Pyrazolo[3,4-d]pyrimidine prodrugs: Strategic optimization of the aqueous solubility of dual Src/Abl inhibitors

Article abstract of DOI:10.1021/ml4000782

Design and synthesis of prodrugs of promising drug candidates represents a valid strategy to overcome the lack of favorable ADME properties, in particular aqueous solubility and bioavailability. We report herein the successful application of this strategy with two representative pyrazolo[3,4-d]pyrimidine derivatives (1 and 2), which led to the development of the corresponding and highly water-soluble antitumor prodrugs (7 and 8). In vitro studies confirmed a significant improvement of aqueous solubility and, for compound 8, good plasma stability, suggesting superior in vivo bioavailability. As expected, the uncleaved water-soluble prodrugs 7 and 8 showed no activity toward the enzymatic targets (c-Src and c-Abl) but revealed promising antiproliferative activity in myeloid cell lines, as a consequence of the in vitro hydrolysis of the selected solubilizing moiety, followed by the release of the active compounds (1 and 2).

Full text of DOI:10.1021/ml4000782

Letter
pubs.acs.org/acsmedchemlett
Pyrazolo[3,4‑d]pyrimidine Prodrugs: Strategic Optimization of the
Aqueous Solubility of Dual Src/Abl Inhibitors
Giulia Vignaroli,† Claudio Zamperini,† Elena Dreassi,† Marco Radi,†,‡ Adriano Angelucci,§ Patrizia Sanita,§
̀
Emmanuele Crespan,∥ Miroslava Kissova,∥ Giovanni Maga,∥ Silvia Schenone,⊥ Francesca Musumeci,⊥
and Maurizio Botta*,†,#
†Dipartimentodi Biotecnologie, Chimica e Farmacia, Universita
‡Dipartimento di Farmacia, Universita
degli Studi di Parma, Viale delle Scienze 27/A, 43124 Parma, Italy
§Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Universita
dell’Aquila, Via Vetoio, 67100 Coppito, L’Aquila, Italy
∥Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
⊥Dipartimento di Scienza Farmaceutiche, Universita
̀
degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy
̀
̀
̀
degli Studi di Genova, Viale benedetto XV 3, 16132 Genova, 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: Design and synthesis of prodrugs of promising drug candidates
represents a valid strategy to overcome the lack of favorable ADME properties,
in particular aqueous solubility and bioavailability. We report herein the
successful application of this strategy with two representative pyrazolo[3,4-
d]pyrimidine derivatives (1 and 2), which led to the development of the
corresponding and highly water-soluble antitumor prodrugs (7 and 8). In vitro
studies confirmed a significant improvement of aqueous solubility and, for
compound 8, good plasma stability, suggesting superior in vivo bioavailability.
As expected, the uncleaved water-soluble prodrugs 7 and 8 showed no activity
toward the enzymatic targets (c-Src and c-Abl) but revealed promising
antiproliferative activity in myeloid cell lines, as a consequence of the in vitro
hydrolysis of the selected solubilizing moiety, followed by the release of the
active compounds (1 and 2).
KEYWORDS: Prodrug, pyrazolo[3,4-d]pyrimidine, aqueous solubility, dual c-Src and c-Abl inhibitor
yrazolo[3,4-d]pyrimidines represent a promising class of
compounds capable of inhibiting several oncogenic
able to induce apoptosis and reduce cellular proliferation in
different solid tumor cell lines (A431, 8701-BC, SaOS-2, and
PC3).4−7
Other 4-amino-substituted pyrazolo[3,4-d]pyrimidines are
known to inhibit the proliferation of Bcr-Abl-positive human
leukemia cell lines (K-562, KU-812, and MEG-01), to reduce
Bcr-Abl tyrosine phosphorylation and to promote apoptosis of
Bcr-Abl expressing cells.8
P
tyrosine kinases, which represent an attractive target for the
development of new therapeutic agents against cancer.1 In this
context, 4-amino-substituted pyrazolo[3,4-d]pyrimidines, ex-
tensively studied and regularly synthesized in our laboratories
(general structure A, Figure 1), were found to inhibit c-Src and
c-Abl activities in cell-free assays, some of them with nanomolar
activity.2,3 Furthermore, several members of this family were
The biological activity exhibited by this family of compounds,
as for other tyrosine kinases inhibitors, is associated with low
aqueous solubility; this could influence the pharmacokinetic
parameters and cause issues in the future development of these
putative drug candidates.9,10
Early assessment of pharmaceutical properties such as
solubility, metabolic stability, and permeability has become a
key step in the drug discovery process11 since it is estimated
Received: February 25, 2013
Accepted: May 20, 2013
Figure 1. Pyrazolo[3,4-d]pyrimidines: general structure A and selected
compounds 1 and 2.
