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Biologically Driven Synthesis of Pyrazolo[3,4d]pyrimidines As

Protein Kinase Inhibitors: An Old Scaold As a New Tool for


Medicinal Chemistry and Chemical Biology Studies
Silvia Schenone,*
,
Marco Radi,

Francesca Musumeci,

Chiara Brullo,

and Maurizio Botta


,

Dipartimento di Farmacia, 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

Dipartimento di Biotecnologie, Chimica e Farmacia, Universita 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 N 12th Street, Philadelphia, Pennsylvania 19122, United States
CONTENTS
1. Introduction B
2. Synthesis of 1H-Pyrazolo[3,4-d]pyrimidines B
2.1. Synthesis Starting from the Pyrazole Ring B
2.1.1. Synthesis of 1-Substituted/1-Unsubsti-
tuted Compounds B
2.1.2. Synthesis of 3-Substituted and 1,3-
Disubstituted Compounds D
2.1.3. Synthesis of 6-Substituted Compounds G
2.2. Synthesis Starting from the Pyrimidine Ring J
3. Kinases as Target Enzymes for Anticancer
Therapy L
4. 1H-Pyrazolo[3,4-d]pyrimidines As Serine-Threo-
nine Kinase Inhibitors M
4.1. mTOR Inhibitors M
4.1.1. Synthesis and Biological Properties of 4-
Morpholino Derivatives O
4.1.2. Molecular Modeling Studies and Sub-
stitution Optimization O
4.1.3. Other mTOR Inhibitors S
4.2. 70-kDa Ribosomal Protein S6 Kinase
(p70S6K) Inhibitors S
4.3. PI3K Inhibitors and Dual PI3K/mTOR Kinase
Inhibitors T
4.3.1. Synthesis and Biological Properties T
4.4. Mitogen Activated Protein Kinase (MAPK)
Inhibitors U
4.4.1. Raf Inhibitors U
4.4.2. p38 MAPK Inhibitors V
4.5. Glycogen Synthase Kinase-3 (GSK-3) Inhib-
itors W
4.5.1. Synthesis and Biological Properties W
4.6. Other Serine-Threonine Inhibitors Y
4.6.1. Cyclin-Dependent Kinase (CDK) Inhib-
itors Y
4.6.2. Aurora Kinase Inhibitors Y
5. 1H-Pyrazolo[3,4-d]pyrimidines As Receptor Tyro-
sine Kinase (RTK) Inhibitors AA
5.1. Epidermal Growth Factor Receptor (EGFR)
Inhibitors AA
5.1.1. Synthesis and Biological Properties AA
5.2. Insulin-Like Growth Factor 1 Receptor (IGF-
1R) Inhibitors AC
5.2.1. Synthesis and Biological Properties AD
6. 1H-Pyrazolo[3,4-d]pyrimidines As Cytoplasmic
Tyrosine Kinase Inhibitors AE
6.1. Src Family Kinase Inhibitors AE
6.1.1. Synthesis and Biological Properties of
Src Inhibitors AE
6.1.2. Synthesis and Biological Properties of
Src-Engineered Kinase Inhibitors AE
6.1.3. Synthesis and Biological Properties of
Other Src and Lck Inhibitors AF
6.2. Dual Src/Abl Tyrosine Kinase Inhibitors AG
6.2.1. Synthesis and Biological Properties of
Dual Src/Abl Inhibitors AH
6.2.2. Molecular Modeling Studies and Opti-
mization of the Substituents AJ
6.3. Type II (DGF-out Binding) Dual Src/Abl
Inhibitors AN
7. 1H-Pyrazolo[3,4-d]pyrimidines As Inhibitors of
Other Kinases AO
8. Multitargeted Inhibitors AQ
9. Conclusions AR
Author Information AS
Corresponding Author AS
Notes AS
Biographies AS
Acknowledgments AT
References AT
Received: May 16, 2013
Review
pubs.acs.org/CR
XXXX American Chemical Society A dx.doi.org/10.1021/cr400270z | Chem. Rev. XXXX, XXX, XXXXXX
1. INTRODUCTION
Nitrogen-containing heterocycles are widely distributed in
nature and essential for life, playing a vital role in the
metabolism of all living cells. Among the many nitrogen-
containing heterocycles, the pyrazolo[3,4-d]pyrimidine nucleus
(Figure 1) is an important drug-like scaold that is present in
many pharmacologically active compounds. This structure is an
isostere of adenine, which is fundamental for every aspect of
cell life as a constituent of DNA and RNA. Due to continuing
interest in pyrazolo[3,4-d]pyrimidine derivatives as drugs with
the potential to modulate purine activity or metabolism and
purinergic receptor activation, researchers have dedicated much
eort to investigating new approaches for the synthesis of these
derivatives.
The antitumor activity of pyrazolo[3,4-d]pyrimidines was
initially reported many years ago,
1
but it was only recently
shown that this eect is primarily dependent on tyrosine or
serine/threonine kinase inhibition. Due to the large role of
kinases in several diseases (especially cancer), chemical
modication of the pyrazolo-pyrimidine scaold to improve
the biological activity and kinase selectivity of these compounds
has been extensively studied.
The most common methods for the synthesis of pyrazolo-
[3,4-d]pyrimidines are described in the rst section of this
review, beginning with the work of Robins, which occurred
when the understanding of the biological behavior of these
compounds was in an embryonic state, and extending to
present day. Classic approaches and alternative strategies,
starting from either the pyrimidine or the pyrazole nucleus, are
reviewed.
The body of the article is focused on pyrazolo[3,4-
d]pyrimidines as serine/threonine and tyrosine kinase inhib-
itors. Information on the biological targets is also reported to
provide useful insight into the synthesis of novel drug
candidates and to suggest specic functionalities that could
be introduced onto the scaold to improve biological activity.
Examples of this target-based synthesis of specic pyrazolo[3,4-
d]pyrimidines are given along with a description of how the
evolution of chemical methodologies, chemical biology,
pharmacology and medicine have led to many new drug
candidates.
Many pyrazolo-pyrimidine inhibitors have been synthesized,
and it is almost impossible to exhaustively report the known
derivatives. Most of the compounds described here were
reported in the most important medicinal chemistry journals,
and some representative examples from the patent literature are
also included.
2. SYNTHESIS OF 1H-PYRAZOLO[3,4-d]PYRIMIDINES
2.1. Synthesis Starting from the Pyrazole Ring
2.1.1. Synthesis of 1-Substituted/1-Unsubstituted
Compounds. In the mid-1950s, Robins performed the
synthesis of dierent substituted pyrazolo[3,4-d]pyrimidines
and initiated their biological evaluation, particularly as
antitumor agents.
Reaction of ethoxymethylenemalononitrile 1 with hydrazine
monohydrate without solvent or with dierent substituted
hydrazines in reuxing ethanol produced 5-amino-1H-pyrazole-
4-carbonitriles 2ad and similar derivatives, which were then
hydrolyzed with concentrated sulfuric acid to the correspond-
ing amides 3ad. These amides were subsequently cyclized
into the corresponding 1H-pyrazolo[3,4-d]pyrimidin-4-ols 4a
d by treatment with boiling formamide. C4 chlorination of the
latter compounds with POCl
3
gave 5ad, which underwent
nucleophilic displacement of the chlorine atom by primary or
secondary amines in reuxing alcoholic solution to aord the 4-
amino derivatives 6 (Scheme 1).
2,3
Notably, the reaction of monosubstituted hydrazines R-NH-
NH
2
with 1,3-difunctional compounds (,-unsaturated
ketones, esters, acids and nitriles) can lead to dierent pyrazole
regioisomers depending on the nature of the hydrazine, the 1,3-
difunctional compound, and the reaction medium. The
regioselectivity of this reaction has been extensively inves-
tigated.
4
By reacting ,-unsaturated nitriles with monosub-
stituted hydrazines, the 5-amino-pyrazolo 7 or the 3-amino-
pyrazolo derivatives 8 (Figure 2) can be obtained.
5
However, as
reported in several examples in this article, the 5-amino
derivatives are usually obtained as the main products under
neutral conditions.
The fusion of the N1-unsubstituted derivative 3a with urea
or thiourea aorded 1H-pyrazolo[3,4-d]pyrimidin-4,6-diol 9a
or the corresponding 6-mercapto derivative 10, respectively.
Treatment of the latter with methyl iodide yielded 6-
methylthio-derivative 11, which was subjected to the
chlorination/amination protocol previously described to aord
4-amino-substituted 6-methylthio derivatives 13 (Scheme 2).
2
Figure 1. 1H-Pyrazolo[3,4-d]pyrimidine.
Scheme 1. Synthesis of 4-Amino-Substituted 1H-
Pyrazolo[3,4-d]pyrimidines
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Reaction of 5-amino-1H-pyrazole-4-carbonitrile 2a with
formamide, urea or thiourea produced the following corre-
sponding 4-NH
2
derivatives: adenine isostere 1H-pyrazolo[3,4-
d]pyrimidin-4-amine 14 (4-APP), 4-amino-1H-pyrazolo[3,4-
d]pyrimidin-6-ol 15 or 4-amino-1H-pyrazolo[3,4-d]pyrimidin-
6-thiol 16, respectively (Scheme 3).
2
Chlorination of 9, accomplished using POCl
3
and N,N-
diethylaniline, aorded 4,6-dichloro-pyrazolo[3,4-d]pyrimidines
17 in good yield (Scheme 4). Selective replacement of the
chlorine atoms with amines under carefully controlled
conditions gave a variety of 4-amino- and 4,6-diamino-
substituted derivatives.
6,7
Generally, treatment of 17 with
ethanolic solutions of primary or secondary amines with steam
bath heating for 15 min aorded the corresponding 4-amino-6-
chloro derivatives 18, whereas longer reaction times in the
presence of a large excess of amines usually produced the
disubstituted derivatives 19.
Biological studies with the pyrazolopyrimidines synthesized
by Robins showed that the compounds possessed interesting
activity, including inhibition of xanthine oxidase
8
and
antiproliferative activity in adenocarcinoma and leukemia cell
lines and in vivo tumor models.
9,10
Following a similar synthetic approach, Schmidt and Druey
reported the synthesis of several pyrazolo[3,4-d]pyrimidines
starting from the 5-aminopyrazole-4-carboxylates 21a,c, which
were obtained from ethyl(ethoxymethylene)cyanoacetate (20)
and the appropriate hydrazine.
11
The 4-hydroxy-derivatives 4a
and 4c were then directly obtained by reacting 21 with boiling
formamide (Scheme 5). The replacement of the C4 cyano
group with an ester moiety within the pyrazole structure
allowed the sometimes tricky CN group hydrolysis to be
avoided. This shortening of the procedure by one step is
particularly useful when an NH
2
at C4 is not required.
Chlorination of 4 and subsequent substitution of the chlorine
atom with amines led to products 6.
In a dierent synthetic approach, Nagahara and Robins
reported the synthesis of compounds 4be by reaction of the
corresponding pyrazolo-amides 3be with ethoxymethylene-
malononitrile (1) in reuxing DMF, thus aording the desired
products in satisfactory yields (Scheme 6).
12
The pyrazolo[3,4-d]pyrimidine ring can also be synthesized
by reacting pyrazole derivatives with isocyanates or isothiocya-
nates, which react in a manner similar to other cyclic
enaminonitriles and ortho-aminonitriles.
13
The reaction of
ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (21c) with
chlorosulfonyl isocyanate and subsequent treatment with
aqueous KOH produced 22 (Scheme 7).
14
4-Amino-1-
phenyl-5H-pyrazolo[3,4-d]pyrimidin-6-one 23 was synthesized
in 68% yield by Quinn and colleagues in a one-pot reaction
involving the condensation of 5-amino-1-phenyl-1H-pyrazole-
4-carbonitrile (2c) with benzoyl isocyanate followed by
annulation with ammonia (Scheme 7).
15
Taylor and Patel utilized an aza-Wittig-mediated pyrimidine
annulation reaction to synthesize 6-phenylamino-substituted
pyrazolo[3,4-d]pyrimidines and other fused-pyrimidine deriva-
Figure 2. Structures of 5-amino-pyrazolo derivatives 7 and of 3-amino-
pyrazolo derivatives 8.
Scheme 2. Synthesis of 6-Thiomethyl-Substituted 1H-Pyrazolo[3,4-d]pyrimidines
Scheme 3. Synthesis of 1H-Pyrazolo[3,4-d]pyrimidin-4-
amine 14, 4-Amino-1H-pyrazolo[3,4-d]pyrimidin-6-ol 15,
and 4-Amino-1H-pyrazolo[3,4-d]pyrimidin-6-thiol 16
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tives starting from aminonitriles and aminoesters.
16
The
reaction of pyrazolo-nitrile 2b or pyrazolo-ester 21b with
dibromotriphenylphosphorane, generated in situ by the slow
addition of bromine to a cold solution of triphenylphosphine
(PPh
3
) in DCM, aorded the corresponding iminophosphor-
anes 24 and 25, which undergo aza-Wittig reaction with phenyl
isocyanate to produce the corresponding carbodiimides 26 and
27 in good yields. Treatment of the latter with ammonia
followed by heating in methanol or ethanol gave, via the
guanidine intermediates, the nal cyclized derivatives 28 and
29. In the case of 29, the cyclization was greatly facilitated by
the addition of sodium ethoxide (Scheme 8).
2.1.2. Synthesis of 3-Substituted and 1,3-Disubsti-
tuted Compounds. Several procedures have been reported
for the synthesis of pyrazolo[3,4-d]pyrimidines bearing dier-
ent substituents at the 3 position of the pyrazole ring. Robins
and colleagues reported the synthesis of 3-methyl-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine 32 through reaction of (1-ethoxy-
ethylidene)-malononitrile 30 and hydrazine monohydrate
followed by treatment of the formed cyanopyrazole 31 with
boiling formamide (Scheme 9).
3
Taylor and colleagues prepared a number of new 4-amino-
pyrazolo[3,4-d]pyrimidines bearing a C3 cyanomethyl or cyano
group.
17,18
An example of the synthesis of a 3-cyanomethyl
derivative is the reaction of 5-amino-3-(cyanomethyl)-1-phenyl-
1H-pyrazole-4-carbonitriles 33b,c with ethyl orthoformate to
give the intermediates 34b,c. The latter were nally cyclized to
35b,c by treatment with ethanol saturated with ammonia
(Scheme 10).
(4-Amino-6-mercapto-1H-pyrazolo[3,4-d]pyrimidin-3yl)-
acetonitrile (36) was synthesized by reacting pyrazole derivative
33a with benzoyl isothiocyanate followed by acidication
(Scheme 10).
19
The synthesis of 3-cyano-derivatives was performed starting
with tetracyanoethylene (37), which reacted easily with
monosubstituted hydrazines to give 5-amino-3,4-dicyanopyr-
azoles 38.
20
Pyrazoles 38 were converted to 1-substituted-3-
cyano-pyrazolo[3,4-d]pyrimidines 39 with the protocol used
for the synthesis of 35b,c (Scheme 11).
21
Tominaga and colleagues synthesized a variety of pyrazolo-
[3,4-d]pyrimidines starting from ketene dithioacetals, partic-
ularly from bis(methylthio)methylene malononitrile 40a and
bis(methylthio)methylene cyanoacetamide 40b and from
anilino(methylthio)methylene malononitriles 41a and the
corresponding amides 41b.
22
These last two derivatives, 41a
and 41b, were prepared by reacting 40a and 40b with suitable
anilines. The reaction of 40a,b and 41a,b with substituted
hydrazines under reux gave the corresponding 5-amino-
pyrazoles 42a,b and 44a,b, which were subsequently treated
with formamide to give the corresponding 3-substituted
pyrazolo[3,4-d]pyrimidine derivatives 43a,b and 45a,b. The
4-amino-pyrazolo-pyrimidines were obtained from 4-cyano-
pyrazoles 42a and 44a, and the 4-hydroxy-pyrazolo-pyrimidines
were obtained from the pyrazolo-4-carboxamides 42b and 44b
(Scheme 12).
Tominaga also reported the synthesis of other 3-substituted
derivatives.
22
The N1 phenyl pyrazolo-pyrimidines 46 and 47
were prepared by reacting the N1 phenyl-substituted
compounds of the 42a series with guanidine carbonate or
urea (Scheme 13). Similarly, starting from the pyrazolo-
carboxamide of the 42b series, the 4,6-dihydroxy derivatives
48 were synthesized, with yields higher than 90%. Moreover,
the reaction of the aminocarboxamides 42b with carbon
disulde in the presence of potassium hydroxide aorded the 6-
methylthio analogues 49 in reasonable yield (Scheme 13).
Treatment of the 4-hydroxy derivatives 48 and 49 with
phosphorus oxychloride gave 4-chloro-pyrazolo[3,4-d]-
pyrimidines, which are key intermediates for the synthesis of
the 4-amino substituted derivatives.
The synthesis of highly functionalized dicyanoethylenes 52
and their application to the preparation of C3-substituted
pyrazolo[3,4-d]pyrimidines were reported in a dierent article
from the same authors.
23
The reaction of 50 with tetracyano-
ethylene oxide (51) gave the corresponding dicyanoethylene
compounds 52, which were subsequently reacted with
hydrazine or phenyl hydrazine to obtain 5-aminopyrazolo-4-
carbonitriles 53 (Scheme 14). The latter compounds were then
Scheme 4. Synthesis of 4,6-Disubstituted 1H-Pyrazolo[3,4-
d]pyrimidines
Scheme 5. Synthesis of 1H-Pyrazolo[3,4-d]pyrimidin-4-ol Derivatives
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cyclized with formamide to aord the corresponding C3-
substituted 4-amino-pyrazolo[3,4-d]pyrimidines.
Southwich and Dhawan prepared 3-aryl- or 3-benzyl-
pyrazolo[3,4-d]pyrimidines 58.
24
In their synthesis, malononi-
trile 54 was reacted with the appropriate acyl chloride and TEA
in benzene or, alternatively, with sodium ethoxide and acyl
chlorides to give compounds 55. These intermediates were
transformed into the corresponding methoxy derivatives 56 by
methylation with dimethylsulfate and sodium bicarbonate. The
aryl- or benzyl-substituted methoxymethylenemalononitriles 56
reacted with a suitable hydrazine to provide pyrazole
intermediates 57, which were then cyclized to pyrazolopyr-
imidines 58.
The authors also prepared a number of 3-substituted 4,6-
diamino-pyrazolo[3,4-d]pyrimidines 59 by modifying a proce-
dure previously reported by Davoll and Kerridge for the one-
step synthesis of 4,6-diamino-pyrazolo[3,4-d]pyrimidine.
25
In
the modied method, 1- and/or 3-substituted-5-amino-4-
cyanopyrazoles 57 were maintained under reduced pressure
during fusion with guanidine carbonate to reduce exposure of
the mixture to atmospheric oxygen and remove the water
formed in the reaction. The reaction temperature was kept 10
30 C above the melting point of the pyrazole derivative for 2
3 h. In a few cases, the authors used triethanolamine as the
reaction medium (Scheme 15).
24
Similar 1,3-disubstituted pyrazolo[3,4-d]pyrimidines have
been synthesized from 1-alkyl-5-amino-3-aryl-4-cyanopyrazoles
60 in a one-pot synthesis. To a THF solution of malononitrile
54 sodium hydride, acyl chloride, dimethylsulfate and nally
alkylhydrazine dihydrochloride in the presence of TEA were
added in succession to give the expected intermediates in 21
58% yield.
