Exploring the chemical space around the privileged pyrazolo[3,4-d] pyrimidine scaffold: Toward novel allosteric inhibitors of T315I-mutated Abl

Article abstract of DOI:10.1021/co500004e

A library of pyrazolo[3,4-d]pyrimidines, endowed with a high level of molecular diversity, has been developed applying a synthetic sequence that allowed C3, N1, C4, and C6 substitution. The enzymatic screening of this "privileged scaffold"-based compound collection, validated the use of a diversity-oriented approach in a field characteristically explored by target-oriented synthesis. In fact, several compounds showed high activity against the selected kinases (i.e., Src, Abl wt, and T315I mutated-form), furthermore and interestingly a new compound has emerged as an allosteric inhibitor of the T315I mutated-form of Abl, opening up new opportunities for the development of a novel class of noncompetitive inhibitors of Abl (T315I).

Full text of DOI:10.1021/co500004e

Research Article
pubs.acs.org/acscombsci
Exploring the Chemical Space around the Privileged
Pyrazolo[3,4‑d]pyrimidine Scaffold: Toward Novel Allosteric
Inhibitors of T315I-Mutated Abl
Giulia Vignaroli,† Martina Mencarelli,† Deborah Sementa,† Emmanuele Crespan,‡ Miroslava Kissova,‡
Giovanni Maga,‡ Silvia Schenone,§ Marco Radi,†,∥ and Maurizio Botta*,†,⊥
†Dipartimento di Biotecnologie, Chimica e Farmacia, Universita
‡Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
§Dipartimento di Scienza Farmaceutiche, Universita
degli Studi di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
∥Dipartimento di Farmacia, Universita
degli Studi di Parma, Viale delle Scienze 27/A, 43124 Parma, Italy
̀
degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, 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: A library of pyrazolo[3,4-d]pyrimidines, endowed with a high level of
molecular diversity, has been developed applying a synthetic sequence that allowed C3,
N1, C4, and C6 substitution. The enzymatic screening of this “privileged scaffold”-based
compound collection, validated the use of a diversity-oriented approach in a field
characteristically explored by target-oriented synthesis. In fact, several compounds
showed high activity against the selected kinases (i.e., Src, Abl wt, and T315I mutated-
form), furthermore and interestingly a new compound has emerged as an allosteric
inhibitor of the T315I mutated-form of Abl, opening up new opportunities for the
development of a novel class of noncompetitive inhibitors of Abl (T315I).
KEYWORDS: pyrazolo[3,4-d]pyrimidine, diversity-oriented synthesis, dual Src/Abl inhibitors, Abl T315I, allosteric inhibitors
amino-substituted pyrazolo[3,4-d]pyrimidines, has been
achieved (Figure 1).13
INTRODUCTION
■
The pyrazolo[3,4-d]pyrimidine nucleus represents an interest-
ing scaffold for the development of drug-candidates aiming at a
wide range of biological targets1,2 and has been exploited in the
development of kinase inhibitors,3,4 antiviral agents,5,6
adenosine antagonists,7,9 glutamate modulators,10 and anti-
tubercular agents.11 Because of its structure, isosteric to that of
purine, this nitrogen-containing heterocycle behaves as a
“privileged” scaffold,12 acting in antagonism with natural
purines and showing different specificity for the biological
targets depending on its chemical functionalization. In this
context, our research group has long focused on the design and
synthesis of 4-amino-substituted pyrazolo[3,4-d]pyrimidines
active on different oncogenic tyrosine kinases and cancer cell
lines depending on the nature and position of substituents on
the heterocyclic core.13−22
Recent literature showed how approaches directed toward
the creation of libraries endowed with high chemical variability
(such as combinatorial chemistry,23 diversity-oriented synthesis
(DOS),24 biology-oriented synthesis (BIOS)25) could lead to
interesting and unexpected biological results.
Fascinated by the potential results achievable by applying this
kind of approach to the pyrazolo[3,4-d]pyrimidine privileged
scaffold, in the present work we focused on the development of
a divergent synthetic strategy for the construction of a highly
functionalized library of pyrazolo[3,4-d]pyrimidines to be
tested against oncogenic kinases.