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
that 40% of potential drug candidates fail to reach the market
due to poor physicochemical properties.12,13
In this context, the determination of solubility represents an
important and crucial aspect, indeed a low aqueous solubility
could possibly be responsible for irreproducible and inaccurate
in vitro results.14,15 This is why a coordinated and in-parallel
optimization of biological activity and pharmaceutical proper-
ties, such as water solubility, likely represents a valid tactic to
develop a new potential drug candidate. Herein, the use of
prodrugs, chemically modified versions of the pharmaceutically
active drug, which after undergoing in vivo transformations
release the active drug, is reported. This approach represents a
well established strategy to improve the physicochemical,
biopharmaceutical, or pharmacokinetic properties of potential
drug candidates.16,17
In the present study, a prodrug strategy was planned with the
aim of validating a novel and useful approach to overcome the
lack of aqueous solubility that characterizes the pyrazolo[3,4-
d]pyrimidine class of compounds. Suitable prodrugs of
pyrazolo[3,4-d]pyrimidine compounds, should display im-
proved aqueous solubility in order to enhance pharmacokinetic
properties, avoid problems faced during in vitro assays,9,10 and
facilitate in vivo distribution. Furthermore, the development of
a rapid and versatile synthesis, applicable to a wide range of
previously synthetized final compounds, was an appealing goal.
To fulfill these aims, we first selected the most suitable position
on the pyrazolo[3,4-d]pyrimidine core, on which to affix the
prodrug functionality, and second chose the appropriate
enzymatically cleavable linker and solubilizing moiety. Sticking
to the plan to design the most versatile protocol, the secondary
amine in the C-4 position was selected, as a consequence of
being the moiety that most of the previously synthetized
pyrazolo[3,4-d]pyrimidines shared. Indeed, the NH group on
C-4 position represents an essential feature, able to create
favorable interactions within the ATP-binding site of tyrosine
kinases, as previously demonstrated by structure−activity
relationships (SAR) and computational studies.18 Furthermore,
the high variability of the secondary amino moieties introduced
in C-4 position could allow for the validation of a highly
adaptable synthetic protocol.
A thorough literature search suggested the O-alkyl carbamate
moiety, easily cleavable in vivo by hydrolases, as a viable linker
to connect the secondary amino group at C-4 with the
solubilizing group.19,20
Taken the water solubility issue into account, a N-
methylpiperazino moiety, protonated at physiological pH
(pKa 9.27 0.1)21 and characterized by a high water solubility
(molar solubility 9.98 mol L−1, pH 7), was chosen in order to
increase the water affinity of the resulting prodrugs (7 and 8) in
comparison with the starting drugs (1 and 2). Figure 2 shows
the three final components assembled to give the prodrug: (I)
pyrazolo[3,4-d]pyrimidine core, (II) O-alkyl carbamate linker,
and (III) the solubilizing moiety.
Two representative derivatives (1 and 2), previously
synthetized in our laboratories, were chosen on the basis of
their structural frameworks and biological activities. First, the
different alkylamino or arylamino moieties on C-4 position
(compound 1 and 2, respectively) allowed for good structural
variability; this was essential and required to initiate a highly
adaptable synthesis. Second, they are associated with a
remarkable ability to inhibit c-Abl and c-Src tyrosine kinases
(Table 1).22−25
Figure 2. Prodrug’s components.