26
The structure of 60 was conrmed by NOE
experiments, as cyclization could have also resulted in the
corresponding 1-alkyl-3-amino-5-aryl-4-cyanopyrazoles. The
aminonitriles 60 were converted into pyrazolo-pyrimidines 61
Scheme 6. Dierent Synthesis of 1H-Pyrazolo[3,4-d]pyrimidin-4-ol Derivatives
Scheme 7. Synthesis of the N1-Phenyl Derivatives 22 and 23
Scheme 8. Synthesis of 4,6-Disubstituted Derivatives 28 and 29
Scheme 9. Synthesis of 3-Methyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine 32
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in high yield (8298%) by treatment with formamide at reux
(Scheme 16).
The synthesis of 3-(p-toluenesulfonylamino)-1H-pyrazolo-
[3,4-d]pyrimidines was performed by Tominaga
27
using N-
bis(methylthio)methylene-p-toluenesulfonamide 62 as the
starting material. Reaction with malononitrile 54 or cyanoace-
tamide 63 in the presence of potassium carbonate in DMSO
aorded the corresponding displacement products 64a or 64b
in yields exceeding 90%. The reaction of 64 with various
hydrazines aorded the 5-aminopyrazole derivatives 65, which
were reacted with formamide to give the desired pyrazolo-
pyrimidines 66 (Scheme 17).
A dierent approach to C3-substituted pyrazolo[3,4-d]-
pyrimidines has been reported by Schultz and Ding.
28
The
synthesis starts with the 3-bromo derivative 67, which was
obtained from Robins compound 5a by treatment with N-
bromosuccinimide (NBS) under microwave irradiation. The
application of microwaves gave high yields of the desired
product (more than 95%), provided only a small amount of
Scheme 10. Synthesis of 3-Cyanomethyl-1H-pyrazolo[3,4-d]pyrimidines
Scheme 11. Synthesis of 3-Cyano-1H-pyrazolo[3,4-d]pyrimidines
Scheme 12. Synthesis of 3-Methylthio or 3-Amino-
Substituted 1H-Pyrazolo[3,4-d]pyrimidines
Scheme 13. Synthesis of the 3-Methylthio-1-phenyl-
Substituted 1H-Pyrazolo[3,4-d]pyrimidines 46-49
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side products, and required short reaction times. Compound 67
was then subjected to a one-pot, two-step process involving
sequential S
N
Ar displacement of the C4 chlorine atom with
dierent amines under mild acidic conditions (acetic acid)
followed by a Suzuki coupling with dierent boronic acids to
aord the C3-substituted derivatives 68 (Scheme 18).
Interestingly, this approach permitted straightforward
functionalization of the C3 position in the presence of relatively
reactive functional groups (amine, amide, alcohol) that would
have required protection from the strong acidic or basic
conditions used in the previously reported procedures.
Recently, the synthesis of 6-oxo or 6-thio-substituted
derivatives bearing a phenyl ring at C3 has been reported.
29
Ethyl benzoylacetate (69) was reacted with trichloroacetonitrile
in ethanol in the presence of sodium acetate to aord acrylate
intermediate 70. The reaction of 70 with hydrazine hydrate in
reuxing ethanol aorded 5-amino-3-phenyl-1H-pyrazole-4-
carboxylic acid ethyl ester 71. The reaction of 71 with benzoyl
isothiocyanate aorded intermediate 72, which was treated with
ethanolic sodium ethoxide to aord 3-phenyl-6-thioxo-1,5,6,7-
tetrahydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (73b). The fu-
sion of 71 with urea or thiourea aorded pyrazolo-
pyrimidinones 73a and 73b in good yield (Scheme 19). The
pyrazolo-pyrimidinones thus obtained represent advanced
intermediates for further functionalization.
Todorovic and colleagues reported the microwave-assisted
synthesis of 1,3-disubstituted-pyrazolo[3,4-d]pyrimidine deriv-
atives, including compounds 76, starting from 1H-pyrazolo[3,4-
d]pyrimidin-4-amine 14 (Scheme 20).
30
Iodination of 14 was achieved by treatment with N-
iodosuccinimide (NIS) under microwave irradiation. Com-
pound 74 was alkylated at N1 using Mitsunobu chemistry,
involving treatment with an alcohol in the presence of PPh
3
and
diisopropyl azodicarboxylate (DIAD), thus providing 75 in
good yield. However, the need for repeated chromatography to
remove triphenylphosphine oxide encouraged the development
of an alternate alkylation method, which involved treating 74
with K
2
CO
3
and bromobutane or isobutyl bromide. It should
be noted that these two approaches were only suitable for the
introduction of primary alkyl groups at N1.
30
Arylation at C3 was achieved via Suzuki cross-coupling using
a Pd catalytic system including the 1,3,5,7-tetramethyl-2,4,8-
trioxa-6-phenyl-6-phosphaadamantane (PA-Ph) ligand devel-
oped by the same authors. Microwave irradiation of 75 with a
variety of boronic acids i n the presence of tris-
(dibenzylideneacetone)dipalladium(0) [Pd
2
(dba)
3
] and the
PA-Ph ligand aorded the desired N1- and C3-substituted
pyrazolo[3,4-d]pyrimidines 76. The same authors also
synthesized two N1-tert-butyl derivatives 80a,b and the
corresponding N1-unsubstituted derivatives 81 starting from
N1-substituted compound 77 using microwave-assisted proce-
dures. In this case, the C3 halogenation was performed using
bromine in water (Scheme 21).
30
Recently, other authors used MAOS (microwave-assisted
organic synthesis) for the preparation of pyrazolo[3,4-d]-
pyrimidine compounds in high yield. Heravi and colleagues
reported the synthesis of pyrazolo[3,4-d]pyrimidines 58 using
Keggins heteropolyacids, such as phosphotungstic acid
(H
3
PW
12
O
40
) or molybdophosphoric acid (H
3
PMo
12
O
40
),
and microwave irradiation starting from the usual 5-amino-4-
cyanopyrazoles 57 and formamide.
31
The microwave-solid acid
combination resulted in a convenient catalytic synthesis with
yields higher than 61% and reduced reaction times compared to
traditional methods (Scheme 22).
2.1.3. Synthesis of 6-Substituted Compounds.
Although the synthesis of a few C6 substituted compounds
has been described above, this section focuses on the synthetic
approaches generally used to obtain C6 alkyl or aryl derivatives.
One of the rst examples of the synthesis of 6-alkyl-
substituted (particularly, 6-methyl and 6-ethyl) pyrazolo[3,4-
Scheme 14. Synthesis of Functionalized Pyrazoles Starting
from Tetracyanoethylene Oxide
Scheme 15. Synthesis of 1,3-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidin-4-amines 58 and of 1,3-Disubstituted 1H-Pyrazolo[3,4-
d]pyrimidin-4,6-diamines 59
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d]pyrimidines 84 was reported by Robins and colleagues.
32
5-
Amino-4-cyanopyrazoles 2ad were acylated by acetic or
propionic anhydride to give the corresponding 5-acylamino-4-
cyanopyrazoles 82, which were then treated with an alkaline
solution of hydrogen peroxide to give 6-alkyl-4-hydroxy-
pyrazolo-pyrimidines 83. Finally, C4 chlorination with
phosphorus oxychloride and subsequent nucleophilic displace-
ment with a primary or secondary amine gave the desired
compounds 84 (Scheme 23).
The Taylor group, during a study of the base-catalyzed
condensation of aromatic and heterocyclic ortho-aminonitriles
with nitriles, synthesized a series of 4-amino-pyrazolo[3,4-
d]pyrimidines 85 by the reaction of 4-cyano-5-amino-pyrazoles
2ac with dierent nitriles in methanolic ammonia at 200 C
for 20 h in a steel hydrogenation bomb (Scheme 24).
33
Subsequently, Baker and Kozma synthesized 6-aryl-substituted
derivatives 86 by reaction of 2a with substituted benzamidine
hydrochlorides and sodium acetate at 200 C under solvent-
free conditions (Scheme 24).
34
A method for obtaining 1,6-alkyl/aryl disubstituted 1,5-
dihydro-pyrazolo[3,4-d]pyrimidin-4-ones 87 is represented by
the straightforward reaction of carboxamides 3b,c with dierent
esters in the presence of sodium ethoxide in ethanol. The
resulting derivatives 87 can subsequently be chlorinated with
POCl
3
and reacted with amines, as previously reported, to give
the 4-amino analogues (Scheme 25).
35
Interestingly, 6-triuoromethyl derivatives 92 were synthe-
sized by other authors using a similar procedure.
36
Cyanopyr-
azole 2a was reacted with triuoroacetic anhydride (TFAA) to
aord intermediate 88, which was hydrolyzed to the
corresponding amide 89. The latter compound was cyclized
at 200 C to give the pyrazolo-pyrimidine 90, which was
Scheme 16. Synthesis of Other 1,3-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidin-4-amines
Scheme 17. Synthesis of 3-(p-Toluenesulfonylamino)-1H-
pyrazolo[3,4-d]pyrimidine Derivatives
Scheme 18. Synthesis of C3-Substituted 1H-Pyrazolo[3,4-
d]pyrimidines
Scheme 19. Synthesis of 3-Phenyl-Substituted 1H-Pyrazolo[3,4-d]pyrimidin-4-ones
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chlorinated to give 91 and underwent subsequent nucleophilic
substitution with NH
3
or dierent amines (Scheme 26).
The amino-pyrazole carboxamides 3ac were condensed
with dierent aromatic aldehydes to provide 5-(N-
arylideneamino)pyrazolo derivatives 93, which were cyclized
with HCl or para-toluenesulfonic acid (p-TSA) to give the
corresponding 6-substituted-1H-pyrazolo[3,4-d]pyrimidin-4-
ones 94. These compounds can also be obtained directly by
reaction of 3ac with aromatic carboxylic acids in heated
polyphosphoric acid (PPA) or polyphosphate ester (PPE)
(Scheme 27).
37
Derivatives 94 are useful intermediates for the
synthesis of 4-amino-substituted compounds following the
procedures mentioned above.
Similar compounds, 95, have recently been obtained through
a one-pot procedure reacting derivatives 2 with aliphatic acids
in the presence of POCl
3
.
38
The latter reagent acted as both a
chlorinating agent, generating the acyl chloride in situ from the
corresponding acid, and also as a hydrolyzing agent to convert
the cyano group to the corresponding amide. The reaction of
the amide with the acyl chloride formed an intermediate that
cyclized into target compounds 95 (Scheme 28).
Scheme 20. Microwave-Assisted Synthesis of 1,3-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidines
Scheme 21. Synthesis of 3-Aryl-1H-pyrazolo[3,4-
d]pyrimidin-4-amines 80 and 81
Scheme 22. Synthesis of 1,3-Disubstituted 1H-pyrazolo[3,4-
d]pyrimidines Using Keggins Heteropolyacids
Scheme 23. Synthesis of 6-Methyl or 6-Ethyl 1H-
Pyrazolo[3,4-d]pyrimidines
Scheme 24. Synthesis of 6-Aryl and 6-Alkyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amines
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Other pyrazolo[3,4-d]pyrimidines bearing dierent 6-sub-
stitutions, including heterocyclic rings, have been obtained
using MAOS. In a modication of the Taylor preparation,
33
5-
amino-1H-pyrazole-4-carbonitriles 2, aromatic nitriles and t-
BuOK were irradiated in an open vessel for 10 min in the
absence of solvent to give the nal derivatives 96 in 4085%
yield (Scheme 29).
39
2.2. Synthesis Starting from the Pyrimidine Ring
The pyrazolo[3,4-d]pyrimidine scaold can be prepared
starting from pyrimidines, although this procedure has been
used less frequently than cyclizations starting from pyrazolo
intermediates. This synthesis generally involves reaction of
hydrazine derivatives with pyrimidines possessing a carbonyl
function at the C5-position.
40
2-Amino-4,6-dichloro-pyrimidine-5-carbaldehyde 97 was cy-
clized with hydrazine hydrate to give 4-chloro-1H-pyrazolo[3,4-
d]pyrimidin-6-ylamine 98 in good yield (Scheme 30).
41
Recently, Quiroga and colleagues reacted 97 with amines to
obtain N
4
-substituted-2,4-diamino-6-chloro-5-carbaldehydes
99, which were transformed into the corresponding pyrazolo-
[3,4-d]pyrimidines 100 by reaction with hydrazine hydrate
under microwave irradiation in the absence of solvent.
42
The
authors performed the reaction of equimolar amounts of
aldehyde 97 with a variety of aliphatic, aromatic and
heterocyclic amines in a basic medium in reuxing ethanol.
When the amination of 97 was performed in the presence of
two equivalents of amines and TEA, the disubstituted products
101 were obtained (Scheme 31).
4-Amino-2-dimethylamino-6-chloro-5-cyanopyrimidine 102
was cyclized with methyl hydrazine to produce the correspond-
ing 1-methyl-pyrazolo[3,4-d]pyrimidine 103 in high yield with
no trace of the 2-methyl regioisomer (Scheme 32).
43
A new synthesis of 1-alkyl or 1-aryl-pyrazolo[3,4-d]-
pyrimidines has been reported by Waring and colleagues.
44
The reaction of 4,6-dichloropyrimidine-5-carbaldehyde 104
with a number of substituted hydrazines provided the nal
compounds 105 in a single step. The N2-substituted
regioisomer has also been isolated from the reaction, but it is
readily separable using chromatography (Scheme 33).
Scheme 25. Synthesis of 1,6-Disubstituted 1H-Pyrazolo[3,4-
d]pyrimidin-4-ones
Scheme 26. Synthesis of 6-Triuoromethyl-Substituted 1H-
Pyrazolo[3,4-d]pyrimidines
Scheme 27. Synthesis of 6-Substituted 1H-Pyrazolo[3,4-
d]pyrimidin-4-ones
Scheme 28. Synthesis of 6-Substituted 1H-Pyrazolo[3,4-
d]pyrimidin-4-ones
Scheme 29. MW-Assisted Synthesis of 1,6-Disubstituted 1H-
Pyrazolo[3,4-d]pyrimidin-4-amines
Scheme 30. Synthesis of 4-Chloro-1H-pyrazolo[3,4-
d]pyrimidin-6-ylamine 98 Starting from 2-Amino-4,6-
dichloro-pyrimidine-5-carbaldehyde 97
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Recently, Webb and colleagues
45
synthesized a library of 4,6-
diamino-substituted pyrazolo[3,4-d]pyrimidines 109 starting
from 2,4,6-trichloropyrimidin-5-carbaldehyde 106, which re-
acted with anilines in the presence of catalytic amounts of the
phase transfer catalyst tetrabutylammonium iodide (TBAI) to
give the intermediates 107 in moderate to high yields. These
compounds were subsequently treated with dierent anilines
under the same basic conditions to give the substituted diamino
intermediates 108 in a regioselective manner. Finally, reaction
of 108 with hydrazines gave pyrazolo[3,4-d]pyrimidines 109
(Scheme 34).
After the development of this stepwise protocol, the authors
also perfected a two-pot protocol that allowed the preparation
of compounds 109 without the need for chromatographic
purication of any synthetic intermediates. Compound 107 was
prepared rst, and dierent anilines and substituted hydrazines
were then added to 107 in sequence. The two-pot protocol
allowed the preparation of a large number of compounds in a
high-throughput manner, with the aim of obtaining sucient
pure material. The average reaction yield was 35%. The major
disadvantage was the unsuccessful results obtained with alkyl
hydrazines, which did not cyclize to form the pyrazolopyr-
imidine moiety, most likely due to steric interactions.
45
Jones and colleagues described an ecient three-step, one-
pot synthesis of 1-aryl-pyrazolo[3,4-d]pyrimidines under mild
Scheme 31. Synthesis of 4,6-Diamino-Substituted 1H-Pyrazolo[3,4-d]pyrimidines
Scheme 32. Synthesis of N
6
,N
6
,1-Trimethyl-1H-
pyrazolo[3,4-d]pyrimidine-3,4,6-triamine 103
Scheme 33. Synthesis of N1-Substituted 4-Chloro-1H-
pyrazolo[3,4-d]pyrimidines
Scheme 34. Synthesis of 1,4,6-Trisubstituted-1H-
pyrazolo[3,4-d]pyrimidines Starting from 2,4,6-
Trichloropyrimidin-5-carbaldehyde 106
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reaction conditions.
46
The nucleophilic substitution of the
commercially available 4,6-dichloropyrimidine-5-carboxalde-
hyde 104 with anilines was performed in acetonitrile at 0 C.
Hydroxylamine-O-sulfonic acid was then added; after dilution
with dichloromethane and aqueous 1 M sodium hydroxide
solution, a biphasic mixture was obtained. The desired N1-aryl-
pyrazolo-pyrimidines 110 were obtained in yields ranging from
30 to 69% (Scheme 35).
Very recently, Moon and colleagues reported the synthesis of
1-substituted 4-chloro-pyrazolo[3,4-d]pyrimidines by the re-
action of 4,6-dichloropyrimidine-5-carboxaldehyde 104 with
various hydrazines, which were sometimes utilized as their
hydrochloride salts.
47
For aromatic hydrazines, the reaction was performed in the
absence of an external base, which led to the exclusive
formation of hydrazone 111. The hydrazones were sub-
sequently cyclized by heating at 200 C in acetonitrile under
MW irradiation or in dioxane with conventional heating to
form the desired pyrazolo[3,4-d]pyrimidine products 112. For
aliphatic hydrazines, the reaction sequence proceeded in a
single step in the presence of an external base to form
derivatives 113 (Scheme 36).
In further studies of the reactivity of 104 with hydrazines, the
same authors optimized a one-pot reaction that provided 4-
chloro-pyrazolo[3,4-d]pyrimidines bearing aromatic moieties at
N1.
48
The reaction was performed by reacting 104 with an
aromatic hydrazine (as the hydrochloric salt) in acetonitrile or
THF, while heating to 200 C for a short time (115 min) or
by using microwave irradiation. In this manner, the authors
prepared 4-chloropyrazolo[3,4-d]pyrimidines 112.
3. KINASES AS TARGET ENZYMES FOR ANTICANCER
THERAPY
In one way or another, protein phosphorylation is involved in
and regulates every cellular process. Even complex functions
such as memory can ultimately be traced to the phosphor-
ylation of a specic protein.
49
The family of human protein kinases, of which there are 538
members, represents the third largest and the most important
enzyme class; they are estimated to be responsible for
modifying one-third of the human proteome.
50,51
These
enzymes catalyze the transfer of the gamma phosphate group
from ATP to specic serine, threonine or tyrosine hydroxy
groups on target protein substrates involved in a number of cell
signaling pathways. It has been conclusively demonstrated that
kinase alterations (especially hyperactivation, hyperproduction,
or mutations) leading to the disruption of cell signaling
cascades play important roles in several diseases, including
cancer, inammation, neurological disorders and diabetes.
52
Thus, kinases represent important targets for drug develop-
ment. The catalytic domain is highly conserved among kinases
and consists of a bilobed structure with Mg-ATP situated in a
deep cleft located between the N- and C-terminal lobes. The
majority of small-molecule kinase inhibitors developed thus far
target the ATP binding site, and the kinase adopts a
conformation almost identical to that used to bind ATP (the
active conformation; Figure 3A). This is not surprising because,
historically, most inhibitors have been discovered using
biochemical screening based on activation loop-phosphorylated
recombinant kinase catalytic domains (at very low concen-
trations of ATP), conditions in which hydrophobic compounds
are most likely to interact with the active conformation of the
ATP cleft. Compounds that target the active state of the
enzyme are classied as type I inhibitors and bind to the ATP
Scheme 35. Three-Steps One-Pot Synthesis of 1-Aryl-4-
chloro-1H-pyrazolo[3,4-d]pyrimidines
Scheme 36. Synthesis of 1-Alkyl or 1-Aryl-4-chloro-1H-pyrazolo[3,4-d]pyrimidines
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binding site through the formation of hydrogen bonds to the
kinase hinge residues and through hydrophobic interactions
in and around the region occupied by the adenine ring of
ATP.