Among the different synthetic strategies commonly used for
the synthesis of functionalized pyrazolo[3,4-d]pyrimidines, the
most used are those starting from the pyrimidine or,
alternatively, the pyrazole core.26 In our research group, these
compounds have been synthesized starting from commercial
Our target-based approach, aimed toward the improvement
of the tyrosine kinase inhibitory activity, always considered the
computational analysis of the target-inhibitor complexes to
design and synthesize compounds with improved affinity via
point-modification of the pyrazolo[3,4-d]pyrimidine scaffold.
On the basis of this approach, the synthesis of several active 4-
Received: January 14, 2014
Revised: March 2, 2014
Published: March 5, 2014
© 2014 American Chemical Society
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Figure 1. Selected 4-amino-substituted pyrazolo[3,4-d]pyrimidines synthesized in our laboratories, active toward different tyrosine kinases (RET,
Src, Hck, Abl wt, and T315I mutated-form).
phenyloxiranes (A), following the route to construct the
pyrazole core (B)14−22 or, alternatively, from allopurinol
(D)13e,27 (Figure 2, routes a and b, respectively). However,
the main drawback of the approach exemplified in route a is
represented by its poor versatility in developing N1 substituted
analogues: the chemical diversity in the N1 side chain is in fact
limited by the commercial availability and substitution of the
starting phenyloxiranes (A). Recently, we proposed a more
versatile approach that allowed obtaining C6 unsubstituted
compounds, based on the use of allopurinol (D) as the starting
material. Although this strategy allowed to generate different
1,4-disubstituted pyrazolo[3,4-d]pyrimidines, C6 substituted
compounds cannot be obtained, partially limiting its versatility
(Figure 2, route b). In the present work, we focused on the
development of a divergent synthetic approach (Figure 2, route
c) for the generation of highly functionalized pyrazolo[3,4-
d]pyrimidines: substitution of C3 and C6 position on the core,
plus the diversification in the N1 and C4 position was obtained,
giving access to a library of 4-amino substituted pyrazolo[3,4-
d]pyrimidines, characterized by a high level of molecular
diversity.
The synthesis and the subsequent biological evaluation of
this diversified collection have been performed with the aim of
understanding if a diversity-oriented approach based on a
privileged scaffold could quickly lead to interesting and
promising hit compounds in a field characteristically explored
Figure 2. Three different routes developed in our laboratories for the
by the more elaborate target-oriented approach. The obtained
synthesis of pyrazolo[3,4-d]pyrimidines.
results proved valid the application of this strategy to the
privileged pyrazolo[3,4-d]pyrimidine nucleus. The synthesized
compounds were in fact screened in cell-free assays to identify
novel compounds able to inhibit selected tyrosine kinases (Src,
Abl wild type and T315I mutated-form) involved in the
development and progression of tumors, such as chronic
myeloid leukemia (CML). As a result, potent inhibitors of the
selected kinases were identified and, more interestingly, a novel
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a
Scheme 1. Synthetic Procedure for 4-Amino-Substituted Pyrazolo[3,4-d]pyrimidines
a
Reagents and conditions: (i) KOH, MeI, H2O, reflux, 3 h; (ii) POCl3, DMF, reflux, 12 h; (iii) TEA, N2H4·H2O, dioxane, rt, 5 h; (iv) POCl3, reflux,
12 h; (v) LDA, −78 °C, anhydrous THF, PhCHO; (vi) MnO2, toluene, 1 h, Dean−Stark trap; (vii) TEA, N2H4·H2O, dioxane, rt, 1 h; (viii) R2OH,
Ph3P, DIAD, anhydrous THF, μW 100 °C, 3 min; (ix) amine, EtOH, reflux, 12 h; (x) m-CPBA, DCM, rt, 2 h, then nucleophile, THF, 40 °C, 12 h;
(xi) H2, Pd(OH)2/C, MeOH, 2 h; (xii) BnCl, NaN3, CuSO4·5H2O, sodium ascorbate, μW 125 °C, 10 min.