Starting from the final pyrazolo[3,4-d]pyrimidine com-
pounds (1 and 2), a classical carbamate synthesis was
performed using triphosgene and sodium bicarbonate in
CH2Cl2.26 The resulting mixture, after filtration and evapo-
ration under reduced pressure, gave the chlorocarbonate
intermediates (3 and 4), which were immediately used in the
next step without further purification. The subsequent addition
of 2-N-methylpiperazinethanol (6), activated by sodium
hydride, resulted in the formation of the desired prodrugs (7
and 8) (Scheme 1). In our laboratories, several in vitro
experiments were initially conducted in order to define some
ADME properties of the newly synthesized prodrugs (7 and 8)
and compare them to those shown by parent drugs (1 and 2).
Furthermore, these studies allowed us to establish the stability
of the O-alkyl carbamate linker introduced into the parent
drugs.
Aqueous solubility, stability in water, methanol, phosphate
buffer (PBS, pH 7.4), and plasma were investigated, in addition
to metabolic stability in presence of human liver microsomes
(HLM) and permeability in parallel artificial membrane
permeability assay (PAMPA). Solubility, stability, and perme-
ability data obtained for the synthetized prodrugs (7 and 8) and
their parent drugs (1 and 2) are shown in Table 1.
Thermodynamic solubility was tested in water by adding 1
mL of H2O to 1 mg of each compound; the resulting mixture
was then shaken at ambient temperature for 24 h. The
concentration of compounds (1, 2, and 8) was determined by
UV/LC−MS, following filtration through a 0.45 μm nylon
filter.27 Prodrug 8, when compared to its parent drug 2, showed
an encouraging enhancement of aqueous solubility. In fact, the
solubility increased from less than 0.01 to 6 μg mL−1, thus
demonstrating that the prodrug approach could be the correct
choice for overcoming the lack of aqueous solubility of these
types of compounds. Solubility of compound 7 could not be
tested by the in vitro assay, due to the fast rate of hydrolysis
shown in aqueous medium; the value reported is calculated
using Discovery Studio 3.0 software.28 Nevertheless, the
predicted solubility is 17.7 μg mL−1, which represents an
improvement compared to the solubility of the parent
compound 2 (0.25 or 0.05 μg mL−1, predicted and
experimental values, respectively). The stability of prodrugs
(7 and 8) was determined by UV/LC−MS in three polar media
in order to evaluate if the contribution of chemical hydrolysis
induced by water, methanol, and PBS to the overall rate of
hydrolysis in subsequent plasma stability test is minimal. A 500
μM solution was prepared by dissolving the compounds (7 and
8) in PBS (12.5 μM, pH 7.4), water, and methanol,
respectively. Twenty microliter aliquots were taken during 48
h (see Supporting Information for details with regards to
B
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Table 1. Characterization of Compounds 1, 2, 7, and 8: Solubility, Stability, and Membrane Permeability
a
Stability
a
ae
PAMPA Papp 10−6 cm sec‑1
,
H2O solubility
H2O
T1/2
PBS pH 7.4
MeOH
T1/2
human plasma
metabolic
ad
af
,
,
compd
μg mL−1
T1/2
T1/2
stability
%
(% MR)
b
c
c
c
c
f
f
1
2
7
8
0.05 (0.25)
ND
ND
ND
ND
91.5
95.1
5.99 (44.9)
0.01 (80.4)
b
c
c
c
c
0.01 (0.02)
ND
ND
ND
ND
b
c
c
(17.7)
30 min
>48 h
63 min
>48 h
125 min
>48 h
28 min
ND
ND
b
f
6.47 (1.97)
193 min
99.9
2.11 (67.3)
a
b
c
Determined by UV/LC−MS. Data represent mean values of at least two experiments. Calculated by Discovery Studio 3.0. Not determined.