53
Serendipity, in combination with structureactivity relation-
ship (SAR)-guided medicinal chemistry, has allowed the
identication of type II kinase inhibitors, whose members
preferentially bind to the inactive conformation of the kinase,
thereby preventing its activation.
54
Type II inhibitors bind to
the ATP binding cleft and also exploit an adjacent hydrophobic
pocket, known as an allosteric site, created by the activation
loop (which contains the conserved DFG motif) positioned in
an out conformation. The binding mode of a type II inhibitor
is shown in Figure 3B: the compound shown is imatinib, one of
the rst type II inhibitors identied and the rst kinase inhibitor
approved for cancer therapy.
55
Type III non-ATP competitive inhibitors exclusively bind to
the allosteric site adjacent to the ATP binding region. More
recently, type IV inhibitors, which bind to allosteric sites several
angstroms away from the nucleotide binding region, have been
identied. They act by inducing conformational changes that
modulate enzymatic activity. Type III and type IV inhibitors are
often referred to as allosteric inhibitors.
56,57
Finally, type V inhibitors are covalent inhibitors that target
the catalytic site of the kinase.
58
Approximately half of the current research and development
budget of the pharmaceutical industry is spent on kinases and,
in particular, on their inhibitors. Synthetic small molecules are
the most prominent family of kinase inhibitors. In the last ten
years, ten small molecule inhibitors of dierent kinases have
received FDA approval and entered clinical therapy for the
treatment of solid and hematological malignancies, starting in
2001 with imatinib for chronic myeloid leukemia (CML). Many
other compounds are being tested in preclinical and clinical
trials. All of these inhibitors contain at least one nitrogen
heterocycle but belong to very dierent chemical families.
The pyrazolo[3,4-d]pyrimidine-based inhibitor ibrutinib was
very recently approved by the FDA for the treatment of
lymphomas, and a large number of other derivatives have
recently shown promising preclinical activity. The following
paragraphs provide selected examples of pyrazolo-pyrimidines
as kinase inhibitors as well as a description of some important
features of the specic biological targets.
Some examples of the most active compounds cited in this
section are reported in Table 1.
4. 1H-PYRAZOLO[3,4-d]PYRIMIDINES AS
SERINE-THREONINE KINASE INHIBITORS
4.1. mTOR Inhibitors
The serine-threonine kinase mammalian target of rapamycin
(mTOR) is a member of the phosphatidylinositol 3-kinase-
related kinase (PIKK) family.
59
mTOR is a critical component
of the phosphatidylinositol 3-kinase (PI3K) pathway, the
protein kinase B (Akt/PKB) pathway and the Ras/extracellular
signal regulated kinase (ERK) pathway, all which are
fundamental for cell growth, survival, motility, proliferation,
protein synthesis, and transcription.
60
These signaling cascades
are frequently deregulated in human cancer.
61,62
Intracellular
mTOR is present in two complexes, in which it is associated
with dierent proteins: mTOR complex 1 (mTORC1) and
mTOR complex 2 (mTORC2). The rst is regulated by growth
factors (i.e., IGF1 and IGF2) through the PI3K and Akt/PKB
pathways and by nutrients in a complex network of interactions.
Specically, Akt is an upstream enzyme in the signaling cascade
that leads to mTORC1 activation.
63
In turn, mTORC1
stimulates cell growth and proliferation, and its activity is
mediated by two major downstream targets, i.e., the p70 S6
ribosomal kinase 1 (p70S6K1)
64
and the eukaryotic initiation
factor 4E-binding protein 1 (4E-BP1).
65
Moreover, mTORC1
is involved in the regulation of dierent proteins, including
cyclins, cyclin dependent kinases, protein phosphatases and
RNA polymerases.
66
The second complex, mTORC2, is involved in the regulation
of cytoskeleton functions by stimulating actin bers, paxillin,
RhoA, Rac1 and protein kinase C (PKC).
67
Importantly,
mTORC2 activates Akt, which is upstream of mTOR in the
mTORC1 pathway and downstream in the mTORC2 pathway.
Similarly to mTORC1, mTORC2 appears to be activated by
growth factors, perhaps in an Akt-independent manner.
mTOR activity is deregulated in human diseases, particularly
in malignancies, in which increased levels and/or the
Figure 3. A. Binding mode of ATP in the active kinase conformation. B. Binding mode of a type II inhibitor (imatinib). Type II inhibitors bind to
both the ATP binding site and an immediately adjacent hydrophobic pocket, an allosteric site, which is created when the DFG motif is in the out
conformation, thus freezing the kinase in the inactive state.
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phosphorylation of its downstream targets have been correlated
with tumor aggressiveness and poor prognosis.
68
The macrolide
rapamycin and its derivatives are selective and allosteric
inhibitors of mTOR. They form a complex with the ubiquitous
intracellular protein FK506-binding protein-12 (FKBP12); this
complex binds to the FRB (FKBP-rapamycin binding) domain
of mTOR, located near the kinase domain,
69
and inhibits
enzymatic functions.
70
However, rapamycins inhibit the
enzyme in the mTORC1 complex, but not in mTORC2,
which was originally dened as rapamycin insensitive. It has
subsequently been shown that rapamycins suppress mTOR
activity in both mTORC1 and mTORC2 complexes but at very
dierent concentrations; indeed, whereas mTORC1 is inhibited
by low nanomolar range concentrations of rapamycin,
mTORC2 inhibition requires low micromolar concentrations.
71
However, low doses of rapamycin have been shown to induce
feedback mechanisms that lead to an IGF1 receptor-dependent
mTORC2 activation and subsequent increased Akt phosphor-
ylation. The dierential inhibitory activity of rapamycins against
the two mTOR complexes may limit their eectiveness as
antitumor agents; indeed, the overall objective response rates
achieved with rapamycin therapy for solid tumors have been
modest. In preclinical and clinical settings, the treatment of
certain tumors with rapamycin increased PI3K/Akt activity,
thus decreasing the therapeutic potential of mTORC1
inhibition.
72
The discovery that mTORC2 directly phosphor-
ylates Akt, which is usually hyperactivated in tumors, suggests
that mTORC2 inhibitors could be valuable anticancer drugs.
67
Furthermore, recent studies in cancer biology have suggested
that mTORC2 activity is essential for the survival of several
cancer cells but is less necessary in normal cells. For all of these
reasons, several small molecule ATP-competitive inhibitors that
target the kinase domain of mTOR, in both mTORC1 and
mTORC2, have been developed in the past few years with the
aim of circumventing the problems associated with allosteric
inhibition by rapamycins.
68
The inhibition of signaling through
mTORC1 is usually detected by a decrease in the
phosphorylation of proteins such as 4E-BP1 and p70S6K1,
whereas the inhibition of mTORC2 is indicated by decreased
Akt Ser473 phosphorylation.
Table 1. Examples of Pyrazolo-Pyrimidines, Their Target Kinases, Pharmaceutical Company or Author, and Main Activities
target kinase key compound research group biological activity
mTOR 141a Wyeth IC
50
of 0.21 nM in enzymatic assays.
WYE-132
82
IC
50
of 2 nM in LNCaP cells.
antitumor in vivo activity.
p70S6K 147 Exelixis IC
50
of 2 nM in enzymatic assays.
XL418
89
IC
50
values of 30 and 55 nM in A549 and PC-3 cells, respectively.
antitumor in vivo activity.
PI3K 152
97
Genentech IC
50
of 3 nM for PI3K in enzymatic assays.
PI3K/mTOR 155
98
Wyeth IC
50
values of 1, 68, and 13 nM against PI3K, PI3K and mTOR, respectively, in
enzymatic assays.
Raf 159
110
Genentech IC
50
of 0.9 nM against B-Raf V600E in enzymatic assays.
p38 MAPK 162a
111
Bristol-Myers
Squibb
IC
50
of 5 nM in enzymatic assays.
169c
112
Novartis IC
50
of 6 nM for the inhibition of TNF production in human peripheral blood
mononuclear cells.
IC
50
of 0.6 nM in enzymatic assays.
96% inhibition of LPS-induced TNF release in mice
GSK-3 189a
130
GlaxoSmith pIC
50
of 8 in enzymatic assays.
Kline
CDK2 192
136
Bristol-Myers
Squibb
IC
50
of 18 nM in enzymatic assays.
CDK9 198
138
Exelixis Inc. IC
50
of 16 nM in NCI H460 cells.
antitumor in vivo activity.
IC
50
of 1 nM in enzymatic assays.
Aurora kinase 205
141
Biogen IC
50
values of 4, 5, and 22 nM against Aurora kinase A, Aurora kinase B and CDK1,
respectively.
IC
50
of 10 nM in HCT116 cells.
EGFR 222a
157
AstraZeneca IC
50
values of 1 and 5 nM against ErbB2 and EGFR, respectively, in enzymatic assays.
IC
50
of 17 nM in BT474C cells.
IGF-1R 226a, A-928605
166
Abbott IC
50
of 37 nM in enzymatic assays.
IC
50
of 90 nM in A431 cells.
antitumor in vivo activity.
Src and other SFK
members
230a PP1, and
230b PP2
176
Hanke et al. IC
50
values in the nanomolar range in enzymatic assays.
Engineered Src 233a Shokat et al. antiproliferative activity in several cell lines.
1NM-PP1
183
IC
50
of 1.5 nM against engineered Src (I338G v-Src).
Lck 244 Abbott IC
50
of 147 nM in enzymatic assays.
A-770041
193
Abl 2633h
209,214
Schenone et al. K
i
of 80 nM in enzymatic assays.
Antiproliferative activity on CML cell lines.
Btk 281a Pharmacyclics Inc. IC
50
of 0.5 nM in enzymatic assays.
PCI-32765 ibrutinib
233
approved November 2013 for the treatment of mantle cell lymphomas.
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The largest family of mTOR inhibitors is the pyrazolo[3,4-
d]derivatives.
4.1.1. Synthesis and Biological Properties of 4-
Morpholino Derivatives. Zask and colleagues from Wyeth
have synthesized several potent mTOR inhibitors, including
117 (WAY-600), 121a (WYE-687), and 123 (WYE-354), by
preparing the pyrazolo[3,4-d]pyrimidine scaold starting from
the pyrimidine ring or the pyrazole ring.
73
The synthesis of a
number of compounds starts with 2,4,6-trichloropyrimidin-5-
carbaldehyde 106 (Scheme 37).
The reaction of 106 with the appropriate hydrazine aords
4,6-dichloropyrazolo-pyrimidine 114. The chlorine atom in C4
is selectively displaced by morpholine, leading to 115. Suzuki
coupling with the appropriate boronic acid gives compound
116. Then, the benzyl protecting group on the piperidinyl
nitrogen is removed by hydrogenation, and the resulting
secondary amine is reductively alkylated to give 117 (WAY-
600).
For the synthesis of other derivatives, compound 115 was
debenzylated with -chloroethyl chloroformate (ACE-Cl) to
aord the free piperidino derivative 118, which is converted
into picolyl-functionalized intermediate 119 by reductive
amination. When 119 was subjected to Suzuki-Miyaura
coupling conditions, the 6-anilino-derivative 120 was produced,
which was treated with triphosgene and TEA and subsequently
with a suitable alcohol or amine to aord carbamoylphenyl or
ureidophenyl derivatives 121, including 121a (WYE-687;
Scheme 38).
74
Product 118 was treated with a suitable carbamoyl chloride
to aord intermediates 122, which were subjected to the
reaction sequence presented for 121 to aord the ureas or
carbamates 123, including 123a (WYE-354; Scheme 39).
75
Inhibitors 117 (WAY-600), 121a (WYE-687) and 123a
(WYE-354) are the most studied compounds.
76
These
compounds inhibit mTOR with IC
50
values in the 59 nM
range, show signicant selectivity (>100-fold) over PI3Ks in
enzymatic assays, and block substrate phosphorylation by
mTORC1 and mTORC2 in vitro in response to growth factors,
amino acids and PI3K/Akt hyperactivation. These pyrazolo-
pyrimidines inhibited the phosphorylation of p70S6K1 and Akt
and showed antiproliferative activity associated with G1 cell
cycle arrest, apoptosis induction and the inhibition of protein
synthesis in rapamycin-sensitive and rapamycin-resistant cancer
cell lines. Importantly, 123a (WYE-354) is also active in vivo,
showing antitumor activity when injected into mice bearing
PTEN-null U87MG glioma.
4.1.2. Molecular Modeling Studies and Substitution
Optimization. Molecular modeling studies have demonstrated
the importance of a urea moiety in position C6, showing that
this group interacts more favorably with the ATP-binding site
of the enzyme than the corresponding carbamoylphenyl
group.
7375
Indeed, the same Wyeth researchers built an mTOR
homology model, starting from the known X-ray crystal
structure of PI3K, which was based on the highly similar
sequences of the ATP-binding sites of the two enzymes.
The X-ray structure of a pyrazolo-pyrimidine inhibitor bound
to PI3K (PDB code 3IBE) was used as the basis for docking
studies with the mTOR homology model and showed that the
urea group of the inhibitor makes three hydrogen bond
contacts within the ATP-binding pocket, two between the urea
NHs and Asp2195 and one between the urea carbonyl and
Lys2187. The binding mode of these inhibitors showed the
importance of the C4 morpholino substituent, which
participates in a critical interaction with Val2240 in the hinge
region of mTOR. Moreover, the picolylpiperidine tail in the
front of the binding site interacts with the region containing
Leu2249 and Ala2248 (Figure 4).
Scheme 37. Synthesis of the mTOR Inhibitor 117 (WAY-
600)
Scheme 38. Synthesis of the mTOR Inhibitor 121a (WYE-
687)
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Based on the promising results obtained with this class of
compounds, other potent 4-morpholino-pyrazolo-pyrimidine
mTOR inhibitors with a ureidophenyl substituent at the 6-
position have been synthesized.
6-Ureidophenyl derivatives can be obtained with the
procedures reported above as well as a dierent synthetic
approach in which the appropriate ureidophenylboronate 124,
obtained by reaction of the commercially available 4-
isocyanatophenylboronic acid pinacol ester with amines,
undergoes Suzuki-Miyaura coupling with aryl chloride 115 to
aord 125 (Scheme 40).
74
Structure-based drug design suggested the preparation of
several 6-arylureidophenyl-1H-pyrazolo[3,4-d]pyrimidines sub-
stituted with water-solubilizing groups on the arylureido
moiety; compound 126a (Figure 5), with a 4-hydroxymethyl-
phenyl group, showed an IC
50
value of 0.08 nM against mTOR,
as well as increased water solubility and cellular potency.
75
Derivatives bearing ethyl, pyrrolidino, morpholino or
methylpiperazino groups as solubilizing moieties on the para
position of the urea phenyl ring showed mTOR inhibition and
unprecedented antiproliferative activity in cell assays; com-
pound 126b (Figure 5) showed an IC
50
value of 0.7 nM in an
enzymatic assay and an IC
50
value <1 nM in LNCaP cells.
74
The eects of C4 substitution on the potency and selectivity
of these pyrazolo-pyrimidines were also investigated.
77
Chiral
and achiral methyl-substituted morpholines as well as chiral and
achiral bridged morpholines were introduced at C4 of specic
C6-phenylurea derivatives. Of the dierent groups introduced
at C4, the bridged morpholino substituents resulted in very
active and selective mTOR inhibitors. For example, the
phenylethyl urea 127a (Figure 6), substituted with a 2,6-
ethylene bridged morpholine, is a potent mTOR inhibitor. It
showed an IC
50
value of 0.2 nM for mTOR and an extremely
high selectivity (26665-fold) against PI3K. Moreover, it
Scheme 39. Synthesis of the mTOR Inhibitor 123a (WYE-354)
Figure 4. Schematic interactions between an ureidophenyl pyrazolo-
pyrimidine and mTOR in the homology model of mTOR ATP-
binding pocket. Hydrogen bonds are indicated as dotted lines.
Scheme 40. Synthesis of 4-Morpholino-6-ureidophenyl-1H-
pyrazolo[3,4-d]pyrimidines Active as mTOR Inhibitors
Figure 5. Structures of the mTOR inhibitors 126a and 126b.
Figure 6. Structures of the mTOR inhibitors 127a and 127b, with
bridged morpholines in C4.
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exhibited a strong antiproliferative activity in LNCaP cells.
Methyl derivative 127b (Figure 6), substituted with a 3,5-
ethylene bridged morpholine, showed similar biological
behavior.
Docking studies provided an explanation for the exceptional
mTOR selectivity of the bridged morpholine analogues: the
presence of a leucine in mTOR instead of the larger
phenylalanine in PI3K at residue 961 creates a deeper pocket
that can accommodate the additional ethylene moiety of the
bridged morpholines without losing any of the hydrogen bond
interactions with the backbone amino acids.
77
The 2,6-
ethylene-bridged morpholine at C4 was also combined with
Scheme 41. Synthesis of N1-Triuoroethyl-Substituted 1H-Pyrazolo[3,4-d]pyrimidines mTOR Inhibitors
Scheme 42. Synthesis of Other Triuoroethyl Analogues Bearing a 3,6-Dihydro-2H-pyran Group at C4
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dierent N1 substituents, leading to very active and selective
mTOR inhibitors, such as the triuoroethyl analogues 133a and
133b.
78
The synthesis of these compounds is presented in Scheme
41.
The starting product is the triuoroethylpyrazole 128, which
was acylated with para-nitro-benzoyl chloride to aord
intermediate 129. Intermediate 129 was cyclized with H
2
O
2
in an alkaline medium to give pyrazolo-pyrimidine 130. The
latter was chlorinated with POCl
3
in the presence of DMF to
aord the C4-chloro derivative 131, which was reacted with 8-
oxa-3-azabicyclo[3.2.1]octane hydrochloride in the presence of
TEA to give 132 after reduction of the nitro group in position
C6. The treatment of 132 with triphosgene, followed by
reaction with the appropriate amines, gave desired compounds
133a and 133b (Scheme 41).
78
Compound 133a showed an IC
50
value of 0.23 nM for
mTOR with an mTOR/PI3K selectivity of approximately 12
000-fold. The methylpiperazino derivative 133b exhibited IC
50
values of 0.6 and <0.8 nM in enzymatic and LNCaP cell-based
assays, respectively, as well as favorable pharmacokinetic
properties. Moreover, the compound caused inhibition of
tumor growth and was well tolerated in an MDA-361 nude
mouse xenograft model at low doses (1025 mg/kg).
Importantly, intermittent (every fth day) intravenous
administration of a 20 mg/kg dose resulted in complete
tumor stasis.
78
Other N1-triuoroethyl analogues have a 3,6-dihydro-2H-
pyran (DHP) group at C4 as an alternative to morpholine or
bridged-morpholine for the hinge region binding motif.
79
In the
synthesis of these compounds, the starting product is 4-chloro-
6-(4-nitrophenyl) derivative 131, which is hydrogenated in the
presence of di-tert-butyldicarbonate (BOC
2
O) to aord 134.