family of N1 substituted 1,2,3-triazol-4-yl derivatives turned out
to act as allosteric inhibitors against T315I-mutated Abl, which
confers resistance to the drugs currently available for CML
treatment.28
pathway that could allow a sequential functionalization of the
C3, N1, C4, and C6 position of this core, enabling the synthesis
of a wide variety of compounds, in a timely fashion, with easy
purification, from cheap and commercially available starting
materials, frequently using well-known and solid procedures
(Scheme 1). This approach showed higher versatility in the
decoration of the N1 position and gave the chance of accessing
RESULTS AND DISCUSSION
■
When planning the development of this collection of
pyrazolo[3,4-d]pyrimidines, our aim was to generate a synthetic
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Table 1. Structure and in Vitro Src, Abl wt, and T315I-Mutated Form Kinase Inhibitory Activities of Compounds 9a−p, 10a−g,
11a−c, and 13a−c
a
Ki [μM]
R1
R2
CH2CH2C6H5
R3
R4
Abl wt
>100
Abl T315I
d
Src
0.09
9a
9b
9c
9d
9e
9f
H
NHC6H4mCl
NHcyclohexyl
NHC6H4mOH
NHC6H5
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
ND
H
H
H
H
H
H
H
H
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
CH2C6H5
1.45
0.003
0.01
6.84
1.90
0.92
0.01
5.1
0.036
1.89
0.178
0.056
0.165
1.354
4.32
NHC6H4pOCF3
NHcyclohexyl
NHC6H4mOH
NHC6H4mOH
0.552
0.145
0.013
0.462
9g
9h
9i
CH2C6H5
0.15
1.23
5.52
CH2CH(OMe)C6H5
CH2CH(OMe)C6H5
NHCH24-
pyridine
>100
>100
9j
H
H
H
H
CH2CH(OMe)C6H5
CH2CCH
NHC6H4pOCF3
NHC6H4mOH
N(Me)2-pyridine
NHC6H4pOCF3
NHMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
NHnPr
1.047
0.92
0.51
>100
0.066
>100
0.074
0.56
9k
>100
0.24
>100
9l
CH2CCH
1.375
d
9m
9n
CH2CCH
N.D.
C6H5 CH2CH2C6H5
0.077
0.004
0.444
0.019
0.49
1.95
1.416
0.18
9o
C6H5 CH2CH(OMe)C6H5
C6H5 CH2CH(OMe)C6H5
NHMe
0.006
0.028
0.004
0.18
9p
NH2
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
13a
H
H
H
H
H
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
NHC6H4mCl
NHC6H4mCl
NHcyclohexyl
NHC6H4pOCF3
NHC6H4mCl
NHMe
0.04
NHCH2CH2morpholine
0.75
OCH2CH2morpholine
1.42
0.2
0.47
NHnPr
NHMe
NHMe
NHMe
H
0.06
0.02
0.701
1.07
0.003
0.122
0.124
0.004
0.047
C6H5 CH2CH2C6H5
2.426
10.437
0.48
C6H5 CH2CH(OMe)C6H5
NHMe
0.022
d
H
H
CH2CH2C6H5
CH2CH2C6H5
NHC6H4mCl
NHC6H4pOCF3
NH2
>100
ND
H
0.105
0.037
>100
C6H5 CH2CH(OMe)C6H5
H
0.26
0.562
1.113
b
b
b
b
,
c
c
b
H
H
H
1-benzyl-1H-1,2,3-triazol-4-yl) NHC6H4mOH
methyl
SMe
0.29 (14.7)
0.067 (3.16)
9.48 (151.7)
b
,
c
b
13b
13c
1-benzyl-1H-1,2,3-triazol-4-yl) N(Me)2-pyridine
methyl
SMe
SMe
0.29 (33.4)
0.67 (14.7)
0.41 (3.63)
1.19 (35.0)
b,
b
1-benzyl-1H-1,2,3-triazol-4-yl) NHC6H4pOCF3
methyl
0.077 (8.66)
2.19 (19.0)
a
Data represent the mean of three independent experiments. Standard deviation (SD) was lower than 5%. Ki values toward isolated kinases
b
c
calculated according to the eq 2 (see Supporting Information). ID50 values are reported in parentheses. ID50 values are not dependent on the
concentration of ATP and the peptide substrate. ND: not determined
d
C6 and C3 substituted and unsubstituted compounds from the
same starting material.
acidification with HCl 1 N allowed the recovery of pure 2 as a
white precipitate.29
Compounds 4 and 7, differing only in the functionalization
of C3, represent the key intermediates, which allowed creating a
wide range of derivatives through a sequence of chemical
modifications. First, the R2 at the N1 position can be
introduced through N-alkylation or Mitsunobu reaction, using
either alcohols or halogenated alkyl-chains; second, the chlorine
on the C4 position can be displaced by various nucleophiles to
introduce different R3 substituents. Third, the thiomethyl
moiety on C6 can be converted, via oxidation, in a leaving
group that can be easily displaced by different nucleophiles to
introduce various substituent (R4) in C6 position.
