An alternative synthetic approach for the synthesis of biologically relevant 1,4-disubstituted pyrazolo[3,4-d]pyrimidines

Article abstract of DOI:10.1016/j.tetlet.2013.07.069

A versatile approach for the synthesis of 1,4-disubstituted pyrazolo[3,4-d]pyrimidines has been developed. Starting from commercially available allopurinol, TBAF mediated N1-functionalization and subsequent C4 nucleophilic substitution, under microwave assisted- or standard heating conditions, granted access to highly functionalized pyrazolo[3,4-d]pyrimidines of potential biological interest.

Full text of DOI:10.1016/j.tetlet.2013.07.069

Accepted Manuscript
An alternative synthetic approach for the synthesis of biologically relevant 1,4-
disubstituted pyrazolo[3,4-d]pyrimidines
Marco Radi, Vincenzo Bernardo, Giulia Vignaroli, Annalaura Brai, Mariangela
Biava, Silvia Schenone, Maurizio Botta
PII:
S0040-4039(13)01218-5
DOI:
http://dx.doi.org/10.1016/j.tetlet.2013.07.069
TETL 43267
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
6 June 2013
3 July 2013
9 July 2013
Please cite this article as: Radi, M., Bernardo, V., Vignaroli, G., Brai, A., Biava, M., Schenone, S., Botta, M., An
alternative synthetic approach for the synthesis of biologically relevant 1,4-disubstituted pyrazolo[3,4-
d]pyrimidines, Tetrahedron Letters (2013), doi: http://dx.doi.org/10.1016/j.tetlet.2013.07.069
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An alternative synthetic approach for the
synthesis of biologically relevant 1,4-
disubstituted pyrazolo[3,4-d]pyrimidines
Leave this area blank for abstract info.
Marco Radi, Vincenzo Bernardo, Giulia Vignaroli, Annalaura Brai, Mariangela Biava, Silvia Schenone and
Maurizio Botta

1
Tetrahedron Letters
journal homepage: www.elsevier.com
An alternative synthetic approach for the synthesis of biologically relevant 1,4-
disubstituted pyrazolo[3,4-d]pyrimidines
Marco Radi,a,b Vincenzo Bernardo,a Giulia Vignaroli,a Annalaura Brai,a Mariangela Biava,c Silvia
Schenoned and Maurizio Bottaa,e∗
a Dipartimento di Bioteconologie, Chimica e Farmacia, Università degli Studi di Siena Via Alcide de Gasperi, 2, 53100 Siena, Italy
b Dipartimento di Farmacia, Università degli Studi di Parma, Viale delle Scienze, 27/A, 43124 Parma, Italy
c Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, Piazzale Aldo Moro 5, 00185-Roma, Italy
d Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
e Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife
Science Building, Suite 333, 1900 N 12th Street, Philadelphia, PA, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A versatile approach for the synthesis of 1,4-disubstituted pyrazolo[3,4-d]pyrimidines has been
developed. Starting from commercially available allopurinol, TBAF mediated N1-
functionalization and subsequent C4 nucleophilic substitution, under microwave assisted- or
standard heating conditions, allowed to access to highly functionalized pyrazolo[3,4-
d]pyrimidines of potential biological interest.
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
Pyrazolo[3,4-d]pyrimidines
N1-Functionalization
Microwave
Antitumor
Nitrogen-containing heterocycles are widely distributed in
nature and play a key role in the metabolism of all living cells.
Among the many nitrogen-containing heterocycles, the
pyrazolo[3,4-d]pyrimidine nucleus represents a very interesting
and versatile scaffold for the synthesis of potential drug
candidates acting on a wide range of biological targets.1 Among
their many applications, pyrazolo[3,4-d]pyrimidines have been
used as kinase inhibitors,2 antiviral agents,3 adenosine
antagonists,4 glutamate modulators5 and antitubercular agents.6
The two most common synthetic approaches for the preparation
of pyrazolo[3,4-d]pyrimidine derivatives build the bicyclic
scaffold starting alternatively from the pyrimidine7 and the
pyrazole nucleus.1a However, several steps are needed in most
cases to construct and functionalize the core scaffold. In the last
few years, our research group has developed several
pyrazolo[3,4-d]pyrimidines (general structure VI, Scheme 1)
which proved to be promising antitumor agents active on
different cancer cell lines depending on the nature and position of
substituents on the heterocyclic core.8 These compounds were
synthesized starting from commercial phenyloxiranes (I) which
were converted to the ethyl esters of 5-amino-1H-pyrazole-4-
carboxylic acids (III) in two steps. Reaction of the latter
intermediates with ethyl(ethoxymethylene)cyanoacetate followed
———
∗ Corresponding author. Tel.: +39-0577-234306; fax: +39-0577-234333; e-mail: botta.maurizio@gmail.com; maurizio.botta@unisi.it