Compound 134 was treated with tributyl(3,6-dihydro-2H-
pyran-4-yl)stannane under Stille conditions
80
to give the
coupling product 135. The removal of the BOC protecting
group with triuoroacetic acid (TFA) gave aniline intermediate
136, which was treated with triphosgene, followed by the
addition of selected amines to furnish the nal ureas 137
(Scheme 42). Despite the fact that this synthetic procedure was
not optimized by the authors, the products were obtained in
sucient yield and purity for medicinal chemistry purposes.
Compound 137a,
81
with a uoroethyl substituent on the
urea moiety, possessed an IC
50
value of 1 nM in enzymatic
assays and an IC
50
value of 72 nM in an antiproliferative assay
in LNCaP cells, which are comparable to the IC
50
values for the
corresponding morpholino derivative. However, 137a showed
improved selectivity and increased microsomal stability.
Molecular modeling studies showed similar binding modes
for the inhibitors containing the morpholine and DHP groups.
The oxygen atoms of these C4 substituents were overlapped,
similarly participating in H-bonds to the hinge region valine.
Nevertheless, the DHP-substituted compounds were less active
than the corresponding 2,6-ethylene bridged morpholino
derivative previously reported. The authors examined other
N1 substitutions, focusing particularly on cyclic groups
structurally related to piperidine, which was present in most
of the previously reported compounds. One of the most
interesting compounds, 141a, was obtained from 1,4-cyclo-
hexanedione monoethyl ketal 138. This compound was reacted
with t-butyl carbazate, and the t-Boc group was then removed
in reuxing water. The obtained hydrazine 139 was reacted
with aldehyde 106 and 2,6-ethylene bridged morpholine to
aord chloro derivative 140. The procedure reported for the
synthesis of similar derivatives gave nal compounds 141,
including 141a (WYE-132; Scheme 43).
82
WYE-132 showed IC
50
values of 0.21 nM and 2 nM in
enzymatic and LNCaP cell-based assays, respectively, with
5619-fold selectivity over PI3K and good stability in both
mouse and human microsomes.
82
Importantly, it possesses
antitumor activity in a number of cancer models in vitro and in
vivo. The oral administration of WYE-132 in tumor-bearing
mice resulted in potent antitumor activity against MDA361
breast, U87MG glioma, A549 and H1975 lung, and A498 and
786-O renal tumors. In a nude mouse xenograft model of
MDA361 cells, a dose of 10 mg/kg/day resulted in tumor
Scheme 43. Synthesis of the mTOR Inhibitor 141a (WYE-132)
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stasis, whereas a dose of 20 mg/kg/day led to the complete
inhibition of tumor growth without evident toxicity. Compared
with the rapamycin derivative temsirolimus/CCI-779, WYE-
132 demonstrated substantially stronger inhibition of cancer
cell growth and survival, protein synthesis, metabolism, and
adaptation to hypoxia.
83
4.1.3. Other mTOR Inhibitors. Shokat and colleagues have
reported selective mTOR inhibitors that are pyrazolo[3,4-
d]pyrimidines, namely 144 (PP242) and 145 (PP30; Scheme
44), bearing an NH
2
group at C4, an isopropyl substituent at
N1 and heterocyclic substituents at C3.
84
Unlike other
pyrazolo-pyrimidines reported by the same authors that target
multiple kinases (including TKs, PI3Ks and mTOR), these two
compounds exhibited exceptional mTOR selectivity with
respect to a panel of 219 kinases (including PI3Ks). PP242
and PP30 inhibited mTOR with IC
50
values of 8 and 80 nM,
respectively, both mTORC1 and mTORC2 in an ATP-
competitive fashion, and displayed more dramatic eects on
cell growth, proliferation, cell cycle and cap-dependent
translation than rapamycin. Inhibition of mTORC2 results in
the blocking of Akt phosphorylation at Ser473, preventing full
activation of the kinase. However, the authors demonstrated
that mTORC2 inhibition is not responsible for the enhanced
activity of these compounds compared to rapamycin. In
particular, they showed that the improved activity of PP242
depends on the inhibition of mTORC1 functions that are
insensitive to rapamycin but eectively targeted by ATP-
competitive mTOR inhibitors. Whereas both rapamycin and
PP242 inhibited p70S6K1 phosphorylation, only PP242 fully
inhibited the phosphorylation of 4EBP1 at Thr36/45 and
Ser65, which was only modestly decreased by rapamycin.
85
In a
recent study, Janes and colleagues found that PP242, but not
rapamycin, induces the death of mouse p190 transformed cells
and K562 human leukemic cells harboring the Philadelphia
chromosome translocation, the hallmark of CML. PP242 delays
leukemia onset in vivo and increases the eects of the current
front-line tyrosine kinase inhibitors, such as dasatinib, more
eectively than rapamycin. Unexpectedly, PP242 has much
weaker eects than rapamycin on the proliferation and function
of normal lymphocytes.
86
The starting compound for the synthesis of 144 (PP242) and
145 (PP30) was 1H-pyrazolo[3,4-d]pyrimidin-4-amine 14,
which was treated with NIS under standard conditions to
give 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine 74. The
latter intermediate was converted into the corresponding 1-
isopropyl derivative 142 by reaction with isopropyl bromide.
Compound 142 was then reacted with a protected indolyl
boronic acid derivative via palladium-catalyzed Suzuki coupling
to provide 143. Removal of the protecting groups yielded the
nal product 144 (PP242). Similarly, for the synthesis of 145
(PP30), 142 was coupled with the appropriate boronic acid
(Scheme 44).
84,87
4.2. 70-kDa Ribosomal Protein S6 Kinase (p70S6K)
Inhibitors
The 70-kDa ribosomal protein S6 kinase (p70S6K) is a serine-
threonine kinase in the PI3K/Akt/mTOR pathway. It has been
implicated in cancer; thus, there is much research toward
p70S6K inhibitors. Researchers at Exelixis recently identied a
family of pyrazolo-pyrimidines, including 146 (Figure 7), that
are nanomolar inhibitors of p70S6K in enzymatic and cell-
based assays (A549 and PC-3 cells).
88
Moreover, compound
146 proved to be selective for p70S6K over other kinases,
showed a reasonable ADME (adsorption, distribution, metab-
olism, and excretion) prole and was ecacious in the PC3
xenograft model.
Scheme 44. Synthesis of 144 (PP242) and of 145 (PP30)
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The same authors also synthesized 3-bromo derivative 147
(Figure 7), with a C4-piperazine moiety, as a dual Akt/p70S6K
inhibitor. The Akt inhibition of this derivative results in
improved ecacy in xenograft studies compared to 146. Due to
its excellent in vitro and in vivo activity, combined with good
oral bioavailability in higher species, compound 147 entered
phase I clinical trials for solid tumors and hematological
malignancies under the name XL418.
89
Unfortunately, this
study has suspended participant recruitment due to low drug
bioavailability.
4.3. PI3K Inhibitors and Dual PI3K/mTOR Kinase Inhibitors
The PI3K (phosphatidylinositide 3-kinase) family includes 15
lipid kinases with distinct substrate specicities and expression
patterns. PI3Ks belong to the same signaling pathway as mTOR
and are classied as IA, IB, II, or III based on their substrate
specicity and sequence homology.
90
These kinases phosphor-
ylate the 3-hydroxy position of phosphatidylinositol 4,5-
diphosphate, a membrane phospholipid, to form phosphatidy-
linositol 3,4,5-triphosphate, which regulates cell shape, motility,
growth, dierentiation, and survival via Akt activation. In
particular, class IA PI3Ks are heterodimers that contain one of
three PI3K catalytic subunits (PI3K, PI3K, PI3K) and a p85
regulatory subunit. The gene encoding the PI3K subunit is
mutated and/or overexpressed in breast, ovarian, colorectal,
brain (glioblastoma), and gastric cancers. Thus, PI3K has
emerged as an attractive target for cancer therapy, and selective
PI3K inhibitors have been synthesized and tested in
preclinical and clinical trials. Along with their potential
therapeutic interest, these inhibitors have been extremely
helpful tools for exploring the role of PI3K enzymes in signal
transduction and downstream physiological and pathological
processes.
91
It has also been demonstrated that the PI3K pathway is
paradoxically activated following selective mTOR inhibition,
thus providing a rationale for developing dual PI3K/mTOR
kinase inhibitors.
92
Indeed, given the complexity of the mTOR
signaling pathway, with its multiple feedback systems and
crosstalk with other pathways, compounds targeting more than
one enzyme could be more eective in therapeutic strategies. In
particular, these drugs could completely block the feedback
activation of PI3K/Akt and subsequently neutralize the survival
pathways activated upstream of mTOR by PI3K and Akt.
93
Although mTOR is a serine-threonine kinase and PI3K is a
lipid kinase, they show high sequence homology in the hinge
region (also called the catalytic cleft). For this reason, some
compounds originally discovered as PI3K inhibitors were later
demonstrated to target mTOR and are currently considered
dual PI3K/mTOR inhibitors.
Despite past eorts to develop selective targeted inhibitors
for the treatment of cancer, current strategies are focused on
nding compounds that act on multiple targets to overcome
drug resistance connected to the activation of alternative
signaling pathways. In this context, several molecules developed
in the past few years are dual kinase inhibitors. Usually, target
combinations are suggested by pathway connectivity. For
example, mTOR activates the well-known negative feedback
loop that inhibits the activity of PI3K. mTOR inhibitors block
this negative feedback, resulting in hyperactivation of PI3K.
94
Therefore, dual inhibition of PI3K and mTOR has been
predicted to be more eective than a single target strategy.
4.3.1. Synthesis and Biological Properties. All mTOR
inhibitors reported in this article have also been tested against
PI3K, and the most active compounds are specic for mTOR,
showing an mTOR/PI3K selectivity from 10 to more than
10000. However, in 2010, Wyeth researchers reported a related
series of pyrazolo[3,4-d]pyrimidines that are potent PI3K
inhibitors with good selectivity versus mTOR and PI3K.
Compound 148 (Figure 8), obtained using a synthetic
procedure similar to that reported for other mTOR inhibitors,
proved to be active against PI3K, PI3K, and mTOR with
IC
50
values of 36, 993, and 215 nM, respectively. Moreover, it
possessed antiproliferative activity against dierent cancer cell
lines (e.g., LoVo and PC3). The inhibitory activity for the PI3K
signaling pathway was conrmed by decreased phosphorylation
of Akt, downstream of PI3K, in MDA-361 human breast cancer
cells.
95
Genentech patented a series of pyrazolo[3,4-d]pyrimidines
bearing a 3-methoxy-anilino group at C4 and dierent
heterocyclic rings at C6, which were claimed to be PI3K
(including p110) inhibitors.
96
For example, derivative 150
inhibits these enzymes with IC
50
values in the nanomolar range.
It was synthesized from 149 by Suzuki coupling with 2-amino-
pyrimidine boronic acid pinacol ester (Scheme 45).
Figure 7. Chemical structure of the p70S6K Inhibitors 146 and 147
(XL418).
Figure 8. Chemical structure of compound 148.
Scheme 45. Synthesis of the PI3K Inhibitor 150
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Genentech researchers also synthesized dierent pyrazolo-
[3,4-d]pyrimidines as potent PI3K inhibitors; these were
obtained by modifying a lead compound, following suggestions
obtained from X-ray crystallization and molecular modeling
studies.
97
Compound 152, with a 4-indazolyl substituent at C6
and an aryl ether at C4, is one of the most active compounds,
with an IC
50
value of 3 nM for PI3K and improved
bioavailability with respect to the other members of this series.
The synthesis of 152 starts from 1-methyl-4,6-dichloro-1H-
pyrazolo[3,4-d]pyrimidine 17, which reacts with 4-
(methylsulfonyl)phenol and NaH to form 4-substituted
intermediate 151. A standard Suzuki coupling reaction with
the appropriate boronic acid then provided the nal compound
152 (Scheme 46).
Pyrazolo[3,4-d]pyrimidine mTOR/PI3K inhibitors have
been patented by Wyeth. As an example, compound 155
showed IC
50
values of 1, 68, and 13 nM for PI3K, PI3K, and
mTOR, respectively.
98
The compound was synthesized from
17, which was reuxed with 4-aminobenzamide 153. Then, N-
(4-aminophenyl)-N-pyridin-3-ylurea 154 was added, and the
resulting mixture was reuxed for 3 days, leading to the
formation of the nal compound (Scheme 47).
The 1,4,6-trisubstituted derivative 156 (Figure 9) and its
analogues, all bearing a C6 morpholino group, have been
patented by Tyrogenex Inc. as PI3K inhibitors and show IC
50
values of less than 1 M for dierent PI3K isoforms.
99
4.4. Mitogen Activated Protein Kinase (MAPK) Inhibitors
The MAPK (mitogen-activated protein kinase) pathway
includes several key signaling kinases responsible for
phosphorylation events that play a role in tumorigenesis.
These enzymes transmit extracellular signals that regulate cell
growth, dierentiation, proliferation, apoptosis and migration.
Alteration of the Ras-Raf-MEK-ERK-MAPK (Ras-MAPK)
pathway has been reported in human cancer.
100
In particular, B-Raf, a member of the Raf serine-threonine
kinases, is the main MEK activator, and its gene is one of the
most frequently mutated in malignancies.
101
Small molecule B-Raf kinase inhibitors are being developed
as anticancer compounds
102
on the basis of preclinical and
clinical studies that have led to the approval of sorafenib
tosylate (Nevaxar) (a multikinase inhibitor that also acts on B-
Raf) for the treatment of advanced renal carcinoma and
unresectable hepatocellular carcinoma.
103
In August 2011,
vemurafenib, a pyrrolo-pyridine B-Raf inhibitor that acts only
on the V600E mutated form, was approved by the FDA for the
treatment of late-stage melanomas in patients bearing this
mutation (approximately 60% of the total).
104
However, some members of the complex Ras-MAPK
pathway, e.g., p38MAPKs, are targets for anti-inammatory
agents.
105
Indeed, the serine-threonine kinase p38 family is
involved in the inammatory response
106
and regulates the
production of inammatory cytokines, including TNF-, IL-1,
and IL-6. p38 inhibitors could prove interesting for the
treatment of rheumatoid arthritis, pain and respiratory
diseases.
107
Pyrazolo[3,4-d]pyrimidine compounds are among the high
number of inhibitors targeting dierent members of the MAPK
pathway.
108
4.4.1. Raf Inhibitors. In a recent patent, Genentech
researchers reported a family of pyrazolo[3,4-d]pyrimidines
with a substituted urea moiety at C4 as B-Raf inhibitors.
109,110
Compounds 159 are among the most active derivatives of the
series, with IC
50
values against B-Raf V600E of 0.9 nM when R
= C
2
H
5
and 1.6 nM when R = CH
3
. For their synthesis, 5-
Scheme 46. Synthesis of the PI3K Inhibitor 152
Scheme 47. Synthesis of the mTOR/PI3K Inhibitor 155
Figure 9. Chemical structure of compound 156.
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amino-3-alkoxy-1H-pyrazole-4-carbonitriles 157 and formami-
dine acetate were reacted under microwave irradiation to aord
the corresponding pyrazolo[3,4-d]pyrimidines 158 in 22%
yield. The nal compounds 159 were obtained by reaction of
158 with phenyl 2,6-diuoro-3-[(propylsulfonyl)amino]-
phenylcarbamate (Scheme 48).
4.4.2. p38 MAPK Inhibitors. Bristol-Myers Squibb
synthesized a number of pyrazolo[3,4-d]pyrimidines as potent
and selective p38 MAPK inhibitors that inhibited TNF-
production in cells.
111
For the synthesis of C4/C6 diamino-
substituted compounds, the 4,6-dichloro-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidine (17) was reacted with the appro-
priate aniline in absolute ethanol in the presence of N,N-
diisopropylethylamine (DIPEA). This procedure formed the 4-
anilino derivative 160 exclusively, which gave compound 161
when treated with N-methyl-homopiperazine (Scheme 49).
Following a similar synthetic procedure, the authors obtained
derivatives 162 (Figure 10).
111
An alternative route was used for 6-NH
2
substituted
derivatives. Base-catalyzed condensation of phenyl hydrazine
and 2-amino-4,6-dichloro-pyrimidine-5-carboxaldehyde 97 in
reuxing THF gave intermediate 163, which aorded 164 upon
treatment with the appropriate aniline (Scheme 50).
Several C6 amino-substituted and C6 unsubstituted com-
pounds were shown to be low-nanomolar inhibitors of p38.
SAR optimization of dierent fragments of the pyrazolo-
pyrimidine ring led to the identication of the isoxazole-
substituted compound 162a (Figure 10), which possessed IC
50
values of 5 nM against p38 in the enzymatic assay and 6 nM
against TNF production in human peripheral blood
mononuclear cells. Importantly, the compound is also active
in vivo in inhibiting TNF production in an acute murine
model of lipopolysaccharide (LPS)-induced TNF produc-
tion.
111
Other pyrazolo[3,4-d]pyrimidine p38 MAPK inhibitors are
substituted with amino groups at C6 or C3 and, unlike the
kinase inhibitors above, they do not possess a C4 amino group.
For this reason, they were obtained through a dierent
synthetic approach.
Novartis researchers have reported a number of pyrazolo-
heteroaryls, including pyrazolo[3,4-d]pyrimidines 169, which
have a C6 2,4-diuoro-anilino substituent.
112
The synthesis of compounds169 started from 5-bromo-2-
methoxybenzaldehyde 165, which was reacted with 5-lithio-2,6-
dichloropyrimidine formed in situ from 2,6-dichloropyrimidine
and LDA (lithium diisopropylamide); oxidation of the resulting
alcohol gave ketone 166. Reaction with hydrazine at low
temperature gave pyrazolo-pyrimidine 167, which aorded 168
in high yields upon treatment with 2,4-diuoroaniline.
Sonogashira coupling of 168 with a series of alkynes aorded
the nal compounds 169 (Scheme 51). The most active
compounds showed IC
50
values in the nanomolar range for
p38 inhibition in enzymatic assays and strong (96%)
inhibition of LPS-induced TNF release in mice at an orally
administered dose of 20 mg/kg. The compounds were also
active in animal models of rheumatoid arthritis.
Recently, Roche researchers synthesized a number of 3-
amino-pyrazolo[3,4-d]pyrimidines as p38 inhibitors.
113
The
most active compound, 177 (R6226), was synthesized starting
with the reaction of 5-bromo-2,4-dichloropyrimidine 170 and
2,4-diuorophenoxide to aord 5-bromo-2,4-bis(2,4-diuoro-
phenoxy)-pyrimidine 171. The latter compound was acylated at
C5 via a Grignard reaction with isopropyl magnesium chloride
and CO
2
. Intermediate 172 was reacted with oxalyl chloride
and then (R)-1-methyl-2-methylsulfanyl-ethylamine to give
compound 173, which was subsequently oxidized to the
corresponding sulfone 174. Sulfone 174 was converted to nal
compound 177 by a three-step synthetic sequence. Specically,
intermediate 174 was reacted with 2,6-lutidine and TFAA to
give compound 175, which possessed a triuoroacetate group
Scheme 48. Synthesis of the Raf Inhibitor 159
Scheme 49. Synthesis of the p38 MAPK Inhibitor 161
Figure 10. Structure of compounds 162.
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that was displaced with t-butyl carbazate to obtain 176. Finally,
176 was cyclized by treatment with TFA in DCM to aord the
nal compound 177 (Scheme 52).
Compound 177 showed an IC
50
of 0.109 M for p38 in an
enzymatic assay and high selectivity in a panel of 363 kinases.
114
It also showed an IC
50
value of 0.039 M against the LPS-
induced production of interleukin-1 in human whole blood as
well as good bioavailability in vivo.