S-methyl thiobarbituric acid (2), was treated with the in situ
generated Vilsmeier reagent to give 4,6-dichloro-2-
(methylthio)pyrimidine-5-carbaldehyde (3a)30 or alternatively
with phosphorus(V) oxychloride to obtain the 4,6-dichloro-
derivative 3b.31 4,6-Dichloro-2-methylsulfanyl-pyrimidine-5-
carbaldehyde (3a) gave access to C3 unsubstituted compounds,
whereas 4,6-dichloro-2-methylsulfanyl-pyrimidine (3b) was
used for the synthesis of C3 substituted pyrazole[3,4-
d]pyrimidines. 4,6-Dichloro-2-(methylthio)pyrimidine-5-car-
baldehyde (3a) was reacted with hydrazine monohydrate in
dioxane, using triethylamine as base to give the C3
unsubstituted pyrazole[3,4-d]pyrimidine core (4) in quantita-
tive yield.32
The planned synthetic approach started from cheap, stable
and commercially available thiobarbituric acid (1). As first step,
an alkylation of the thiol moiety was performed using methyl
iodide in aqueous media with potassium hydroxide as base,
4,6-Dichloro-2-methylsulfanyl-pyrimidine (3b), characterized
by an acid proton ortho to the chloro substituents, was treated
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Figure 3. Kinetic analysis of kinase reactions of T315I-mutated Abl in the presence of different concentration of compound 13a (0, 0.8, 2.0, and 4.0
μM). (a) Variation of the reaction velocity of T315I-mutated Abl as a function of ATP concentration at different fixed concentrations of compound
13a. (b) Variation of the reaction velocity of T315I-mutated Abl as a function of peptide substrate concentration at different fixed concentrations of
compound 13a. (c) Effect of compound 13a at various concentrations (0−4 μM) on the Km and Vmax values.
with LDA at −78 °C to generate the nucleophile able to react
with aromatic aldehydes, such as benzaldehyde, giving the
secondary alcohol. The alcohol 5 was oxidized to the
corresponding ketone 6 using manganese dioxide in toluene;
the subsequent reaction with hydrazine gave access to the C3
substituted pyrazole[3,4-d]pyrimidine core (7).33
The pyrazolo[3,4-d]pyrimidine nucleus of 4 and 7 was
substituted at the N1 position using a microwave irradiated
Mitsunobu reaction, using commercially available alcohols
(such as 2-phenyl- and 2-methoxy-2-phenyl-ethanol, benzyl-
and propargyl alcohol). This approach allowed us to obtain a
set of compounds (8a−f), with short reaction times and
moderate to excellent yields, introducing different R2
substituents. In the next step, C4 functionalization was achieved
by nucleophilic substitution of the chlorine with primary and
secondary amines in order to obtain a first set of potentially
active pyrazolo[3,4-d]pyrimidines (9a−p).
To introduce additional variability in the C6 position, we
performed an oxidation of the thiomethyl moiety of
compounds 9a, b, e, n, and o using m-chloroperbenzoic acid
in dichloromethane to obtain a mixture of sulfone and
sulfoxide. The latter mixtures were then treated with
nucleophiles such as n-propylamine, 2-morpholinoethanol, 4-
(2-aminoethyl)morpholine and methylamine to obtain deriva-
tives 10a−g.32
In addition, starting from S-methyl-substituted compounds
9a, e, and p, the C6 unsubstituted products 11a−c were
obtained by applying a hydrogenation protocol using palladium
hydroxide as catalyst in methanol. To add a wider variability
regarding the N1 substitution, thus enhancing the structural
novelty of this diversity-oriented library with comparison to the
previously synthesized ones, we decided to perform a Huisgen
reaction on the alkyne chain of compound 9k−m, to synthesize
a novel family of 1,2,3-triazol-4-yl derivatives 13a−c (Scheme
1). Benzylazide, generated in situ from benzylchloride and
sodium azide, was reacted with the propargylic moiety of
compound 9k−m using CuSO4·5H2O and sodium ascorbate as
catalyst under microwave-assisted conditions to give com-
pounds 13a−c in high yields after only 10 min.34 To
demonstrate the versatility of the click reaction on our
pyrazole[3,4-d]pyrimidine compounds, the same reaction was
also performed on the C4-unfunctionalized compound 8d to
give the triazolo intermediate 12, finally converted into 13a−c
by nucleophilic aromatic substitution with different amines.