d
e
f
Expressed as percentage of unmodified drug. PAMPA; see Supporting Information for experimental details. Membrane retention (MR) expressed
as percentage of compound unable to reach the acceptor compartment.
a
Scheme 1. Synthesis of Prodrugs 7 and 8
a
Reagents and conditions: (i) triphosgene, NaHCO3, CH2Cl2; 3 h, 0 °C to r.t.; (ii) 2-bromoethanol, toluene, r.t., overnight then 80 °C, 2 h; (iii)
NaH, CH2Cl2.
sampling) and analyzed by HPLC. Prodrug 8 displayed a good
stability profile in all polar solutions tested and is thus regarded
as chemically stable for the subsequent evaluation of its
susceptibility to enzymatic hydrolysis. However, the stability of
prodrug 7 was low in all the polar solutions tested (especially in
water, with a half-life of 30 min); this high rate of hydrolysis is
the reason why this compound could not be tested with regards
to its aqueous solubility, metabolic stability, and permeability
(these evaluations require higher water stability, for at least 1 h;
see Supporting Information for experimental details). As
already highlighted, plasma stability plays an important role
in drug discovery and development, especially for groups
susceptible to plasma enzyme hydrolysis, that could give rise to
unstable fragments, which tend to have rapid clearance and a
short half-life, resulting in poor in vivo performance. In order to
understand the behavior of the O-alkyl carbamate linker toward
plasma hydrolysis, an in vitro experiment using pooled human
plasma was designed. The prodrugs 7 and 8 were incubated at
37 °C in human plasma, the disappearance of the substrate and
the consequent formation of 1 and 2, respectively, was
monitored by HPLC analyses (Table 1). In the case of
compound 7, the plasma half-life confirmed the data obtained
by water stability assay (half-life of 28 min) highlighting that
the contribution of solvent-mediated hydrolysis in the cleavage
of prodrug 7 can not be ignored. Compound 8 showed a half-
life in plasma of about three hours, which is a good and
encouraging value to guarantee an appropriate in vivo
distribution.11,29,30 Furthermore, for produg 8 passive mem-
brane permeability could be evaluated performing a PAMPA
assay.31,32 The obtained passive permeability value (2.11 × 10−6
cm s−1) showed a good enhancement with respect to the value
obtained for parent drug 2 (0.01 × 10−6 cm s−1), indicating that
applying the prodrug approach could represent an interesting
way to increase passive membrane permeability. Moreover, the
prodrug strategy permitted to reduce the percentage of
membrane-trapped compound; in fact, prodrug 8 showed a
membrane retention (MR) percentage of 67.3% lower than the
value obtained for parent drug 2 (80.4%).33 In order to
simulate phase I metabolism, compounds 1, 2, and 8 were
incubated at 37 °C for one hour, with 5 μL of man-pooled
HLM. After this time the percentage of drugs and metabolites
was determined by LC−MS analysis: all tested compounds
showed good metabolic stability (higher than 90%). Parent
drug 1, in a previously performed cell-free assay, showed Ki
values of 0.60 and 0.32 μM toward c-Src and c-Abl, respectively,
whereas 2 exhibited a high affinity toward c-Src (Ki value of
0.02 μM) and micromolar activity against c-Abl (1.07 μM)
(Table 2). However, both parent drugs 1 and 2 showed activity
as dual c-Src and c-Abl inhibitors. The newly synthetized
prodrugs 7 and 8 were inactive when tested against both
enzymes since the prodrug concept involves the chemical
modification of a parent drug into a structurally different
molecule with an altered set of pharmaceutical and
pharmacokinetic properties.34 In our example, the introduction
of the highly aqueous soluble N-methylpiperazino group masks
the C-4 secondary amino group needed to create favorable
interactions within the ATP-binding site.18 This is the reason
why the modifications used to design our prodrugs (7 and 8)
C
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ACS Medicinal Chemistry Letters
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(1 and 2) to overcome the lack of good aqueous solubility
shown by this promising class of compounds, which are able to
inhibit c-Src and c-Abl in myeloid cell lines. We developed a
versatile synthesis, which allowed the introduction of a N-
methylpiperazino group linked by an O-alkyl carbamate chain.