Other potent p38 MAPK inhibitors from the same company
are 178,
115,116
which had an IC
50
of 10 nM, and the 4-amino-3-
aryl-substituted derivatives 179
117
and 180
118
(Figure 11),
which had IC
50
values of 2 and 4 nM, respectively.
4.5. Glycogen Synthase Kinase-3 (GSK-3) Inhibitors
Glycogen synthase kinase-3 (GSK-3), which exists in the two
isoforms GSK-3 and GSK-3, is a serine-threonine kinase that
is ubiquitously expressed and involved in the regulation of
several processes. First discovered in 1980 as a regulatory
kinase for glycogen synthase (GS), GSK-3 has since been
identied as a kinase for over 40 dierent proteins in a variety
of pathways.
119
GSK-3 has recently been the subject of much research
because it has been implicated in a number of diseases,
including type 2 diabetes, Alzheimers disease (AD), bipolar
disorders, inammation, and cancer.
120
In particular, GSK
activity and expression are higher in type 2 diabetic patients
than in healthy subjects.
121
For these reasons, GSK-3 inhibitors
may be useful for the treatment of type 2 diabetes mellitus.
GSK-3 phosphorylates the cytoskeletal protein Tau, which if
hyperphosphorylated, is involved in the onset of neuro-
generative diseases, including AD.
122
Inhibitors of GSK-3
could have therapeutic applications in such pathologies.
123
GSK-3 also participates in cancer-related pathways, such as the
Wnt pathway
124
and nuclear factor B pathway.
125
These recent ndings have shown the potential utility of
GSK-3 inhibitors as anticancer agents.
126,127
New studies also
focus on the specic contributions of GSK-3 and GSK-3 to
AD pathology and acute myeloid leukemia.
128
4.5.1. Synthesis and Biological Properties. Thomson
and colleagues at GlaxoSmithKline identied a novel lead
pyrazolo-pyrimidine structure as a GSK-3 inhibitor and
proceeded to optimize this family of compounds. These
authors synthesized a number of hydrazones of the general
structure of 181 with dierent aromatic or heteroaromatic N1
substituents and usually a 4-pyridyl group on the hydrazone
moiety.
129
The compounds were obtained by reaction of the 4-
chloro-pyrazolo-pyrimidine intermediates 5 with hydrazine
followed by condensation with 4-pyridyl aldehyde (Scheme
53).
Compound 181a is a potent GSK-3 inhibitor with a pIC
50
of
8.2 and low nanomolar ecacy for the stimulation of glycogen
synthesis in vitro.
130
In light of SAR studies and structural
analysis of the GSK-3 binding pocket, the authors incorporated
a benzimidazole group at N1 and obtained derivatives 189
(Scheme 54).
Scheme 50. Synthesis of the p38 MAPK Inhibitor 164
Scheme 51. Synthesis of C4-Unsubstituted 1H-Pyrazolo[3,4-d]pyrimidine p38MAPK Inhibitors
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Scheme 52. Synthesis of the p38 MAPK Inhibitor 177 (R6226)
Figure 11. Structure of other Roche p38 inhibitors.
Scheme 53. Synthesis of GSK-3 Inhibitors
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The treatment of 4-amino-benzidimidazole 182 with BF
3
-
Et
2
O and isoamyl nitrite aorded the intermediate diazonium
salt, which was subsequently reduced with SnCl
2
to the
aryl hydrazine 183. Heating a solution of 183 and
(ethoxymethylene)malononitrile in reuxing ethanol aorded
N1 substituted pyrazole 184, which upon treatment with
trimethyl ortho-formate, gave the imino-ether 185. A Dimroth
rearrangement was then performed by heating in the presence
of excess of hydrazine monohydrate. The rearrangement occurs
via the formation of the cyclic intermediate 186, followed by
ring-opening to 187 and ring-closure to pyrazolo[3,4-d]-
pyrimidine derivative 188.
18,131,132
Compound 188 was reacted
with various aldehydes and a catalytic amount of pyrrolidine in
reuxing ethanol to aord the nal compounds 189. Derivative
189a, with a 4-uoro-phenyl substituent, showed similar activity
to 181a and improved cellular ecacy in stimulating glycogen
synthesis in rat skeletal muscle L6 cell line.
130
4.6. Other Serine-Threonine Inhibitors
4.6.1. Cyclin-Dependent Kinase (CDK) Inhibitors. The
cyclin-dependent kinase (CDK) family is composed of at least
nine highly homologous serine-threonine kinases, which in
association with specic regulatory subunits (cyclins), control
cell cycle progression.
133
The importance of these kinase
pathways is highlighted by the fact that the genes encoding
CDKs, their cyclin partners, or their endogenous peptide
inhibitors (CKIs) are frequently mutated in human tumors.
134
For these reasons, small-molecule CDK inhibitors are being
evaluated as anticancer agents.
135
Markwalder and colleagues synthesized several pyrazolo[3,4-
d]pyrimidin-4-ones as potent inhibitors of CDKs. One of the
most active compounds in the series was 192.
136
Its synthesis
was accomplished starting with pyrazole 190, which reacted
with para-amino-phenylacetic methyl ester in the presence of
sodium ethoxide to aord 6-(4-aminobenzyl)-3-ethyl-1-(2,4,6-
trichlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one 191 in high yield. Finally, the hydrochloride salt of 192 was
prepared by reaction of 191 with N,N-dimethylglycine in the
presence of TEA and 1-ethyl-(3-dimethylaminopropyl)-
carbodiimide (EDC), followed by treatment with 4 N HCl in
dioxane (Scheme 55).
Compound 192 is a potent CDK2 inhibitor with
antiproliferative eects against NCI H460 and other cell lines
and shows activity in mouse xenograft models.
136
Compound 194 is a substituted pyrazolo[3,4-d]pyrimidin-4-
one patented by GCP Biotech AG as a CDK inhibitor and
shows activity in the nanomolar range.
137
In addition, it shows
high selectivity for CDKs when tested against a panel of non-
CDK kinases.
Compound 194 was synthesized from intermediate 193 by
reaction with 3-hydroxy-4-(pyrrolidinomethyl)phenylacetic
acid ethyl ester in the presence of potassium tert-butoxide
(Scheme 56).
In 2010, researchers at Exelixis Inc. patented a number of
substituted pyrazolo[3,4-d]pyrimidines as CDK inhibitors.
138
Some of these compounds are particularly active against CDK9,
which plays a role in immune cell function, showing IC
50
values
in the low nanomolar range.
One of the most active CDK9 inhibitors is 198, which is
synthesized from 67. The C4 chlorine atom was replaced with a
dimethylamine to form 195. The pyrazole-N1 was then
protected with 2-trimethylsilyl)ethoxymethyl chloride
(SEMCl) to give 196, which was subjected to Suzuki coupling
to produce 197, which was nally deprotected to 198 (Scheme
57).
4.6.2. Aurora Kinase Inhibitors. Aurora kinases are
mitotic serine-threonine kinases that play important roles in
the development and progression of cancer by acting as key
regulators of cell proliferation. Small molecule inhibitors
targeting both Aurora kinases and CDKs could present a
useful opportunity in cancer therapy.
139
Biogen researchers
recently reported pyrazolo-pyrimidine derivatives possessing
this dual activity.
140
In particular, compound 204 possessed
nanomolar IC
50
values against Aurora kinase A and CDK1 and
Scheme 54. Synthesis of GSK-3 Inhibitors 189
Scheme 55. Synthesis of the CDK2 Inhibitor 192
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showed ecacy in an HCT116 colon cancer xenograft model
(Scheme 58).
The synthesis of this monosulfonamidophenyl pyrazolo-
pyrimidine compound and its congeners started with the
reaction of thiomethyl pyrazolo-pyrimidine 199 and cyclo-
heptanol. The oxidation of 200 using Oxone aorded methyl
sulfone 201, which was subsequently converted into the aniline
intermediate 202 by reaction with 1,4-diaminobenzene under
standard conditions. Treatment of 202 with methanesulfonyl
chloride under mild basic conditions (DIPEA) resulted in
monomethylsulfonamide 203. The nal product 204 was
prepared by reacting 203 with the appropriate halide in the
presence of K
2
CO
3
.
Docking studies claried the key interactions between
compound 204 and the ATP binding site of Aurora kinase A.
Two hydrogen bond contacts were found between the
pyrazolo-pyrimidine core and the hinge region, specically
the backbone of Ala213. The sulfonamide moiety is able to
Scheme 56. Synthesis of the CDK Inhibitor 194
Scheme 57. Synthesis of the CDK9 Inhibitor 198
Scheme 58. Synthesis of the Aurora Kinase Inhibitor 204
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participate in a hydrogen bond interaction with the backbone of
Thr217. Compound 204 was also predicted to interact in the
same manner with the hinge region of CDK1 because this
kinase shows high sequence homology with Aurora kinase A.
140
Biogen also recently synthesized derivatives such as 205
(Figure 12), which have a substituted pyrrolidine amide as a
polar tail. This compound inhibited Aurora kinase A, Aurora
kinase B and CDK1 with IC
50
values of 4, 5, and 22 nM,
respectively; it showed the desired AKA/CDK1 inhibition in
HCT116 cells but only showed moderate activity in animal
models.
141
5. 1H-PYRAZOLO[3,4-d]PYRIMIDINES AS RECEPTOR
TYROSINE KINASE (RTK) INHIBITORS
Tyrosine kinases (TKs) are enzymes that catalyze the transfer
of the terminal phosphate of ATP to specic tyrosine residues
present on a target substrate, and they regulate growth,
dierentiation, migration, adhesion and apoptosis in mamma-
lian cells.
142
TKs have been subdivided into two families: the rst is the
receptor tyrosine kinases (RTKs), which are activated by ligand
binding to the extracellular receptor domain;
143
the second
family includes the cytoplasmic or nuclear nonreceptor tyrosine
kinases (nRTKs), which are activated by dierent mecha-
nisms.
144
In the human genome, the two families are estimated
to account for 58 and 32 genes, respectively.
145
RTKs include epidermal growth factor (EGF) receptor,
platelet-derived growth factor (PDGF) receptor, vascular
endothelial growth factor (VEGF) receptor, broblast growth
factor (FGF) receptor, nerve growth factor (NGF) receptor,
insulin receptor, Eph, Axl, and Tie. They are single-pass
transmembrane receptor proteins containing three parts: an
extracellular portion, which binds ligands, including growth
factors, cytokines and hormones; a transmembrane hydro-
phobic domain, consisting of 2538 residues; and a C-terminal
intracellular portion, which possesses catalytic activity and
regulatory sequences.
146
RTKs are usually monomeric in the absence of the ligand,
but some exist as multimeric complexes. Ligand binding to the
extracellular region causes dimerization of the monomeric
receptor and rearrangement of the multimeric receptors. This
process promotes kinase activation by the autophosphorylation
of a specic tyrosine located in the cytoplasmic region, thus
allowing signal transduction within the cell.
147
Approximately
20 dierent RTK classes have been identied. RTKs have been
shown not only to be key regulators of normal cellular
processes but also to have a critical role in the development and
progression of many types of cancer.
148,149
5.1. Epidermal Growth Factor Receptor (EGFR) Inhibitors
EGFR (or ErbB1) is a receptor tyrosine kinase belonging to the
ErbB family, which also includes ErbB2, ErbB3 and ErbB4.
150
The overexpression of EGFR and ErbB2 has been implicated in
the development of various types of epithelial cancers and in
proliferative disorders of the epidermis, such as psoriasis. EGFR
TK was one of the rst TKs described 25 years ago, and it has
been a target for drug discovery programs. Subsequent studies
have demonstrated that inhibitors of this TK have great
therapeutic potential for the treatment of malignant and
nonmalignant epithelial diseases.
151
Despite the well-estab-
lished importance of EGFR in tumor growth, progression and
drug-resistant phenotypes, EGFR-targeted therapy has shown
only modest clinical success in cancer patients, except for those
with nonsmall cell lung cancer (NSCLC) carrying EGFR
activating mutations. Even if almost all patients of this type
eventually developed resistance to small molecule EGFR kinase
inhibitors,
152
the use of EGFR inhibitors in rst-line treatment
has provided an unusually large progression-free survival benet
with negligible toxicity compared with cytotoxic chemo-
therapy.
153,154
The search for new EGFR inhibitors is still an
important eld of study for medicinal chemists.
5.1.1. Synthesis and Biological Properties. Traxler and
colleagues at Novartis have synthesized a large number of 4-
phenylaminopyrazolo[3,4-d]pyrimidines as highly potent and
selective inhibitors of EGFR, and these were identied through
the use of a pharmacophore model prepared during a previous
random screening program.
155,156
The synthesis of derivatives
described in the article is presented in Scheme 59.
The [anilino(methylthio)methylene]malononitrile 206a, ob-
tained using the Tominaga method,
23
was reacted with
benzylhydrazine, resulting in the corresponding 5-amino-4-
cyano-pyrazole 207. Ring closure was achieved by reuxing
with 85% formic acid to give intermediate 208, which was
converted into the corresponding chloride 209 by reaction with
phosphorus oxychloride. The substitution of the C4 chlorine
atom with dierent anilines gave the 4-phenylaminopyrazolo-
pyrimidines 210, which aorded the nal products 211 after the
removal of the benzyl group. The reaction of 206a and 206b
with hydrazine monohydrate leads to pyrazoles 212, which are
the starting materials for an improved shorter synthesis that
avoids benzyl protection. The reaction of 212 with N,N-
dimethylformamide-dimethylacetal (DMF-DMA) in toluene
aorded amidines 213, which were converted directly to the
nal product 215 by reaction with meta-chloroaniline in
reuxing alcohols; the authors reported that the reaction
most likely proceeds via an imino intermediate 214, which was
isolated in some cases, that rearranges into the nal products
(Scheme 59).
The authors also synthesized compound 216 with an
aromatic moiety directly attached to C3 of the pyrazole ring
(Figure 13).
155
Optimization of this class of pyrazolo[3,4-d]pyrimidines
primarily focused on derivatives with a variety of C3
substituents, thereby exploiting the cavity of a large hydro-
phobic pocket (hydrophobic region I) identied in the ATP
binding site of the target enzyme. Quite bulky substituents are
tolerated in such positions, as demonstrated by 3-phenylamino
derivatives 211, benzylamino derivatives 215 and phenyl
derivatives 216. The most active compounds in these series
showed IC
50
values in the low nanomolar range against EGFR
and a selectivity ratio >1000 against a wide panel of kinases. In
an EGF-dependent MK cell-based assay, the compounds
Figure 12. Structure of the Aurora Kinase inhibitor 205.
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inhibited EGFR phosphorylation with IC
50
values below 50
nM, whereas they were nearly inactive against PDGF receptor
phosphorylation.
156
Unfortunately, the compounds showed plasma levels after
oral administration in mice that were not sucient to be of
interest for in vivo testing, and the preclinical development of
these compounds was dismissed for this reason and because of
toxicity.
Ducray and colleagues synthesized a number of 3-alkoxy-4-
anilino-1H-pyrazolo[3,4-d]pyrimidines that showed activity as
EGFR and ErbB2 TK inhibitors.
157
The synthesis started from
the known pyrazole 218,
158
which was obtained by reaction of
1,3-dioxolan-2-ylidenemalononitrile (217) with hydrazine.
Intermediate 218 reacted with DMF-DMA to give the amidine
219, which underwent cyclo-condensation with the appropriate
aniline in acetic acid, most likely via a Dimroth rearrangement,
to aord intermediates 220. The hydroxyl group of 220 was
rst transformed into the corresponding chloride or mesylate
221 and then displaced by a primary or secondary amine to
Scheme 59. Synthesis of 4-Phenylaminopyrazolo[3,4-d]pyrimidines as EGFR Inhibitors
Figure 13. Structure of compounds 216.
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aord the nal product 222 (Scheme 60). One of the most
interesting compounds was 222a, which showed IC
50
values of
1 and 5 nM in enzymatic assays for the inhibition of ErbB2 and
EGFR, respectively, and 17 nM against the BT474C cell line,
which is sensitive to the inhibition of either ErbB2 or EGFR.
The compound also showed acceptable bioavailability in rats
and dogs.
5.2. Insulin-Like Growth Factor 1 Receptor (IGF-1R)
Inhibitors
IGF-1R is another receptor TK belonging to a complex system
of growth factors and receptors, the IGF pathway, which
includes IR (insulin receptor), insulin-like growth factor I/II
(IGF-I/IGF-II), and six insulin-like growth factor-binding
proteins.
159
The membrane-bound receptors of this pathway
exist either as homodimers of IGF-1R or hybrid heterodimers
of IGF-1R and IR, and they are activated by the binding of the
ligands IGF-1 and IGF-2, as well as insulin when the hybrid
receptor is present.
160
In addition to other physiological functions, IGF-1R
regulates growth and development in healthy cells and is
involved in the suppression of apoptosis. Indeed, this receptor
simultaneously activates the antiapoptotic PI3K/Akt pathway
and the mitogenic ERK/MAPK pathway. IGF-1R deregulation
Scheme 60. Synthesis of 3-Alkoxy-4-anilino-1H-pyrazolo[3,4-d]pyrimidines as EGFR and ErbB2 TK Inhibitors
Scheme 61. Synthesis of IGF-1R Inhibitors
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allows cancer cells to avoid apoptosis and induces cell migration
and invasion.
161
For these reasons, IGF-1R has been implicated
in multiple aspects of tumor progression, including oncogenic
transformation, cell proliferation, evasion of apoptosis, tumor
cell invasion, and metastasis.
162
The inhibition of IGF-1R
represents a therapeutic approach for treatment of malignan-
cies.
163
5.2.1. Synthesis and Biological Properties. Abbott
researchers, during high-throughput screening (HTS) of their
compound repository, identied a number of pyrazolo[3,4-
d]pyrimidines as moderate IGF-1R inhibitors and initiated a
research program to identify more potent compounds.
164
The
synthesis of pyrazolo-pyrimidines 226 bearing a substituted
benzimidazole ring at C3 is presented in Scheme 61.
The starting material was 3-iodo-1H-pyrazolo[3,4-d]-
pyrimidin-4-ylamine 74, which was subjected to Mitsunobu
coupling with a suitable ketalalcohol. After keto-group
unmasking, intermediate 223 was obtained
165
and was
subsequently treated with morpholine and formic acid. The
crude product, after recrystallization from DMF/isopropyl
alcohol, aorded the desired trans-diastereoisomer 224 (yield of
25%). Intermediate 224 reacted with boronates under Suzuki
conditions to give 225. The nitro-anilino substituent was nally
cyclized with the appropriate aldehyde in the presence of 1 M
Na
2
S
2
O
4
in a microwave reactor (Scheme 61).
The most active compound was 226a, A-928605, which
inhibited IGF-IR with an IC
50
value of 37 nM in the enzymatic
assay and reduced IGF-1R cellular phosphorylation with an
IC
50
value of 90 nM. Moreover, it displayed promising
pharmacokinetics and activity in a murine model. The
compound was also able to signicantly inhibit the growth of
neuroblastoma xenografts in vivo and demonstrated additive
eects when used in combination with clinically approved
agents, i.e., the EGFR monoclonal antibody cetuximab, in
NSCLC and human pancreatic tumor models.
166
Using the same synthetic approach, the authors synthesized
other compounds with the C3 benzimidazole substituent but a
dierent N1 basic side chain.