Finally, the affinity of the synthesized library toward Src and
Abl (wt and T315I mutated-form) was evaluated in cell-free
assays. Many of the compounds showed interesting dual Src/
Abl activity, with a general micromolar or nanomolar potency,
comparable or higher to that of the analogues we previously
obtained via target-oriented approaches (9a and 10a).13a
Compound 9b showed Ki value of 3 nM, against Abl T315I
mutated-form. The C3 phenyl derivative 9o, exhibited a 4 nM
Ki value against Abl wt and a 6 nM toward T315I mutated-
form. Compound 10e characterized by an N-methylamino C4
moiety showed Ki values of 3 and 4 nM against Abl wt and
T315I mutated-form, respectively (Table 1). In analogy with
previously reported results13e and with the data of several
compounds (9−11) reported in Table 1, also the triazolo
derivatives 13a−c showed a better affinity toward the T315I
mutated-form of Abl with respect to the wild type form.
Considering also to the peculiar structure of compounds
13a−c, we decided to investigate the mechanism of action of
these derivatives in the inhibition of Abl T315I. The most
active compound 13a, was chosen for kinetic studies in order to
elucidate the mechanism of inhibition toward T315I Abl.
Surprisingly, the obtained data revealed that compound 13a
inhibits the Abl T315I with a noncompetitive kinetic with
respect to both the ATP and the peptide substrate, thus
representing an allosteric inhibitor of the T315I Abl mutated-
form (Figure 3). Furthermore, we can suggest that the
structurally and activity related compounds 13b and c act
with similar kinetics; as a consequence these two compounds
(13b and c), together with 13a, could represent a novel class of
so far unexplored allosteric T315I Abl inhibitors whose target is
currently under study.
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was added to the mixture, followed by extraction with EtOAc
(3 × 20 mL). The collected organic phases were dried over
Na2SO4, filtered and the solvent removed under reduced
pressure; this afforded the desired product that was used in the
next step without further purification.
General Procedure 2 (GP2): 8a−f. To an ice-cooled
solution of Ph3P (0.15 mmol, 1.50 equiv) and 4-chloro-6-
(methylthio)-1H-pyrazolo[3,4-d]pyrimidine (4 and 7) (0.10
mmol, 1.00 equiv) in anhydrous THF (2 mL) was added the
appropriate alcohol (0.15 mmol, 1.50 equiv), followed by
dropwise addition of DIAD (0.15 mmol, 1.50 equiv). The
reaction mixture was stirred at 4 °C for 15 min then allowed to
warm to rt. The reaction was irradiated under μW at 100 °C for
3 min; after evaporation of the solvent the oily residue obtained
was purified by flash chromatography using a PE/EtOAc
mixture as eluent.
General Procedure 3 (GP3): 9a−p and 13a−c. The
desired amine (0.45 mmol, 5.00 equiv) was added to a solution
of the appropriate 1-N-substitued 4-chloro-6-(methylthio)-1H-
pyrazolo[3,4-d]pyrimidine (8a−f and 12) (0.15 mmol, 1.00
equiv) in EtOH (2 mL). The reaction solution was stirred
under reflux, until the starting material spot disappeared from
TLC. The solvent was removed under reduced pressure to
obtain the reaction crude, that was purified as described in each
section.
