer
Research AIRC IG_4538 grant and Istituto Toscano Tumori
Grant Proposal 2010.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details of enantiomers separation, a figure for the
evaluation of compound 29 toward SH-SY5Y cell growth, data
collection and refinement statistics of the X-ray complex,
elemental analysis for new synthesized compounds, chromato-
graphic and MS parameters. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (+39) 010 353 8362. E-mail: schensil@unige.it.
■
́
A.; Olivier, A.; Otterbein, L.; Ple, P. A.; Warin, N.; Costello, G. N-(5-
Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-
M
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