167
The substituted piperazino
derivative 227 (Figure 14) is a potent multikinase inhibitor,
possessing IC
50
values of 81, 58, and 54 nM for the enzymatic
inhibition of IGF-IR, EGFR and ErbB2, respectively, and 115
and 94 nM, respectively, in cell-based assays against MiaPaCa-2
and N87 cells. In a murine model, 227 completely inhibited
receptor phosphorylation of both IGF-IR and EGFR.
Because several studies have suggested that the simultaneous
inhibition of IGF-IR and EGFR/ErbB2 could be more
protable than selective inhibition of a single kinase in the
treatment of cancer,
168
the authors continued the research
program on pyrazolopyrimidines and identied a number of
derivatives 229 bearing 4-(benzo[d]oxazol-2-ylamino)phenyl
moieties at C3 by HTS.
169
Reductive amination of ketone 223 aorded a mixture of the
cis/trans isomers of amino derivatives 228 (in an approximately
1:1 ratio), which were separated by chromatography. Suzuki
coupling of 228 under microwave irradiation with the
appropriate pinacol boronates, prepared as described by the
authors, aorded the nal compounds 229 (Scheme 62).
The most active compound, 229a (Figure 15), was obtained
using a slightly dierent procedure in which the Suzuki
coupling precedes the reductive amination. This compound
showed IC
50
values of 12, 31, and 11 nM against IGF-1R,
EGFR, and ErbB2, respectively, in enzymatic assays; in cellular
functional assays (MiaPaCa-2 and BT474 cells), it potently
Figure 14. Structure of the IGF-1R inhibitor 227.
Scheme 62. Synthesis of Dual IGF-IR and EGFR/ErbB2
Inhibitors
Figure 15. Structure of the IGF-IR and EGFR/ErbB2 inhibitor 229a.
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inhibited the phosphorylation of IGF-1R and ErbB2 but not
EGFR.
169
6. 1H-PYRAZOLO[3,4-d]PYRIMIDINES AS
CYTOPLASMIC TYROSINE KINASE INHIBITORS
Cytoplasmic TKs, also called nonreceptor TKs, are located
inside the cell, although many of them function in complexes
tethered to the plasma membrane. They are critical conveyers
of extracellular signals and are involved in the regulation of cell
proliferation, dierentiation, chemotaxis and apoptosis; they are
also important for hematopoiesis and immune development.
Nonreceptor TKs respond to extracellular stimuli as modular
units that have kinase activity and regulatory functions and can
modulate proteinprotein interactions.
170
To date, more than
35 nonreceptor TKs have been identied and classied in
dierent families, including Src, Abl, Tec, Csk, Syk, Ack, Jak,
Fak, and Fer.
145
The aberrant activity of cytoplasmic TKs has
been implicated in cancer growth and progression, the
induction of drug-resistance, tumor neovascularization, and
metastasis. Nonreceptor TK inhibitors, e.g., the Abl inhibitors
imatinib and nilotinib and the dual Src/Abl inhibitor dasatinib,
have been approved during the last ten years for CML therapy
and are being tested in several clinical trials on a wide range of
hematological and solid malignancies.
171
6.1. Src Family Kinase Inhibitors
Src family kinases (SFKs) were the rst discovered and are the
most studied cytoplasmic TKs. This family includes nine
members (Src, Fyn, Yes, Blk, Yrk, Fgr, Hck, Lck, and Lyn) and
regulates multiple signal transduction pathways involved in
growth, proliferation, dierentiation, migration, metabolism,
and apoptosis. In normal cells, Src is only transiently activated
during the multiple cellular events in which is involved. Src is
overexpressed and/or hyperactivated in a large variety of solid
tumors, and it is most likely a strong promoting factor for the
development of metastatic cancer phenotypes.
172,173
Studies
with Src-dominant negative mutants have suggested that SFKs
are involved in the proliferation of CML cell lines. In particular,
Hck and Lyn are overexpressed and/or activated during CML
progression.
174
Several extracellular molecules can activate Src
binding receptors, including growth factor receptors, integrins
and other adhesion receptors, guanosine phosphate binding
protein-coupled receptors (GPCRs), cytokine receptors and
ion channels. Src, after being activated in response to these
extracellular signals, phosphorylates various downstream
targets, thus regulating multiple signal transduction pathways,
such as the Ras/Raf, RhoGAP, and PI3K/Akt pathways.
175
6.1.1. Synthesis and Biological Properties of Src
Inhibitors. The 4-aminopyrazolo[3,4-d]pyrimidines 230a
(PP1) and 230b (PP2) (Figure 16) were the rst SFK
inhibitors and were reported by Pzer researchers in 1996.
176
These compounds inhibit all SFK members with IC
50
values
in the low nanomolar range in an ATP-competitive fashion, as
demonstrated by crystal structures with dierent SFK members,
including the PP1:Hck
176
and PP2:Lyn complexes (Figure
17).
177
Further biological studies have demonstrated that these
compounds, in particular PP1, also inhibit Abl and c-Kit, a
RTK.
178
Even though the two compounds were not used
clinically, likely because of their toxicity, they have been
extensively used for studying the biological pathways aected
by SFKs and for the identication of other SFK functions in
cells, especially during cancer formation and development, but
also in other pathologies (e.g., Alzheimers or Parkinsons
diseases).
179,180
6.1.2. Synthesis and Biological Properties of Src-
Engineered Kinase Inhibitors. Shokat and colleagues have
been working with the pyrazolo[3,4-d]pyrimidine scaold as a
tool for chemical biology studies for years.
181
They applied a
chemical genetic approach to obtain further insight into SFK
functions, specically studying v-Src (viral-Src, the viral variant
of c-Src) and Fyn, another SFK member. They have prepared
engineered kinases, termed analogue-sensitive alleles, bearing
specic mutations, i.e., I338G v-Src and T339G Fyn. In these
new enzymes, a bulky residue (isoleucine or threonine) called
a gatekeeper, which is naturally present in the ATP binding site,
was replaced with a smaller amino acid (glycine), creating a
novel pocket not present in a wild type (WT) kinase.
182
Then,
the authors synthesized a number of the inhibitors 231 and 232
that were specic for the engineered kinases and unable to bind
the corresponding WT kinases. The compounds are pyrazolo-
[3,4-d]pyrimidines analogues of 230 but bear C4 bulky
substituents which occupy the enlarged ATP binding pocket
of the mutated kinases.
The synthesis of these compounds started from 1-tert-butyl-
3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (61),
26
which
was reacted with an excess of an acyl chloride in pyridine to
form amides 231. Reduction of the amides to the
corresponding amines 232 was accomplished with LiAlH
4
(3
equiv) in dry THF under an argon atmosphere at 0 C for 30
min and under reux for 30 min (Scheme 63).
Compounds 231 and 232 inhibited engineered v-Src and
Fyn, but they are only weakly active toward the native enzyme.
The most potent compound is the 4-tert-butyl-3-phenyl
derivative 231, which inhibited I338G v-Src with an IC
50
of
430 nM and T339G Fyn with an IC
50
of 830 nM.
Based on further computational studies, the authors deduced
that a C3 bulky group could improve inhibitor anity, and they
synthesized a new family of C3-derivatized compounds, of
which 233a (1NM-PP1) and 233b (1NA-PP1) (Figure 18)
were extremely active against the engineered Src (I338G v-Src),
possessing IC
50
values of 1.5 and 4.2 nM, respectively (versus 1
and 28 M for the nonengineered kinase).
183
The two
compounds showed the same trend for activity toward
engineered Fyn and Abl. For example, 1NM-PP1 inhibits WT
c-Abl with an IC
50
value of 600 nM and T315A c-Abl with an
IC
50
of 7 nM.
184
The use of small molecules as chemical probes is useful for
elucidating the role of specic genes or proteins in physiology
and pathology and for validating the pharmaceutical potential
of a molecular target.
185
This approach, which was also applied
to single-cell eukaryotes such as yeast,
186
is particularly useful
when the WT kinase can be replaced with the engineered form
in animals. Genetically engineered mouse models that carry a
Figure 16. Structure of compounds 230a, PP1, and 230b, PP2.
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mutant allele in place of the WT kinase have been created,
allowing the rst in vivo assessment of the eects of a specic
inhibitor.
187
Shokat and colleagues continued their research in this eld of
genetic strategies with the aim of obtaining other inhibitors
specic for engineered kinases.
188
They observed that the
introduction of a glycine or alanine gatekeeper residue, as
performed in the previous studies, resulted in diminished kinase
activity and ATP anity in a number of cases. To address this
issue, a new chemical genetic approach was developed based on
covalent complementarity between an engineered gatekeeper
cysteine in Src and an electrophilic inhibitor. The authors found
that Src with a cysteine gatekeeper (T338C c-Src) possesses
WT activity and can be irreversibly inhibited in T338C c-Src-
transformed NIH-3T3 cells by specic vinylsulfonamide-
derivatized pyrazolo-pyrimidine inhibitors, including 236.
To synthesize 236, nitro derivative 234 was reduced to the
corresponding amine 235,
189
which was reacted with 2-chloro-
1-ethane sulfonyl chloride (Scheme 64).
188
A cocrystal structure of T338C c-Src with 236 was solved to
elucidate the inhibitor-binding mode (Figure 19). Remarkably,
a selectivity study on a wide panel of kinases revealed few o-
target activities, making this compound and its analogues the
most selective chemical genetic inhibitors reported to date;
thus, they could be useful tools for the study of protein
kinases.
188
6.1.3. Synthesis and Biological Properties of Other Src
and Lck Inhibitors. Kumar and colleagues synthesized 3-
phenyl-pyrazolo[3,4-d]pyrimidine derivatives substituted with
an alkyl or aryl carboxylic acid at the N1 position, such as 237
and 238 (Figure 20), respectively. These molecules were shown
Figure 17. A. Crystal structure of the Hck:PP1 complex (PDB code: 1QCF). B. Crystal structure of the Lyn:PP2 complex (PDB code: 2ZV9). PP1
and PP2 establish three hydrogen bonds (yellow dashed lines) with Hck and Lyn, respectively. Two hydrogen bond interactions exist between the
C4 amino groups of the inhibitors with the side chains of Thr338 and Thr319, in Hck and Lyn, respectively, and the backbone carbonyl groups of
Glu339 and Glu320 in Hck and Lyn, respectively. Another hydrogen bond is present between the N2 of the inhibitor and the NH backbone of
Met341 and Met322 in Hck and Lyn, respectively. For clarity, only residues interacting with the inhibitors are displayed.
Scheme 63. Synthesis of Compounds 231 and 232, Active As Inhibitors of the Engineered Kinases I338G v-Src and T339G Fyn
Figure 18. A. Structure of compounds 233a (1NM-PP1) and 233b
(1NA-PP1). B. Crystal structure of the Src:1NM-PP1 complex (PDB
code: 4LGH). 1NM-PP1 makes contacts with the hinge region by
forming two hydrogen bonds (yellow dashed lines) involving the NH
and carbonyl backbone of Met341 and Glu339, respectively. For
clarity, only residues interacting with the inhibitor are displayed.
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to be weak inhibitors of c-Src phosphorylation, with IC
50
values
in the high micromolar range.
190
Moreover, N-terminal peptide-substituted conjugates 239
(Figure 21) have been synthesized to improve Src activity. In
particular, two compounds, the 3-phenyl-pyrazolo[3,4-d]-
pyrimidine-CH
2
COCIYKYY and the 3-phenyl-pyrazolo[3,4-
d]pyrimidine-CH
2
CO-YIYGSFK conjugates, inhibited Src
phosphorylation with IC
50
values in the low micromolar
range, showing activities signicantly higher than those of the
acids 237 and 238.
The conjugation of the phenylpyrazolo[3,4-d]pyrimidine
moiety with peptides produced a synergistic inhibition, possibly
through the creation of favorable interactions between the
conjugate and the kinase domain, as shown by molecular
modeling studies.
Lck is a member of the SFK family that is expressed primarily
in T lymphocytes and plays an essential role in the immune
responses. Selective inhibitors of Lck could have therapeutic
applications for the treatment of autoimmune diseases and
organ transplant rejection. Abbott researchers have synthesized
a series of pyrazolo[3,4-d]pyrimidines, such as 242, containing
an extended 3-substituent, as potent Lck inhibitors; in some
cases, these have shown good selectivity toward the other SFK
members and good bioavailability in animal models.
191
To synthesize 242, iodo intermediate 223 underwent
reductive amination with N-methyl piperazine to give 240 in
a 2.5:1 ratio of cis/trans diastereoisomers, which were separated
by silica gel ash chromatography to isolate the desired trans
isomer. Suzuki coupling with the appropriate boronate ester
gave 241. Subsequent deprotection of the carbamate gave the
nal product 242 (Scheme 65).
The same group also synthesized 243 (A-420983)
192
and
244 (A-770041)
193
(Figure 22) using a similar synthetic
approach followed by acylation of the amino group at the para
position of the C3 phenyl ring.
Compounds A-420983 and A-770041 have been studied in
vitro against a large panel of kinases as well as in vivo. A-770041
binds Lck in its inactive conformation and shows selectivity for
Lck against a panel of kinases. This selectivity likely results
because the bulky substituent at C3 favors the inactive
conformation of the protein. This transition may be
thermodynamically favored only for kinases such as Lck.
Importantly, A-770041 is a 147 nM inhibitor of Lck and has
300-fold selectivity against Fyn, the other Src family kinase
involved in T-cell signaling. Doses of A-770041 at or above 10
mg/kg/day prevent the rejection of hearts transplanted
heterotopically in rats for at least 65 days.
194
6.2. Dual Src/Abl Tyrosine Kinase Inhibitors
c-Abl is another cytoplasmic TK that is normally present in
cells and has been implicated in various cellular processes.
Indeed, once activated, Abl phosphorylates several substrates
involved in signal transduction pathways, such as PI3K, Ras, c-
Jun kinase, paxillin, and FAK.
195
It also activates other
nonreceptor TKs, including SFKs (in particular Hck and
Lyn). However, even if Abl hyperactivation is involved in the
development of pathologies, it has been shown that c-Abl
activity is not essential for cell life, making it a very interesting
target for drug discovery.
Abl hyperactivation is primarily due to its fusion with Bcr,
which leads to the formation of an oncoprotein, Bcr-Abl
constitutively activated. This fusion protein is encoded by the
Philadelphia chromosome and maintains the same general
structure as c-Abl, but it presents the N-terminal portion of Bcr
instead of the N-terminal of Abl.
196
The Philadelphia
chromosome is the etiologic agent of CML, a hematological
malignancy. Bcr-Abl inhibitors, such as imatinib and nilotinib,
Scheme 64. Synthesis of Compound 236, Active As Inhibitor
of the Engineered Kinase T338C c-Src
Figure 19. Crystal structure of the Src:236 complex (PDB code:
3SVV). The inhibitor forms two hydrogen bonds with the backbone
carbonyl and amide groups of Glu339 and Met341, respectively. An
oxygen atom of the sulfonamide forms a hydrogen bond directly to the
backbone amide of Asp404. Furthermore, 236 binds the C helix in an
in conformation making contacts with Glu310. For clarity, only
residues interacting with the inhibitor are shown. All hydrogen bonds
are shown as yellow dashed lines.
Figure 20. Structure of compounds 237 and 238.
Figure 21. Structure of N-terminal peptide substituted conjugates 239.
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which have entered in therapy in the last ten years, considerably
improved the life expectancy of CML patients. Nevertheless,
the onset of TK-resistant mutations (among which T315I is the
most dicult to overcome) makes the search for new molecules
a very interesting challenge in medicinal chemistry.
197
Because
Bcr-Abl shares signicant sequence homology and remarkable
structural resemblance to Src family members in its active state,
several ATP-competitive type I inhibitors that were originally
developed as Src inhibitors have also proven to be potent Abl
inhibitors. Based on the similarity between Abl and Src and the
critical role of both enzymes in the pathogenesis of CML,
198
small molecules acting as dual Bcr-Abl/Src inhibitors could
possess enhanced activity against CML compared with
inhibitors uniquely targeting Bcr-Abl. The dual inhibitor
dasatinib has been approved for CML therapy and is being
tested in several advanced clinical trials for solid tumor
treatment.
6.2.1. Synthesis and Biological Properties of Dual Src/
Abl Inhibitors. In the continuing eort to nd new anticancer
agents, we have recently synthesized dierent families of highly
functionalized pyrazolo[3,4-d]pyrimidines, including 250, 251,
256, and 257.
199,200
Synthesis of the compounds started from phenyloxiranes
245, which react with hydrazine monohydrate to form the
corresponding 2-hydrazinoethanols 246. Reaction of the latter
with ethyl(ethoxymethylene)cyanoacetate (20) aorded the
ethyl esters of 5-amino-1H-pyrazole-4-carboxylic acids 247.
Reaction of these derivatives with formamide at 190 C
aorded the pyrazolo-pyrimidinones 248, which were trans-
formed into dichloro derivatives 249 by treatment with the
Vilsmeier complex (POCl
3
/DMF, 1:1). Regioselective sub-
stitution of the chlorine at C4 with an excess of various amines
aorded 250 in good yield (Scheme 73). Alternatively, reuxing
compounds 247 with benzoyl isothiocyanate in THF gave
intermediates 252, which were cyclized to pyrazolo[3,4-
d]pyrimidinones 253 by treatment with 2 M NaOH and then
acidied with acetic acid. Alkylation of the C6 thio group with
the appropriate alkyl iodide in reuxing THF aorded 6-
alkylthio derivatives 254, which was subsequently treated with
the Vilsmeier complex to obtain dihalogenated compounds
255. Reacting these with an excess of various amines provided
products 256 in good yield. Styryl derivatives 251 and 257 have
been obtained from 250 and 256 by treatment with NaOH
under reux (Scheme 66).
Derivatives 259 and 262 have been synthesized using 248 as
the starting material (Scheme 67).
201,202
Compound 258 was prepared following the Beal and Ve liz
procedure
203
by treating the derivative 248 (where X = H) with
a mixture of hexamethylphosphorous triamide/N-bromosucci-
nimide (HMPT/NBS) in acetonitrile at 20 C followed by
the addition of LiBr and reuxing for 5 h. Notably, the
secondary OH on the N1 side chain remains unaltered by this
procedure. The C4 bromo-derivative 258 was treated with the
appropriate amines to give nal compounds 259. Reaction of
pyrimidinone 248 in DMF with a solution of phosphorus
tribromide, pyridine, and toluene at room temperature for 3
days provided intermediate 260, with a bromine atom on the
Scheme 65. Synthesis of the Lck Inhibitor 242
Figure 22. Structure of 243 (A-420983) and 244 (A-770041).
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N1 side chain. Compound 260 was chlorinated at C4 by
treatment with the Vilsmeier complex to aord 261, which was
nally reacted with an excess of various amines to obtain
derivatives 262 in high yield.
Compounds 250 and 256, with a 2-chloro-2-phenylethyl N1
side chain, showed submicromolar to nanomolar activity
toward isolated Src as well as antiproliferative activity toward
epidermoid (A431) and breast cancer (BC-8701) cell lines
(which overexpress Src), in which they blocked Src
phosphorylation and induced apoptosis.
201,202
The most potent compounds of the series reduced the
proliferation, migratory ability and adhesive capacity of PC3
carcinoma cells
204
and inhibited the growth of a human SaOS-2
xenograft tumor in a nude mouse model.
205,206
Moreover,
nanomolar concentrations of these c-Src inhibitors have a
highly selective antiangiogenic eect.