2
Tetrahedron Letters
substitution. Zacharie and coworkers reported the alkylation of
the pyrazolo[3,4-d]pyrimidine scaffold with alkyl iodides in the
presence of cesium carbonate or DBU in dry DMF at 0 °C
leading to a mixture of N1/N2-functionalized derivatives in a 3/1
ratio.11 Better results were later reported by Gundersen et al.
which obtained low yields of the N2-functionalized side products
using different alcohols under Mitsunobu conditions.12
On these basis, we decided to develop an alternative synthetic
approach to synthesize 1,4-disubstituted pyrazolo[3,4-
d]pyrimidine derivatives (such as VI) that could be favorably
employed to increase the chemical diversity in the N1 side chain.
Starting from commercial allopurinol 1, treatment with
POCl3/DMF led to the C4-chloro derivative 2 that could be
Table 1. Substituted pyrazolo[3,4-d]pyrimidine analogues
Entry
Compound
R1
4-F -
4-F
R2
Yield (%)
1
2
4a
7a
Bn
63
68
3
7b
4-Cl
75
4
5
6
7c
7d
8a
4-I
4-Me
4-F
Ph
Ph
70
60
58
Scheme 1. Classical synthetic approach for the synthesis of antitumor
pyrazolo[3,4-d]pyrimidines VI.
by treatment with an excess of formamide at 190 °C gave the N1-
substituted pyrazolo-pyrimidinones (IV). The final products (VI)
were then obtained after chlorination with the Vilsmeier complex
(POCl3:DMF, 1:1) followed by regioselective substitution of the
chlorine at C4 with an excess of different amines. The main
drawback of the above described synthetic approach is
represented by its low versatility, especially for its application in
the development of N1 substituted analogues such as VI: the
chemical diversity in the N1 side chain phenyl ring is in fact
determined in an early phase by the choice of the starting
phenyloxiranes (I) that should be submitted to the five-step
sequence described in scheme 1.
7
8
9b
4-Cl
4-Cl
67
76
10b
directly submitted to N1-functionalization with substituted 2-
bromo 4-acetophenones to give the key intermediates 3 (Scheme
2). Unfortunately, when the N1-alkylation was conducted under
reaction conditions reported by Zacharie et al. (Cs2CO3 or DBU
in DMF), compounds 3 were isolated in a low 30% yield. Among
the different bases used in this reaction, tetrabutylammonium
fluoride (TBAF)13 proved to be the best one for the N1-alkylation
giving access to compounds 3a-d in 60-70% yields with no trace
of the N2-substitued side-products. Treating the latter
intermediates with amines and AcOH/dioxane under microwave
assisted conditions, it was possible to obtain the 1,4-disubsituted
derivatives 4a in acceptable yields after only 10 minutes.
Alternatively, the intermediates 3a-d were reduced with NaBH4
in THF to give carbinols 5a-d which were initially converted to
the dichloro derivatives 6a-d and then submitted to the
microwave assisted C4-functionalization with different amines
using the above described protocol to give the desired
pyrazolo[3,4-d]pyrimidines 7a-d.
Herein we report the application of
a
direct N1-
functionalization strategy on the preformed pyrazolo[3,4-
d]pyrimidine scaffold as an alternative approach for the synthesis
of functionalized analogues of antitumor compounds VI. A few
authors reported the direct N1 substitution of the pyrazolo[3,4-
d]pyrimidine ring, especially with short chain substituents, via
alkylation9 or Mitsunobu10 reaction, which led to the concurrent
formation of the N2 substituted derivatives. The N1/N2
substitution ratio usually depends on the base used to form the
salt and is also related to the halide used in the nucleophilic

3
Scheme 2. Alternative synthetic approach for the synthesis of substituted pyrazolo[3,4-d]pyrimidine analogues.
When amine hydrochlorides were used in the latter step, a
Acknowledgments
different approach was required to accomplish the C4
nucleophilic substitution: the amine salt was suspended in DMF,
This work was partially supported by National Interest
Research Projects (PRIN_2008_5HR5JK and
added with an excess of triethylamine and finally treated with the
o
intermediate 6a under microwave irradiation at 150 C for 10
PRIN_2010_5YY2HL). Financial support has been also received
from the Istituto Toscano Tumori-ITT-Grant proposal 2010. We
are grateful to Lead Discovery Siena Srl and Cost Action
CM1106 “Chemical Approaches to Targeting Drug Resistance in
Cancer Stem Cells”.
minutes. In these conditions, both C4 nucleophilic substitution
and dehydrohalogenation in the N1 linker occurred, giving the
1,4-disubstituted derivative 8a. On the other hands, an interesting
outcome in the C4 nucleophilic substitution was observed when
reacting intermediate 6a with amine 11 depending on the reaction
conditions used. As previously reported, the C4 substituent of
these C6-unsubstitued pyrazolo[3,4-d]pyrimidines seems to be
solvent exposed upon binding to the target kinases and the
hydrophilic amine 11 was prepared accordingly (the synthesis is
reported in the SI file). Since the AcOH/dioxane substitution
protocol was unsuccessful, probably due to piperazine
protonation, and in order to avoid the dehydrohalogenation in
N1, different reaction conditions were used in the coupling with
11: treatment of 6b under-microwave assisted conditions in the
presence of DIPEA/i-PrOH gave, unexpectedly, compound 9b as
result of a dealkylative substitution instead of the expected
derivative 10b. The formation of 9b was proved to be a direct
consequence of microwave irradiation since standard heating
conditions allowed to obtain compound 10b as the only product.
Supplementary Material
Supplementary data associated with this article can be found,
in the online version, at
References and notes
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In summary, a versatile approach for the synthesis of 1,4-
disubstituted pyrazolo[3,4-d]pyrimidines has been developed.
Starting from commercially available allopurinol, TBAF
mediated N1-functionalization and subsequent C4 nucleophilic
substitution under different reaction conditions represented the
key steps for the decoration of the pyrazolo[3,4-d]pyrimidine
scaffold. Microwave irradiation drastically reduced the reaction
time need for C4 functionalization and allowed to obtain
compounds (such as 9b) inaccessible under standard heating
conditions. The exploitation of this alternative synthetic protocol
will allow to expand the chemical diversity around the
pyrazolo[3,4-d]pyrimidine scaffold for the identification of novel
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An alternative synthetic approach for the
synthesis of biologically relevant 1,4-
disubstituted pyrazolo[3,4-d]pyrimidines
Leave this area blank for abstract info.
Marco Radi, Vincenzo Bernardo, Giulia Vignaroli, Annalaura Brai, Mariangela Biava, Silvia Schenone and
Maurizio Botta