The increase of aqueous solubility was demonstrated by in vitro
studies, as well as by the plasma stability and half-life. The
newly synthesized prodrug 8 showed an improved passive
membrane permeability and a lower membrane retention when
compared to parent drug 2. Enzymatic assays confirmed the
expected inactivity of compounds 7 and 8; meanwhile, the
subsequent cellular assay showed a micromolar cytotoxic effect
above the one of the corresponding parent compounds 1 and 2.
The Western blot analysis of produgs (7 and 8), conducted on
murine myeloid cells, showed a reduction of phosphorylated
(active) form of both c-Src and c-Abl, moderately higher than
those associated with parent drugs (1 and 2).
Table 2. Biological Evaluation of Compounds 1, 2, 7, and 8
ab
,
ade
, ,
Ki (μM)
IC50
(SD) (μM)
32D-T315I
compd
c-Src
c-Ablwt
32D-p210
1
2
7
8
0.60
0.02
0.32
1.07
3.5 (0.8)
6.2 (0.8)
1.2 (0.1)
2.8 (1.6)
6.7 (1.2)
5.8 (0.9)
2.4 (0.1)
c
c
NA
NA
c
c
NA
NA
2.6 (0.2)
a
b
Data represent mean values of at least two experiments. Ki values
toward isolated kinases calculated according to eq 2 (see Supporting
c
Information). NA: not active at 100 μM (the highest concentration
d
tested). In vitro experiments conducted with 32D-T315I and 32D-
e
p210 cells. IC50: the half maximal inhibitory concentration of the
effectiveness in reducing the number of viable cells with respect to
untreated cells. SD: standard deviation.
employed an O-alkyl carbamate linker, easily cleavable by
hydrolysis and able to release the parent active compound in
vivo.
The developed prodrug strategy allowed us to obtain
pyrazolo[3,4-d]pyrimidines, endowed with an improved
aqueous solubility profile. This method appears to be a
powerful strategy to overcome the suboptimal pharmacokinetic
properties of this class of compounds and, as consequence,
makes their properties suitable for preclinical studies.
The in vitro cytotoxic effects of the prodrugs 7 and 8 was
then evaluated with a murine myeloid cell line (32D),
expressing the nonmutated Bcr-Abl fusion protein (p210) or
the mutated form (T315I). Cells were treated for 48 h with
increasing concentrations of the inhibitors (0.01−50 μM), and
IC50 values were calculated counting viable cells in comparison
with untreated cells. When compared to the activities of the
parent drugs (1 and 2), both prodrugs (7 and 8) showed an
enhancement of their cytotoxic effects (Table 2).
As demonstrated by the enzymatic assays, prodrugs 7 and 8
were not able to interact directly with c-Src and c-Abl, so the
molecular mechanism of action of these compounds in cells was
still to be elucidated. In order to define whether the activities of
7 and 8 were dependent on the interaction between the
released parent drugs 1 and 2 and tyrosine kinase enzyme c-Src,
Western blot analysis was performed (Figure 3). The treatment
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the synthesis, ADME studies
(solubility, stability, and PAMPA), enzymatic and cellular
assays, and instrument details. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(M.B.) E-mail: botta.maurizio@gmail.com.
Funding
We are grateful to Lead Discovery Siena (LDS), Associazione
Italiana per la Ricerca sul Cancro (AIRC grant IG12084 to
G.M.), Progetti di Ricerca di Interesse Nazionale (PRIN 2010
5YY2HL to S.S. and F.M.), Chemical Approaches to Targeting
Drug Resistance in Cancer Stem Cells (CM1106), and Istituto
Toscano Tumori (ITT grant proposal 2010).
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Arora, A.; Scholar, E. M. Role of tyrosine kinase inhibitors in
cancer therapy. J. Pharm. Exp. Ther. 2005, 315, 971−979.