207
Because compounds acting as Src inhibitors often show
activity toward Bcr-Abl as well,
171
we tested a number of
pyrazolo[3,4-d]pyrimidine derivatives on a panel of three
human leukemia cell lines and toward the Abl enzyme in a cell-
free assay. Many compounds possessed submicromolar IC
50
values in the enzymatic assay. Moreover, they inhibited cell
proliferation with micromolar IC
50
values and promoted
apoptosis in Bcr-Abl-expressing cells, including K-562 CML
cells, KU-812 basophilic leukemia cells, and MEG-01 CML
blastic (megakaryoblastic) cells. These eects were accom-
panied by reduced tyrosine phosphorylation of Bcr-Abl and one
of its downstream signaling eectors (the signal transducer and
Scheme 66. Synthesis of Dual Src/Abl Inhibitors
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activator of transcription 5, STAT-5). The biochemical assay
with human recombinant Abl demonstrated inhibition of Abl-
catalyzed peptide substrate phosphorylation. Several com-
pounds were characterized by a biological prole comparable
to or better than that of the reference compound, PP2, 230b.
6.2.2. Molecular Modeling Studies and Optimization
of the Substituents. Computational studies have also been
performed to analyze the binding mode of our ligands and
elucidate the main chemical features responsible for Abl
anity.
208
Two dierent binding modes were identied,
depending on the presence or absence of the alkylthio
substituent at the C6 position of the pyrazolo-pyrimidine
scaold. Compounds bearing this C6 substituent showed an
interaction pathway, herein referred to as binding mode II,
characterized by the pyrazolo-pyrimidine nucleus being located
in the region usually occupied by the adenine ring of ATP
(adenine region), a hydrogen bond with Met318, and several
hydrophobic contacts (Figure 23A).
The alkylthio substituent shared the hydrophobic region II
(HRII) with the side chain on C4 and had van der Waals
contacts with residues Leu248, Gly249, and Tyr253. For
compounds bearing a long and bulky substituent (phenyl-
ethylamino groups) at C4, simultaneous occupancy of this
group and the alkylthio chain within the hydrophobic pocket II
was not permitted. As a consequence, the heterocyclic nucleus
was involved in a rearrangement that resulted in the
accommodation of substituents at C4 and C6 in hydrophobic
region I (HRI); the N1 side chain was located in HRII.
Scheme 67. Synthesis of Dual Src/Abl inhibitors
Figure 23. A. Proposed binding mode (binding mode II) of C6 thioalkyl-substituted compounds to c-Abl (series 256). The C6-substituent
compounds maintain the N1 and C4 side chains within hydrophobic regions I and II, respectively, allowing the C6 substituent to interact with
hydrophobic region II. B. Proposed binding mode (binding mode I) of C6-unsubstituted compounds in c-Abl (series 250). In detail, the C6-
unsubstituted derivative binds the enzyme by positioning the pyrazolo[3,4-d]pyrimidine core within the adenine region, while the side chains at N1
and C4 are directed toward hydrophobic regions I and II, respectively, establishing the classical hydrogen bonds with the hinge region (Met318). C.
Superimposition of 250 and 256 in c-Abl. All hydrogen bonds are shown as red dashed lines.
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Moreover, a hydrogen bond involving the N2 of the pyrazolo-
pyrimidine scaold and the NH backbone of Met318 was found
in this binding mode.
In the alternative binding mode (Figure 23B), the lack of the
alkylthio substituent at the C6 position induced a reorientation
of the pyrazolo-pyrimidine nucleus (binding mode I) without
changing the location of the C4 and N1 side chains, which were
located in HRII and HRI, respectively (Figure 23C). In
particular, the amino group at C4 was the hydrogen bond
donor for the carbonyl oxygen of Met318, whereas N5 accepted
a hydrogen bond from the amide NH group of the same
residue (this additional hydrogen bond, with respect to the
orientation reported in Figure 23A, was not associated with a
signicant increase in activity). Taken together, these results
highlight the importance of hydrophobic interactions for the
binding of the pyrazolo-pyrimidines to Abl kinase. In fact, even
if dierent binding modes were identied, complete occupancy
of HRI and HRII emerged as a feature common to all the active
compounds (Figure 23C). In this context, the lack of anity for
compounds 259 bearing a hydroxyphenylethyl side chain at N1
could be explained in terms of ineective interactions with HRI,
primarily due to the location of the hydrophilic hydroxyl group
within this region. It should also be noted that results of
docking studies on Abl kinase show some interesting
similarities with results previously reported by our group for
the same set of compounds docked into Src kinase.
201
The results of another molecular modeling study suggested
the insertion of halogen substituents with various substitution
patterns. Accordingly, compounds 263 (Figure 24) were
synthesized, as mentioned above (Scheme 66), and were
subjected to biological testing to evaluate their anity toward
Abl (in a cell-free assay) and their antiproliferative activity.
209
As a result, the cellular activity and enzymatic anity
increased (up to an order of magnitude) in comparison with
the corresponding nonhalogenated parent compounds. As
predicted by previous docking studies, halogenated derivatives
adopted the binding mode observed with C6-unsubstituted
pyrazolo-pyrimidines; halogen substituents were most often
located in regions where Grid analysis found protable
interactions between the halogen atoms and the target. The
immunoblot analysis of K-562 cell lysates treated with 263h
(Figure 24), the most active compound of this series, showed
that the compound markedly reduced Bcr-Abl phosphorylation.
Moreover, 263h was also able to greatly reduce the
phosphorylation of both STAT-5 and Src. It also caused a
marked increase in the Bax/Bcl-xL ratio, conrming the
induction of leukemia cell apoptosis.
We also performed other studies with the aim of further
understanding the activity of our pyrazolo-pyrimidines toward
resistant forms of CML.
210
Initially, we used docking
simulations to predict the most favorable interaction between
the T315I mutated form of Abl (invariably associated with
resistance to the tyrosine kinase inhibitor imatinib mesylate,
IM) and the C6-unsubstituted or C6-substituted pyrazolo[3,4-
d]pyrimidines previously found to be dual Src/Abl inhibitors.
Docking results showed that C6-unsubstituted derivatives had
the same pattern of protable interactions (Binding mode I)
already found in the complexes with WT Abl,
208
whereas the
C6-alkylthio analogues did not reproduce this binding mode. A
number of compounds, both C6-unsubstituted and C6-
substituted, were selected and tested against Ba/F3 cells
transduced with WT p210Bcr-Abl, which is IM-sensitive, or
with three of the most common mutations associated with IM
resistance in vivo (T315I, Y253F, and E255K). These cells were
driven to drug resistance by saturating doses of IL-3 or by the
expression of the Bcr-Abl construct coding for the p185 protein
of acute lymphoblastic leukemia. The C6-unsubstituted
compounds were active against all cell lines assayed (LD
50
range 0.74.3 M), whereas C6-substituted compounds
exhibited lower activity. Notably, the C6-unsubstituted
compounds were also eective against the T315I mutation,
which is insensitive to dual Src/Abl inhibitors. The most
interesting compound is the dihalogenated derivative 263h
(Figure 24), which showed a K
i
value of 80 nM toward isolated
Bcr-Abl and, interestingly, an LD
50
of 3 M in cells expressing
the T315I mutation. The cytotoxic eects of these derivatives
toward IM-sensitive and IM-resistant Ba/F3 cells were
attributable, at least in part, to their pro-apoptotic activity.
Taken together, such ndings suggest that C6-unsubstituted
pyrazolo[3,4-d]pyrimidines may be useful inhibitors in IM-
resistant CML.
210
In addition, some C6-unsubstituted deriva-
tives were very eective against early (CD34
+
) CML
progenitors collected from patients sensitive to imatinib.
More importantly, the compounds were also more eective
against CD34
+
cells collected from imatinib-resistant patients,
including those with the T315I mutation.
211
These pyrazolo[3,4-d]pyrimidines are good dual Src/Bcr-Abl
inhibitors, but they suer from substandard pharmaceutical
properties, in particular, poor aqueous solubility. We tried to
solve this problem using dierent approaches. First, we
prepared unilamellar liposomes of some of the most promising
compounds; these formulations exerted cytotoxic eects on
ARO or other thyroid cancer cell lines (that overexpress Src) at
lower doses and after shorter incubation times than the free
compounds. Inhibition of EGF-stimulated Src and ERK
phosphorylation and reduced ARO cell migration was also
observed. Moreover, the growth of tumor xenografts induced in
severe combined immunodecient (SCID) mice was inhibited
by the i.v. administration of 2550 mg/kg of the liposomal
formulation.
212
In another approach, the aqueous solubility was improved
100 to 1000 fold by solubilization with 2-hydroxypropyl--
cyclodextrin (HPCD).
213
The ratio of inclusion in the
complex was determined using the phase solubility method.
Moreover, some complexed compounds were tested against
dierent leukemic (K-652, KU-812 and HL-60) or osteosarco-
ma (SaOS-2) cell lines and showed a higher activity compared
to the noncomplexed compounds.
Figure 24. General structure of derivatives 263 and the structure of
the most active compound of the series, 263h.
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We then rationally designed and synthesized a series of more
soluble pyrazolo[3,4-d]pyrimidine derivatives, which main-
tained the dual Src/Abl activity of the lead.
214
Molecular
modeling studies highlighted the structural features responsible
for the dual Src/Abl inhibitory activity of our compounds.
Specically, the presence of the C6 substituent induced a
reorientation of the pyrazolo[3,4-d]pyrimidine nucleus within
the Abl binding site, maintaining the N1 and C4 side chains
within HRI and HRII, respectively, and allowing the C6-
substituent to interact with HRII. A hydrogen bond interaction
was also identied between the C4 amino group and Met318.
Within the Src binding pocket, the pyrazolo[3,4-d]pyrimidine
nucleus of a C6-thiomethyl derivative was still accommodated
in the adenine region, but the C4 substituent was located in
HRI; HRII hosted the N1 side chain (binding mode III). Two
hydrogen bond interactions were also found, one involving the
C4-amino group and the side chain of Thr340 and the other
between the N2 of the pyrazolo[3,4-d]pyrimidine nucleus and
the NH-backbone of Met343 (Figure 25).
Considering the binding modes just described, we designed
new compounds by introducing polar moieties in the solvent-
exposed C4 and C6 positions of the pyrazolo[3,4-d]pyrimidine
scaold. The virtual library of compounds was docked in the
ATP pockets of Src and Abl; only those derivatives showing
favorable activity and ADME properties were selected for
synthesis. The predicted ADME properties were calculated
using the QikProp 2.5 protocol in Maestro8.5.
Using standard procedures, we synthesized a series of more
polar derivatives, 264, 265, 266, and 267 (Figure 26).
214
The new more polar compounds showed dual Src/Abl
activity similar to that of the lead 263h and promising
antiproliferative activity toward three dierent leukemia cell
lines (K-562, MEG-01, and KU-812) under both normoxic and
hypoxic conditions. This hypoxic condition is particularly
important because leukemia cells inltrating the bone marrow
in rats have proven to be markedly hypoxic and are able to
proliferate by virtue of their adaptation to this hypoxic
microenvironment.
215
Finally, in vitro ADME properties and potential metabolic
issues of the synthesized compounds were determined, allowing
us to identify compounds with a favorable combination of
ADME properties and biological activity in leukemia cells.
214
Figure 25. A. Structure of 256a. B. Graphical representation of the binding mode (binding mode III) of 256a, in the ATP binding site of c-Src
(magenta). C. The proposed binding mode for 256a in c-Src was imported and superimposed on the binding pose found in c-Abl (right, light blue).
In particular, because Tyr253 partially occupied the ribose pocket in Abl, the 6-methylthio-pyrazolo[3,4-d]pyrimidine cannot assume the same
binding mode in Abl as in c-Src. For clarity, few residues are labeled; nonpolar hydrogen atoms are omitted, and hydrogen bond interactions are
represented by red dashes.
Figure 26. Structure of compounds 264267.
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Pyrazolo[3,4-d]pyrimidines 271 with a C6-dimethylamino
group have been synthesized by our group as dual Src/Abl
inhibitors.
216
5-Amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-car-
bonitrile 268 was reacted with N,N-dimethylaminophosgenei-
minium chloride to give the corresponding dimethylcarbami-
midic chloride 269. The latter was cyclized with anhydrous
hydrochloric acid to 4-chloro-1-(2-chloro-2-phenylethyl)-N,N-
dimethyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine 270 in satisfac-
tory yield. The C4 chlorine atom was then replaced by reaction
with the appropriate amines to give the nal compounds
(Scheme 68).
Considering the great interest in antitumor agents acting
with a dual mechanism (i.e., tyrosine kinase inhibition/anti-
inammatory activity), we decided to investigate the potential
anti-inammatory activity of our pyrazolo-pyrimidines.
217
With
this aim, a three-dimensional chemical library containing 423
variously substituted pyrazolo- and 4-pyrrolo-pyrimidines was
designed and screened in silico against structural models of
both COX-1 and COX-2. Docking experiments suggested that
some pyrazolo-pyrimidines were able to t into the COX-2
binding clefts. Thus, we tested some compounds in enzymatic
assays of COX-1 and COX-2 activity. As a result, compound
271 with R = NHC
3
H
7
showed interesting COX-2 activity and
selectivity compared with three reference drugs. In conclusion,
we have demonstrated that antiproliferative pyrazolo-pyrimi-
dines can exert dual activity with anti-inammatory eects
comparable to known COX inhibitors.
Subsequently, we synthesized pyrazolo[3,4-d]pyrimidines
substituted at N1 with a 2-chloro-2-phenylethyl side chain
and at C6 with a thioalkyl (i.e., cyclopentyl or isopropyl)
218
or
methyl group. The C6-methyl-derivatives were synthesized as
shown in Scheme 66.
219
The starting product for the synthesis of C6 methyl
derivatives 275 was 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-
pyrazole-4-carboxamide 272, which was treated with sodium
ethoxide and ethyl acetate in absolute ethanol at reux to aord
1-(2-hydroxy-2-phenylethyl)-6-methyl-1, 5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one 273. This compound was
chlorinated with the Vilsmeier complex to obtain the dichloro
derivative 274. Reaction of the latter with an aniline in absolute
ethanol at reux for 4 h gave the desired compounds 275
(Scheme 69).
The compounds have been tested against a panel of 11
dierent murine lung tumor progenitor cell lines that express
stem cell markers as well as against A549 human lung
adenocarcinoma cells, HepG2 human hepatoma cells and
CaCo2 human colon cancer cells to obtain insight into the
mode of action of these inhibitors. Treatment with the newly
synthesized dual kinase inhibitors eciently blocked c-Abl and
Scheme 68. Synthesis of Dual Src/Abl Inhibitors Endowed with Anti-Inammatory Activity
Scheme 69. Synthesis of 6-Methyl-Substituted Dual Src/Abl Inhibitors
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c-Src kinase activity in the nanomolar range, induced apoptosis,
reduced cell viability, and caused cell cycle arrest, predom-
inantly at the G0/G1 transition. Western blot analysis
conrmed the repressed expression of c-Abl and c-Src as well
as the interacting partners p38 MAPK, heterogeneous
ribonucleoprotein K and CDK1 and other proteins that are
crucial for tumor progression.
c-Src has been reported to play an important role in the cell
dierentiation, adhesion and survival of neuroblastoma (NB), a
common extracranial pediatric solid tumor. Starting from some
of the dual Src/Abl inhibitors previously shown to be active in
NB cell lines,
220
we recently synthesized derivatives 276
(Figure 27), which are characterized by the presence of a C6
methylthio group but have dierent N1 side chains from those
in our previously reported libraries.
221
Interestingly, the introduction of a 2-phenylethyl or 2-
phenylpropyl group on the pyrazole ring abolished the Abl
activity with simultaneous improvement of the anity and
specicity for c-Src. The synthesis of such compounds was
accomplished as reported above for the 2-chloro-2-phenylethyl
N1 substituted derivatives, starting from (2-phenylethyl)-
hydrazine or (2-phenylpropyl)hydrazine.
Compounds 276 are c-Src selective inhibitors active in the
nanomolar range in enzymatic tests. They reduced neuro-
blastoma cell growth in a time- and concentration-dependent
manner. The antiproliferative eect was evident 48 h after
treatment, and immediate toxic/necrotic events were not
observed. Finally, in vitro ADME properties, including eective
permeability in a blood-brain barrier (BBB) model, water
solubility, and microsomal stability, showed that the most active
derivatives are characterized by good metabolic stability, water
solubility and BBB permeation.
221
Other biological studies of pyrazolo[3,4-d]pyrimidines that
we synthesized indicated that some derivatives possess
antiproliferative activity toward mesothelioma cell lines, in
which the compounds induce apoptosis through p27 nuclear
stabilization,
222
and Burkitt lymphoma cell lines.
223
6.3. Type II (DGF-out Binding) Dual Src/Abl Inhibitors
All pyrazolo[3,4-d]pyrimidines described thus far are type I
ATP-competitive kinase inhibitors and bind to the active
conformation of the enzyme termed DFG-in (indicating the
specic conformation of the DFG motif). This binding mode is
also utilized by the reported c-Src inhibitors because the
opposite conformation (DFG-out) is energetically less
favorable in c-Src.
Dierent biological behavior has been reported and widely
demonstrated for Bcr-Abl. Indeed, a number of its inhibitors,
including imatinib and nilotinib (currently used in CML
therapy), bind to the catalytically inactive conformation (DFG-
out) of the enzyme in an ATP-competitive fashion and are
termed type II inhibitors.
Many data demonstrate that the DFG-out conformation of
Bcr-Abl is thermodynamically stable when complexed with
imatinib, but this conformation requires energetically unfavor-
able interactions in c-Src complexes.
224
For these reasons,
Figure 27. Structure of derivatives 276.
Figure 28. A. Structure of compound 277. B. Crystal structure of the Src:277 complex (PDB code: 3EL8). 277 establishes the classical interactions
with Thr338, Glu339 and Met341 of the hinge region. The urea extension forms a hydrogen bond with the side chain of Glu310 within helix C,
whereas the meta-triuoromethyl phenyl portion lies within a pocket lined by residues Leu317, Leu322, Val402, Met341, and His384. C. Crystal
structure of the Src:imatinib complex (PDB code: 2OIQ). Imatinib makes contacts with the hinge binding region and extends into the pocket
created by the DFG ip. All hydrogen bonds are shown as yellow dashed lines.
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designing dual Src/Bcr-Abl inhibitors that bind with high
anity to the DFG-out conformation of both enzymes seems
unlikely.
However, this task was accomplished by Shokat and
colleagues, who synthesized a number of type II inhibitors
with a pyrazolo[3,4-d]pyrimidine core.
225
Based on the analysis
of the previously reported cocrystal structure of kinase
inhibitors and on molecular modeling studies, they derivatized
the phenyl ring of PP1, 230a, with a meta-triuoromethyl
phenylurea group that could engage the DFG-out pocket. They
also inserted dierent N1 substituents (i.e., methyl, isopropyl,
cyclopentyl).
One of the most active compounds is 277 (Figure 28A), with
IC
50
values of 25 and 41 nM for c-Src and Abl, respectively.
The binding mode was conrmed by cocrystallographic
studies of the c-Src puried kinase domain complexed with 277
(Figure 28B): the compound recognizes the generally
unfavorable DFG-out conguration of c-Src, which is also
recognized by imatinib, although with some dierences (Figure
28C). Interestingly, one distinguishing feature between the 277
and imatinib complexes with c-Src is the interaction of these
inhibitors with the gatekeeper residue. Whereas the C3 phenyl
ring of 277 is rotated away from Thr338, the 2-methyl-
phenylamino portion of imatinib participates in a hydrogen
bond interaction with the same residue. This extra distance
from the gatekeeper Thr suggests that compound 277 and its
congeners may also bind to the drug-resistant gatekeeper
mutants, such as the clinically relevant imatinib-resistant Abl
T315I.