General Procedure 4 (GP4): 5. To a solution of 4,6-
dichloro-2-(methylthio)pyrimidine (3b) (150.0 mg, 0.77 mmol,
1.00 equiv) in anhydrous THF (2 mL) at −78 °C was added
dropwise 2 N solution of LDA (1.39 mmol, 1.8 equiv). The
mixture was stirred at the same temperature for 30 min then
the aldehyde (1.16 mmol, 1.5 equiv) in anhydrous THF (2 mL)
was added dropwise at r.t and the resulting mixture was stirred
for 1 h. NH4Cl s.s. was added and the aqueous phase was
extracted two times with EtOAc. The collected organic phases
were dried over Na2SO4, filtered and the solvent evaporated
under reduced pressure. The oily residue obtained was purified
by flash chromatography, using PE/EtOAc = 98:2 as eluent.
General Procedure 5 (GP5): 6. In a flask equipped with a
Dean−Stark trap, a mixture of the secondary alcohol (5) (0.50
mmol, 1.00 equiv), toluene, and MnO2 was heated to boiling
for 1 h. The mixture was filtered on Celite using EtOAc. The
organic phase was dried over Na2SO4, filtered and the solvent
evaporated under reduced pressure to obtain the product used
without further purification in the next step.
CONCLUSION
■
Focusing on the pyrazolo[3,4-d]pyrimidine scaffold, we built a
diversity-oriented compound collection around this molecular
framework by decorating the core in four different positions.
The developed synthetic pathway allowed for C3, N1, C4, and
C6 substitution, starting from cheap and stable starting material
(thiobarbituric acid, 1). On the basis of this approach, we have
discovered potent inhibitors of three tyrosine kinases (Src and
Abl wt and T315I mutated-form) whose activity is comparable
or higher to that of the analogues we previously obtained via
target-oriented approaches. More significantly, the same study
opened up new opportunities for the development of a novel
class of noncompetitive inhibitors of Abl T315I mutated-form,
which proved to act with an allosteric mechanism and with a
higher affinity for this mutated kinase compared to the Abl wt.
The 1,2,3-triazol-4-yl moiety, that characterizes the N1 position
of this family, represents the structural point of novelty in
comparison to the previously synthesized derivatives. These
results demonstrated that a diversity-oriented approach focused
on a “privileged” scaffold, might be a resourceful method for the
development of new drug candidates. In fact, although the field
of tyrosine kinases, which represent well-characterized targets,
is typically faced by a targeted-oriented approach, our work
proved the validity of a diversity-oriented method for the study
of these known targets. Additional studies to assess structural−
activity relationships regarding selectivity toward different
kinases and mechanisms of action are currently ongoing in
our lab and will be reported in due course.
EXPERIMENTAL PROCEDURES
■
General Information. All commercially available chemicals
were used as supplied without further purification. THF was
dried over Na/benzophenone prior to use, POCl3 was freshly
distilled, while DMF was bought already anhydrous. Anhydrous
reactions were run under a positive pressure of dry Argon.
Microwave irradiation experiments were conducted using a
CEM Discover Synthesis Unit (CEM Corp., Matthews, NC).
The machine consists of a continuous focused microwave
power delivery system with operator-selectable power output
from 0 to 300 W. The temperature of the contents of the
vessels was monitored using a calibrated infrared temperature
control located under the reaction vessel.
Merck precoated TLC plates (silica gel 60 F254, 0.25 mm)
were used. UV detection was monitored at 254 nm.
Chromatographic purifications were performed on columns
packed with Merk 60 silica gel, 23−400 mesh, for flash
technique. 1H NMR and 13C NMR spectra were obtained using
a Bruker Avance DPX400 (Bruker Biospin, Germany).
Chemical shifts were reported in parts per million (ppm)
from tetramethylsilane as internal standard. Multiplicities were
indicated as follow: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), dd (doublet of doublet), dt (doublet
of triplet), td (triplet of doublet), bs (broad singlet). Coupling
constants were reported in hertz (Hz). LC-MS was performed
with the UV detection at 254 nm and a single quadrupole mass
spectrometer using electrospray ionization (ESI) source.