(2) Crespan, E.; Radi, M.; Zanoli, S.; Schenone, S.; Botta, M.; Maga,
G. Dual Src and Abl inhibitors target wild type Abl and the Abl T315I
Imatinib-resistant mutant with different mechanisms. Bioorg. Med.
Chem. 2010, 18, 3999−4008.
Figure 3. Western blot analysis of Src, Abl, and their respective
phosphorylated active forms in murine myeloid cell line 32D-p210.
Cells were treated with 1 μM parental compounds (1 and 2) and
prodrugs (7 and 8). Dasatinib was added asa reference dual inhibitor
compound (ref) and Beta-actin (b-actin) as loading control.
(3) Radi, M.; Dreassi, E.; Brullo, C.; Crespan, E.; Tintori, C.;
Bernardo, V.; Valoti, M.; Zamperini, C.; Daigl, H.; Musumeci, F.;
Carraro, F.; Naldini, A.; Filippi, I.; Maga, G.; Schenone, S.; Botta, M.
Design, Synthesis, Biological Activity, and ADME Properties of
Pyrazolo[3,4-d]pyrimidines Active in Hypoxic Human Leukemia
Cells: A Lead Optimization Study. J. Med. Chem. 2011, 54, 2610−
2626.
(4) Carraro, F.; Pucci, A.; Naldini, A.; Schenone, S.; Bruno, O.;
Ranise, A.; Bondavalli, F.; Brullo, C.; Fossa, P.; Menozzi, G.; Mosti, L.;
Manetti, F.; Botta, M. Pyrazolo[3,4-d]pyrimidines endowed with
antiproliferative activity on ductal infiltrating carcinoma cells. J. Med.
Chem. 2004, 47, 1595−1598.
of myeloid cells with prodrugs 7 and 8 caused an effective
reduction of the active phosphorylated form of c-Src and c-Abl,
suggesting that parent compounds were readily available in the
cell cytoplasm. However, in order to clarify the potential of this
therapeutic strategy, further studies are needed, mainly to verify
if the inhibition of target tyrosine kinases is obtained by a direct
or indirect mechanism.
In conclusion, in the present work we employed a prodrug
approach on previously synthetized pyrazolo[3,4-d]pyrimidines
D
dx.doi.org/10.1021/ml4000782 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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Letter
(5) Carraro, F.; Naldini, A.; Pucci, A.; Locatelli, G. A.; Maga, G.;
Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Brullo, C.; Fossa,
P.; Menozzi, G.; Mosti, L.; Modugno, M.; Tintori, C.; Manetti, F.;
Botta, M. Pyrazolo[3,4-d]pyrimidines as potent antiproliferative and
proapoptotic agents toward A431 and 8701-BC cells in culture via
inhibition of c-Src phosphorylation. J. Med. Chem. 2006, 49, 1549−
1561.
(6) Manetti, F.; Santucci, A.; Locatelli, G. A.; Maga, G.; Spreafico, A.;
Serchi, T.; Orlandini, M.; Bernardini, G.; Caradonna, N. P.;
Spallarossa, A.; Brullo, C.; Schenone, S.; Bruno, O.; Ranise, A.;
Bondavalli, F.; Hoffmann, O.; Bologna, M.; Angelucci, A.; Botta, M.
Identification of a novel pyrazolo[3,4-d]pyrimidine able to inhibit cell
proliferation of a human osteogenic sarcoma in vitro and in a xenograft
model in mice. J. Med. Chem. 2007, 50, 5579−5588.
(7) Angelucci, A.; Schenone, S.; Gravina, G. L.; Muzi, P.; Festuccia,
C.; Vicentini, C.; Botta, M.; Bologna, M. Pyrazolo[3,4-d]pyrimidines c-
Src inhibitors reduce epidermal growth factor-induced migration in
prostate cancer cells. Eur. J. Cancer 2006, 42, 2838−2845.