226
This hypothesis has been subsequently conrmed, as
277 and some analogues are potent inhibitors of Abl T315I
with IC
50
values in the nanomolar range.
225
7. 1H-PYRAZOLO[3,4-d]PYRIMIDINES AS INHIBITORS
OF OTHER KINASES
A number of pyrazolo[3,4-d]pyrimidines have been recently
published or patented as inhibitors of dierent kinases involved
in human diseases, particularly cancer.
Derivatives such as 278 (Figure 29) have been patented
227
as
ephrin type-B receptor 4 (EphB4) inhibitors. Ephrin receptors
are receptor tyrosine kinases, and together with their ligands,
the ephrins mediate numerous developmental processes and
are involved in cancer diusion.
228
Compound 278 proved to
be a potent EphB4 inhibitor in both enzymatic and CHO cell-
based assays.
Dierent substituted derivatives have been patented as
Bruton kinase (Btk) inhibitors.
Btk is a cytoplasmic tyrosine kinase member of the Tec
family that plays essential roles in B-lymphocyte signaling. Btk
is specically required for the B-cell antigen receptor (BCR)
signaling pathway, which contributes to the initiation and
maintenance of B-cell malignancies and autoimmune dis-
eases.
229
Btk inhibitors are being evaluated in clinical trials for the
treatment of chronic lymphocytic leukemia and other B-cell
disorders.
230
Derivatives 281,
231,232
patented by Pharmacyclics Inc., are
irreversible Btk inhibitors that possess IC
50
values in the
nanomolar range with a good selectivity prole in a panel of
other kinases. These compounds have been synthesized from
279, which were reacted with N-Boc-3-hydroxypiperidine via
Mitsunobu reaction to give the protected intermediates 280.
After deprotection with acid, coupling with acryloyl chloride
aorded the nal products (Scheme 70).
The most interesting compound is the 3-(4-phenoxyphenyl)
derivative 281a, PCI-32765, ibrutinib, which binds covalently
to Cys-481 in the active site of BTK, resulting in inhibition of
kinase activity with an IC
50
of 0.5 nM.
233
This compound
inhibited B cell functions and Btk-dependent processes within
the BCR pathway.
234
Several studies and successive clinical trials demonstrated the
oral activity of ibrutinib in B-cell malignancies,
233
including
chronic lymphocytic leukemia,
230,235
Bloodmantle cell lympho-
ma, diuse large B-cell lymphoma, and multiple myeloma.
236
In November 2013, the U.S. Food and Drug Administration
granted accelerated approval to ibrutinib (with the commercial
name of Imbruvica) for the treatment of patients with mantle
cell lymphoma who had received at least one previous
therapy.
237
This irreversible inhibitor is also active against
interleukin-2-inducible T-cell kinase (ITK), a member of the
TEC family that is highly expressed in T cells and contributes
to the pathogenesis of some infectious, autoimmune, and
neoplastic diseases.
238
Following the analogue-sensitive approach rst used by
Shokat
84,85
and previously reported in this review, other
authors identied 282 as a nanomolar inhibitor of a rationally
engineered HOG1 (High Osmolarity Glycerol) serine/
threonine kinase, i.e., HOG1 T100G, which is useful for the
study of various aspects of HOG1 regulation, especially in
response to stress.
239
The HOG pathway of the yeast
Saccaromices cerevisiae is a MAPK signaling pathway and is
the functional homologue of the stress-activated JNK and p38
pathways in mammals. Because of this homology, the HOG
pathway is a good model to study osmotic adaptation processes
Figure 29. Structure of the EphB4 Inhibitor 278.
Scheme 70. Synthesis of Btk Inhibitors
Chemical Reviews Review
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in response to stress. For the synthesis of this inhibitor, 4-
amino-3-iodo-pyrazolopyrimidine 142 underwent a Sonoga-
shira coupling with ethynyl-benzene to produce 282 in high
yield (Scheme 71).
240
Compound 282 also displayed ecient
inhibition of RET (rearranged during transfection), a trans-
membrane RTK involved in thyroid cancer development.
Pyrazolo-pyrimidine 287, PF-4800567, (Figure 30), is a
selective inhibitor of casein kinase (CK) 1 which together with
casein kinase 1, forms a family of closely related serine-
threonine kinases involved in the regulation of the circadian
clock period.
241
PF-4800567 should be useful in exploring the
unique roles of these two kinases in multiple signaling
pathways.
The compound was synthesized from malononitrile 283,
which was reacted with (tetrahydro-pyran-4-yl)-hydrazine
dihydrochloride to give the corresponding pyrazolo intermedi-
ate 284 and then cyclized with formamide to aord the
pyrazolo[3,4-d]pyrimidine 285. The benzyl group on the C3
substituent was removed by catalytic hydrogenation. The
synthesis was completed by reaction of 286 with 3-
chlorophenol under Mitsunobu conditions (Scheme 72).
Proliferation of Toxoplasma gondii (Tg), which causes
toxoplasmosis, is mediated in part by calcium-dependent
protein kinase 1 (CDPK1). Maly and colleagues developed a
family of pyrazolo[3,4-d]pyrimidine ATP-competitive
TgCDPK1 inhibitors that block the invasion of parasites into
host cells, preventing their proliferation.
242
The presence of a
rare glycine gatekeeper residue in TgCDPK1 has allowed the
creation of a series of C3-substituted compounds, including
289, that are potent inhibitors selective for CDPK1 over a
panel of human kinases, including SFKs, Abl, EGFR and CSK.
Indeed, the steric bulk of the substituent at the C3 position of
the pyrazolo-pyrimidine core confers selectivity for kinases that
contain small gatekeeper residues.
183
These potent TgCDPK1
inhibitors do not inhibit the growth of human cell lines, making
them promising candidates as toxoplasmosis therapeutics.
Derivative 289, which is one of the most potent compounds
(IC
50
= 2.5 nM), was synthesized by reaction of 74 with the
mesylate of N-Boc protected 4-hydroxymethyl-piperidine. The
N1-substituted compound 288 underwent Suzuki coupling to
give an intermediate bearing the 6-ethoxynaphthyl group at C3.
The nal step was Boc-deprotection with TFA (Scheme 73).
242
Human Ack1 (activated Cdc42-associated kinase) is a
ubiquitously expressed nonreceptor TK. It is activated by
signals that include growth factors and integrins. Inappropriate
Ack1 activation has been implicated in the development,
progression, and metastasis of several forms of cancer. Thus,
there is an increasing interest in Ack1 inhibitors as potential
drugs.
243
Some pyrazolo[3,4-d]pyrimidines, including 293, are potent
Ack1 inhibitors. Compound 293 was synthesized using a six-
step procedure (Scheme 74).
244,245
Chlorination of acid 290 was achieved with a mixture of PCl
5
and POCl
3
. The resulting acid chloride was coupled with 2,6-
dichloro aniline in the presence of an acid scavenger and
treated with but-3-enyl-hydrazine to provide 291. Cyclization of
291 with PCl
5
in reuxing toluene and subsequent chloride
displacement with aniline provided compound 292, which was
dihydroxylated with a mixture of OsO
4
and N-methyl-
morpholine-N-oxide (NMO) to furnish 293 (Scheme 74).
This compound showed IC
50
values for Ack1 of 10 and 20 nM
in enzymatic and cell-based assays, respectively, as well as a
signicant improvement in oral bioavailability relative to earlier
analogues.
244,245
ZAP-70 is a cytoplasmic TK that is required for T cell
antigen receptor signaling. The importance of ZAP-70 in T cell
development and function is evident from the immunode-
ciency that develops in mice and humans decient for this
kinase. Currently, there is no specic small molecule inhibitor
for ZAP-70. Weiss, Shokat and colleagues generated a mutant
ZAP-70 allele that retains kinase activity but is sensitive to a
specic inhibitor, 294 (3-MB-PP1)
246
(Figure 31), which is a
pyrazolo[3,4-d]pyrimidine similar to PP1 230a but with
Scheme 71. Synthesis of the HOG1 T100G Inhibitor 282
Figure 30. Crystal structure of the CK1:287 complex (PDB code: 4HNI). The C4 amino group and the N5 of the pyrazolo-pyrimidine ring of 287
form two hydrogen bonds (yellow dashed lines) with the hinge region of CK1, and the meta-chlorophenyl moiety is buried deep in the binding site,
forming strong hydrophobic interactions with the side chains of Met80 and Met82.
Chemical Reviews Review
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dierent C3 substitution. It was synthesized following
procedures mentioned above.
183
The compound was used for
further investigation of the function of ZAP-70 in T cells.
247
8. MULTITARGETED INHIBITORS
The complexity of cancer has led to recent interest in
polypharmacological approaches for developing kinase-inhibitor
drugs. Multitargeted kinase inhibitors have proven to be more
useful from a therapeutic point of view than selective inhibitors
acting on a single kinase, especially in cancer therapy, where
dierent pathways are often similtaneously activated. The
clinical success of multikinase inhibitors
248
has stimulated
eorts to identify promiscuous drugs with optimal selectivity
proles. Apsel and colleagues discovered pyrazolo[3,4-d]-
pyrimidine compounds that potently inhibit both tyrosine
kinases and phosphatidylinositol-3-OH kinases (PI3K), two
protein families involved in cancer development.
84
Through
iterative chemical synthesis, X-ray crystallography and kinome-
level biochemical proling, they identied compounds that
Scheme 72. Synthesis of the Casein Kinase 1 Inhibitor 287 (PF-4800567)
Scheme 73. Synthesis of the TgCDPK1 Inhibitor 289 Scheme 74. Synthesis of the Ack1 Inhibitor 293
Figure 31. Structure of 294 (3-MB-PP1).
Chemical Reviews Review
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inhibit a range of new target combinations in these two families.
Such compounds could be particularly interesting because
reactivation of PI3K signaling is a common mechanism of
resistance to tyrosine kinase inhibitors. One of the most active
compounds was 295 (PP121) (Figure 32).
PP121 blocks the proliferation of tumor cells (e.g., U87 and
LN229 glioblastoma and BT549 breast cancer) by direct
inhibition of oncogenic tyrosine kinases, including Src, Abl,
VEGFR2, and PDGFR and phosphatidylinositol-3-OH kinases,
including p110 and mTOR, with IC
50
values in the low
nanomolar range. In particular, the compound overcomes drug
resistance in CML by redundant targeting and retains the
ability to block the proliferation of BaF3 cells expressing Bcr-
Abl T315I, most likely by inhibiting the mTOR signaling
pathway.
84
Compound 296 (CLM-3) (Figure 33), synthesized by Da
Settimo and colleagues, inhibited VEGFR-2, EGFR and RET
and demonstrated a highly signicant antiproliferative and
proapoptotic activity for activated endothelial (HMVEC,
HUVEC) and cancer cells (8305C cells).
249
Moreover, it
showed antitumoral activity in vitro and in vivo in papillary
dedierentiated thyroid cancer.
250
Regarding multitargeted kinase inhibitors, optimal kinase
inhibition proles remain dicult to predict. In an important
study, Dar and colleagues used a RET kinase-driven Drosophila
model of multiple endocrine neoplasia type 2 and kinome-wide
drug proling, and they identied that pyrazolo[3,4-d]-
pyrimidine 277, previously reported as an Src DFG-out binder,
also decreases oncogenic RET-induced lethality, whereas other
RET inhibitors reduced ecacy and enhanced toxicity.
Drosophila genetics and compound proling dened three
pathways accounting for the ecacy and dose-limiting toxicity.
The inhibition of RET, Raf, Src and S6K was required for
optimal animal survival, whereas the inhibition of the
antitarget mTOR led to toxicity due to the release of
negative feedback. Rational synthetic studies aorded 297
(AD80) and 298 (AD81) (Figure 34), which bear additional 2F
and 4Cl substituents, respectively, compared to 277. The
compounds do not inhibit mTor and possess balanced pathway
inhibition, improved ecacy and low toxicity in Drosophila and
mammalian multiple endocrine neoplasia type 2 models. This
study provides a powerful system pharmacology approach
toward developing compounds, particularly pyrazolo[3,4-d]-
pyrimidines, with a maximal therapeutic index.
251
9. CONCLUSIONS
The most common methods for the synthesis of the
pyrazolo[3,4-d]pyrimidine scaold have been reported, starting
from the earlier work of Robins, which occurred when the
biological behavior of these compounds was still in an
embryonic state, and extending to present day. We have
described how the evolution of chemical methodologies, which
have led to many new derivatives, has closely accompanied the
evolution of modern biology, pharmacology and medicine. It is
fascinating to observe how the functionalization of the
pyrazolo[3,4-d]pyrimidine scaold can lead to selective
inhibitors for many dierent biological targets, and the number
of these targets continues to rise.
Understanding of the modes of action of the inhibitors has
been facilitated by detailed visualization of the inhibitor-protein
interactions, which are depicted atom by atom in the X-ray
structures of cocrystals of inhibitors with their biological targets,
and/or by molecular modeling studies. These techniques have
accelerated the design of new, sometimes complicated,
Figure 32. A. Structure of compound 295 (PP121). B. Crystal
structure of the c-Src:295 complex (PDB code: 3EN4). The X-ray
complex reveals that the dual selectivity of 295 (kinases and
phosphoatidylinositol-3-OH kinases) is due to a hydrophobic pocket
conserved in both enzyme classes and accessible through the
substituent at C3. For clarity, only the residues interacting with the
inhibitor are labeled.
Figure 33. Structure of 296 (CLM-3).
Figure 34. Structure of 297(AD80) and 298 (AD81).
Chemical Reviews Review
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compounds using modern synthetic organic chemistry, as
described in the review.
The biological activity of some of these new compounds has
also been predicted through these technologies and then
experimentally conrmed. Biological tests have shown that a
number of compounds simultaneously interact with multiple
kinases. These inhibitors are dened as poly target inhibitors.
As a consequence of these multiple interactions, a dierent
potential focus for research has begun; attempts to inhibit more
than one target with a single drug have been a very interesting
tool for combating tumors. This approach is a new weapon in
experimental medicine, and some of the pyrazolo[3,4-d]-
pyrimidines described have been shown to possess this
capability. Further work in this eld is expected to give even
more interesting results in modern molecular medicine.
As reported in this manuscript, many compounds show
excellent results in preclinical studies, and some are expected to
enter clinical trials in the near future, thus following the path of
ibrutinib, which was just approved by the FDA for the
treatment of mantle cell lymphomas.
AUTHOR INFORMATION
Corresponding Author
*E-mail: schensil@unige.it. Phone: +39 010 3538362. Fax: +39
010 3538358.
Notes
The authors declare no competing nancial interest.
Biographies
Silvia Schenone obtained a degree in Medicinal Chemistry in 1987 and
in Pharmacy in 1988 both cum Laude at the University of Genoa
(Italy). In 1992, she received a Ph.D. in Medicinal Chemistry and
became a researcher in 1992. In 2001, she became associate Professor
of Medicinal Chemistry at the same University. At present, she is
involved in the synthesis of heterocyclic compounds as tyrosine kinase
inhibitors. Moreover she is interested in the preparation of adenosine
receptor antagonists and anti-inammatory agents. Prof. Schenones
team is also involved in structural studies on biological macro-
molecules and molecular modeling applications in medicinal subjects.
She is the author of more than 120 publications. In January 2014 Prof.
Schenone received the National Scientic Qualication for the role of
Full Professor.
Marco Radi graduated cum Laude in 2000 from the University of
Siena (Italy) under the guidance of Prof. M. Botta. In 2004 he earned
his Ph.D. in Medicinal Chemistry at the same University working at
the combinatorial synthesis of new antivirals. He then joined the group
of Prof. C. K. Chu at the University of Georgia (U.S.A.) as
Postdoctoral Research Associate where he was involved in the
synthesis of novel nucleoside analogues as anticancer and antiviral
agents via parallel synthesis and subzero microwave-assisted
conditions. From 2006 to 2011 he worked as a Post Doc at the
University of Siena, focusing on the design and synthesis of antitumor,
antitubercular, and antiviral drug candidates. He is currently Assistant
Professor at the University of Parma (School of Pharmacy). In January
2014 Dr. Radi received the National Scientic Qualication for the
role of Associate Professor. His current research is focused on the
design and synthesis of antiviral and anticancer agents, with particular
attention at the development of novel combinatorial methodologies to
quickly produce highly functionalized scaolds. He has authored about
60 paper and patents in these elds.
Francesca Musumeci was born in Genoa (Italy) on 24/07/1983. In
2008 she graduated cum Laude in Medicinal Chemistry at the
University of Genoa. In the same year, she received a six months
fellowship titled New synthetic approaches towards interesting
therapeutic molecules, working at the Department of Chemistry
and Industrial Chemistry, in Genoa. In February 2012, she obtained
her Ph.D. in Medicinal Chemistry at the University of Genoa. During
her Ph.D. she spent three months at the National University of Ireland
in Galway. From 2012 she has been a Post Doc at the University of
Genoa, working on the design and synthesis of potential antitumor
and antiviral drugs in Prof. Schenones group. She is the author of 15
publications on international journals and of a patent and presented
many poster communications and three oral communications to
congress.
Chemical Reviews Review
dx.doi.org/10.1021/cr400270z | Chem. Rev. XXXX, XXX, XXXXXX AS
Chiara Brullo, born in Genoa (Italy) on 11/05/1974, in 1999
graduated cum Laude in Medicinal Chemistry at the University of
Genoa. In 2003 he received the Ph.D. on Pharmaceutical Science,
working at the development of new antiaggregating derivatives. During
her Post-Doctoral experience (20032010) at the University of
Genoa, she focused her attention mainly on the design and synthesis
of novel antiinammatory and anticancer agents. In 2010 she became
Researcher at the Department of Pharmacy of the University of Genoa.
During of her academic career she had participated in dierent
national and international multidisciplinary research projects. In
January 2014 Dr. Brullo received the National Scientic Qualication
for the role of Associate Professor. She is the author of 45 papers in
peer-reviewed international journals, 1 patent application, 1 book
chapter, and numerous proceeding at congresses.
Prof. Maurizio Botta, Ph.D., is full Professor of Medicinal Chemistry at
the department of Biotechnology, Chemistry and Pharmacy of
University of Siena (Italy), where he was director of the Department
and then Dean of the Faculty. From January 2008 is Adjunct Professor
in Temple Universitys College of Science and Technology, in the
Department of Biology, Philadelphia (USA). He is EU expert for
evaluation in the panel Quality of Life. Projects reviewer for Italian,
Slovenian Research Projects, for the Austrian Research Fund
(Wissenschaftsfonds-FWF), for the Cancer Research U.K. Programme
and currently for Fondazione Roma, for the French National
Research Agency and for Italian Ministery for the Economic
Development. He is Member of the Editorial Board for Chem-
MedChem, Journal of Medicinal Chemistry, and Journal of
Chemical Information and Modeling and from January 2010 is an
Associate Editor for Medicinal Chemistry Letters (ACS). He was
Chairman of many scientic schools and meetings. Since 1988, Prof.
Botta is the owner of research funds granted by University, MIUR,
CNR, EU, and Pharmaceutical Companies. Professor Botta is the
author of about 400 papers, 9 publications on volumes, and 25 patents.
ACKNOWLEDGMENTS
We gratefully acknowledge the National Interest Research
Project PRIN_2010_5YY2HL. This work was partially
supported by the Istituto Toscano Tumori-ITT-Grant proposal
2010. We also gratefully acknowledge Dr. Anna Lucia Fallacara
for her useful assistance in the production of the graphics.
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