General Procedure 1 (GP1): 4 and 7. To an ice-cooled
solution of the appropriate aldehyde or ketone (3a or 6) (0.45
mmol, 1.00 equiv) and TEA (0.45 mmol, 1.00 equiv) in
dioxane was added N2H4·H20 (0.50 mmol, 1.10 equiv). After 5
min the ice bath was removed and the reaction mixture was
stirred 5 h at rt. After this time, 10 mL of 0.5 N HCl solution
General Procedure 6 (GP6): 10a−g. m-CPBA (0.13
mmol, 2.10 equiv) was added to a solution of the appropriate 6-
(methylthio)-pyrazolo[3,4-d]pyrimidin-4-amine (9a, b, e, n,
and o) (0.06 mmol, 1.00 equiv) in DCM. The reaction mixture
was stirred at rt for 2 h, the solvent was evaporated under
reduced pressure. The white solid residue was dissolved in
EtOAc, the organic phase was washed twice with NaHCO3 s.s.,
then dried over Na2SO4, filtered and the solvent removed under
reduced pressure to obtain an orange oily residue (sulfone and
sulfoxide), which was solubilized in THF. The desired
nucleophile (0.30 mmol, 5.00 equiv) was added and the
reaction mixture was stirred at 40 °C o.n. The reaction crude,
obtained after evaporation of the solvent, was purified by flash
chromatography using Hex/EtOAc = 2:1 as eluent.
General Procedure 7 (GP7): 12 and 13a−c. Benzyl-
chloride (0.09 mmol, 2.00 equiv) and NaN3 (0.09 mmol, 2.00
equiv) were suspended in H2O/tBuOH = 1:1 (1 mL), then the
appropriate 6-(methylthio)-1-(prop-2-ynyl)-1H-pyrazolo[3,4-
d]pyrimidine (8d and 9k−m) (0.05 mmol, 1.10 equiv),
173
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Research Article
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4161.
CuSO4·5H2O (0.01 equiv) and sodium ascorbate (0.10 equiv)
were added. The reaction mixture was irradiated under μW at
125 °C for 10 min. The solvent was removed under reduced
pressure and the residue purified by flash chromatography using
DCM/MeOH = 99:1 as eluent.
General Procedure 8 (GP8): 11a−c. The appropriate 6-
(methylthio)-pyrazolo[3,4-d]pyrimidin-4-amine (9a, e, and p)
(0.05 mmol, 1.00 equiv) was dissolved in MeOH (5.0 mL)
under a nitrogen atmosphere. Pd(OH)2/C (0.10 equiv) was
added and the reaction mixture was stirred at r.t for 2 h under
H2 atmosphere. The mixture was filtered on Celite and the
solvent removed under reduced pressure to obtain the products
as a white solids.
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H.; Yin, H.; Conn, P. J.; Lindsley, C. W. Positive allosteric modulators
of the metabotropic glutamate receptor subtype 4 (mGluR4): Part I.
Discovery of pyrazolo[3,4-d]pyrimidines as novel mGluR4 positive
allosteric modulators. Bioorg. Med. Chem. Lett. 2008, 18, 5626−5630.
(11) Trivedi, A. R.; Dholariya, B. H.; Vakhariya, C. P.; Dodiya, D. K.;
Ram, H. K.; Kataria, V. B.; Siddiqui, A. B.; Shah, V. H. Synthesis and
anti-tubercular evaluation of some novel pyrazolo[3,4-d]pyrimidine
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details about instruments, synthesis and charac-
terization of compounds, and enzymatic assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) Welsch, M. E.; Snyder, A. S.; Stockwell, B. R. Privileged scaffolds
for library design and drug discovery. Curr. Opin. Chem. Biol. 2010, 14,
347−361.
AUTHOR INFORMATION
Corresponding Author
*E-mail: botta.maurizio@gmail.com.
■
(13) (a) 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. (b) Tuccinardi,
T.; Manetti, F.; Schenone, S.; Martinelli, A.; Botta, M. Construction
and validation of a RET TK catalytic domain by homology modeling. J.
Chem. Inf. Model. 2007, 47, 644−655. (c) Schenone, S.; Bruno, O.;
Bondavalli, F.; Ranise, A.; Mosti, L.; Menozzi, G.; Fossa, P.; Donnini,
S.; Santoro, A.; Ziche, M.; Manetti, F.; Botta, M. Antiproliferative
activity of new 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine deriva-
tives toward the human epidermoid carcinoma A431 cell line. Eur. J.
Med. Chem. 2004, 39, 939−946. (d) Tintori, C.; Laurenzana, I.; La
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.), 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.
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