(8) Manetti, F.; Pucci, A.; Magnani, M.; Locatelli, G. A.; Brullo, C.;
Naldini, A.; Schenone, S.; Maga, G.; Carraro, F.; Botta, M. Inhibition
of Bcr-Abl phosphorylation and induction of apoptosis by
pyrazolopyrimidine in human leukemia cells. ChemMedChem 2007,
2, 343−353.
(9) Dreassi, E.; Zizzari, A. T.; Mori, M.; Filippi, I.; Belfiore, A.;
Naldini, A.; Carraro, F.; Santucci, A.; Schenone, S.; Botta, M. 2-
Hydroxypropyl-β-cyclodextrin strongly improves water solubility and
anti-proliferative activity of pyrazolo[3,4-d]pyrimidines Src-Abl dual
inhibitors. Eur. J. Med. Chem. 2010, 45, 5958−5964.
(21) Bjerrum, J.; Schwarzenbach, G.; Sillen, L. G. Stability Constants.
Part I: Organic Ligands; Chemical Society Special Publication No. 6;
The Chemical Society: London, U.K., 1957.
(22) Falchi, F.; Manetti, F.; Carraro, F.; Naldini, A.; Maga, G.;
Crespan, E.; Schenone, S.; Bruno, O.; Brullo, C.; Botta, M. 3D QSAR
models built on structure-based alignments of Abl tyrosine kinase
inhibitors. ChemMedChem 2009, 4, 976−987.
(23) Schenone, S.; Brullo, C.; Bruno, O.; Bondavalli, F.; Mosti, L.;
Maga, G.; Crespan, E.; Carraro, F.; Manetti, F.; Tintori, C.; Botta, M.
Synthesis, biological evaluation and docking studies of 4-amino
substituted 1H-pyrazolo[3,4-d]pyrimidines. Eur. J. Med. Chem. 2008,
43, 2665−2676.
(24) Santucci, M. A.; Corradi, V.; Mancini, M.; Manetti, F.; Radi, M.;
Schenone, S.; Botta, M. C6-unsubstituted pyrazolo[3,4-d]pyrimidines
are dual Src/Abl inhibitors effective against imatinib mesylate resistant
chronic myeloid leukemia cell lines. ChemMedChem 2009, 4, 118−126.
(25) Radi, M.; Brullo, C.; Crespan, E.; Tintori, C.; Musumeci, F.;
Biava, M.; Schenone, S.; Dreassi, E.; Zamperini, C.; Maga, G.; Pagano,
D.; Angelucci, A.; Bologna, M.; Botta, M. Identification of potent c-Src
inhibitors strongly affecting the proliferation of human neuroblastoma
cells. Bioorg. Med. Chem. Lett. 2011, 21, 5928−5933.
́
́
chez, J.; Rodrlguez-Mata, M.; Busto, E.; Gotor-
(26) Mangas-San
Fernandez, V.; Gotor, V. Chemoenzymatic synthesis of rivastigmine
́
based on lipase-catalyzed processes. J. Org. Chem. 2009, 74, 5304−
5310.
(27) Avdeef, A. High-throughput Measurements of Solubility
Profiles. In Pharmacokinetic Optimization in Drug Research: Biological,
Physicochemical, and Computational Strategies; Wiley: Lausanne,
Switzerland, 2001; pp 305−325.
(28) Accelerys Software Inc. Discovery Studio Modeling Environment
Release 2.5; Accelerys: San Diego, CA.
(10) Radi, M.; Evensen, L.; Dreassi, E.; Zamperini, C.; Caporicci, M.;
Falchi, F.; Musumeci, F.; Schenone, S.; Lorens, J. B.; Botta, M. A
combined targeted/phenotypic approach for the identification of new
antiangiogenics agents active on a zebrafish model: from in silico
screening to cyclodextrin formulation. Bioorg. Med. Chem. Lett. 2012,
22, 5579−5583.
̀
(29) Simoes, M. F.; Valente, E.; Gomez, M. J. R.; Anes, E.;
Constantino, L. Lipophilic pyrazinoic acid amide and ester prodrugs:
Stability, activation and activity against M. tuberculosis. Eur. J. Pharm.
Sci. 2009, 37, 257−263.
(11) Kerns, E. H.; Di, L. Pharmaceutical profiling in drug discovery.
Drug Discovery Today 2003, 8, 316−323.
(30) Wang, Y. J.; Pan, M. H.; Cheng, A. L.; Lin, L. I.; Ho, Y. S.;
Hsieh, C. Y.; Lin, J. K. Stability of curcumin in buffer solutions and
characterization of its degradation products. J. Pharm. Biomed. Anal.
1997, 15, 1867−1876.
(12) Di, L.; Kerns, E. H. Profiling drug-like properties in discovery
research. Curr. Opin. Chem. Biol. 2003, 7, 402−408.
(13) Lipinski, C. A. Drug-like properties and the causes of poor
solubility and poor permeability. J. Pharmacol. Toxicol. Methods 2000,
44, 235−249.
(31) Wohnsland, F.; Faller, B. High-throughput permeability pH
profile and high-throughput alkane/water log P with artificial
membranes. J. Med. Chem. 2001, 44, 923−930.
(14) Alsenz, J.; Kansy, M. High throughput solubility measurement in
drug discovery and development. Adv. Drug Delivery Rev. 2007, 59,
546−567.
(32) Sugano, K.; Hamada, H.; Machida, M.; Ushio, H. High
throughput prediction of oral absorption: improvement of the
composition of the lipid solution used in parallel artificial membrane
permeation assay. J. Biomol. Screening 2001, 6, 189−196.
(33) Avdeef, A.; Strafford, M.; Block, E.; Balogh, M. P.; Chambliss,
W.; Khan, I. Drug absorption in vitro model: filter-immobilized
artificial membranes. 2. Studies of the permeability properties of
lactones in Piper methysticum Forst. Eur. J. Pharm. Sci. 2001, 14, 271−
280.
(15) Popa-Burke, I. G.; Issakova, O.; Arroway, J. D.; Bernasconi, P.;
Chen, M.; Coudurier, L.; Galasinski, S.; Jadhav, A. P.; Janzen, W. P.;
Lagasca, D.; Liu, D.; Lewis, R. S.; Mohney, R. P.; Sepetov, N.;
Sparkman, D. A.; Hodge, C. N. Streamlined system for purifying and
quantifying a diverse library of compounds and the effect of compound
concentration measurements on the accurate interpretation of
biological assay results. Anal. Chem. 2004, 76, 7278−7287.
(16) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.;
(34) Rautio, J., Ed. Prodrugs and Targeted Delivery: Towards Better
ADME Properties; Wiley-VCH: Berlin, Germany, 2011.
Jarvinen, T.; Savolainen, J. Prodrugs: design and clinical applications.
̈
Nat. Rev. 2008, 7, 255−270.
(17) Huttunen, K. M.; Raunio, H.; Rautio, J. Prodrugs-from
Serendipity to Rational Design. Pharmacol. Rev. 2011, 63, 750−771.
(18) Tintori, C.; Magnani, M.; Schenone, S.; Botta, M. Docking, 3D
QSAR studies and in silico ADME prediction on c-Src tyrosine kinase
inhibitors. Eur. J. Med. Chem. 2009, 44, 990−1000.
(19) Persson, G.; Pahlm, O.; Gnosspelius, Y. Oral bambuterol versus
terbutaline in patients with asthma. Curr. Ther. Res. 1995, 56, 457−
465.
(20) Slatter, J. G.; Schaaf, L. J.; Sams, J. P.; Feenstra, K. L.; Johnson,
M. G.; Bombart, P. A.; Cathcart, K. S.; Verbug, M. T.; Pearson, L. K.;
Compton, L. D.; Miller, L. L.; Baker, D. S.; Pesheck, C. V.; Lord, R. S.
Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11)
following I.V. infusion of [14C]CPT-11 in cancer patients. Drug Metab.
Dispos. 2000, 28, 423−433.
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