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Pyrazolo[3,4-d]pyrimidine based scaffold derivatives targeting kinases as

anticancer agents

Article  in  Future Journal of Pharmaceutical Sciences · March 2016

DOI: 10.1016/j.fjps.2016.02.002

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Accepted Manuscript

Pyrazolo[3,4-d]pyrimidine based scaffold derivatives targeting kinases as anticancer

agents

Nasser S.M. Ismail, Eslam M.H. Ali, Diaa A. Ibrahim, Rabah A.T. Serya, Dalal. A.
Abou El Ella

PII: S2314-7245(15)30016-9
DOI: 10.1016/j.fjps.2016.02.002
Reference: FJPS 14

To appear in: Future Journal of Pharmaceutical sciences

Received Date: 12 August 2015

Accepted Date: 28 February 2016

Please cite this article as: Ismail NSM, Ali EMH, Ibrahim DA, Serya RAT, Abou El Ella DA, Pyrazolo[3,4-
d]pyrimidine based scaffold derivatives targeting kinases as anticancer agents, Future Journal of
Pharmaceutical sciences (2016), doi: 10.1016/j.fjps.2016.02.002.

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 ACCEPTED MANUSCRIPT

Review article

Pyrazolo[3,4-d]pyrimidine based scaffold derivatives targeting

kinases as anticancer agents
Nasser S. M. Ismail a.b*, Eslam M. H. Ali d, Diaa A. Ibrahim c, Rabah A.T. Serya a and Dalal. A.

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Abou El Ella a

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a: Pharmaceutical Chemistry Department, Faculty of Pharmacy Ain Shams University, Abbassia,
Cairo 11566, Egypt (Phone: +01015643036, Fax: +02 024051107, E-mail:
saadnasser2003@yahoo.com).

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b: Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and
Pharmaceutical Industries, Future University, Cairo 12311, Egypt

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c: National Organization for Drug Control & Research, P.O. Box: 29, Cairo, Egypt
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d: Medical Union Pharmaceuticals company, Fax : (+20) (064) 3400363, Abu Sultan, Al
Ismaillia, Egypt
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Abstract
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Pyrazolopyrimidines are fused heterocyclic ring systems which structurally can consider as
bioisosteres of adenine, which is fundamental for every aspect of cell life. Pyrazolo[3,4-
d]pyrimidines derivatives have been explored for their inhibitory activity towards various protein
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kinase enzymes and their role as anticancer agents. The present review to the best of our
knowledge is the first compilation on synthesis and medicinal aspects including structure–
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activity relationships of pyrazolo[3,4-d]pyrimidines reported to date.

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Keywords:

Protein kinase, Bioisostere, Pyrazolo[3,4-d]pyrimidine, Synthetic strategy, SAR.

1. Introduction
Several pyrazole derivatives received great attention due to their biological and
pharmacological activities, not only as potential inhibitors of HIV-1[1], pesticides[2],

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fungicides[3], analgesic drugs[1], antihypertensive agents[4] and anticancer agents[5], but they
are also important and useful as starting materials for the synthesis of other fused heterocyclic
systems. One of the most important fused pyrazoloes is prazolopyrimidine derivative which
possess a wide variety of biological activities. In the current work we will put highlights on the
most important aspect of pyrazolopyrimidines, in particular pyrazolo[3,4-d]pyrimidines,

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bioisosteres of purines. Pyrazolo[3,4-d]pyrimidines are reported to encompass pharmacological
potential as antiviral,[6-9] anticoccidials,[10, 11] antimicrobial,[12-15] antitumor,[16-19]

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herbicidal, antileukemic,[20, 21] pesticides,[22] CNS agents,[23] tuberculostatic,[24-26]
antileishmanial,[27-29] radioprotectant,[30] antiinflammatory[31] and cardiovascular

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activities.[32, 33] The present review outlines synthetic strategies and anticancer activity of
pyrazolo[3,4-d]pyrimidines as protein kinase inhibitors. In addition, the radioactive and active

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oxygen species generation activities of pyrazolo[3,4-d]pyrimidines were discussed in this article.
Protein kinases are the most important human enzyme classes. These enzymes are
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responsible for protein phosphorylation by catalyzing the transfer of the ɤ-phosphoryl group
from a nucleoside triphosphate, usually ATP, to the side chain of an amino acid residue in the
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substrate protein.[34, 35]

Based upon their catalytic specificity, the enzyme classes can be subdivided into three
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categories:
I. Tyrosine kinases (TK): These kinases regulate several physiological mechanisms,
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including cell proliferation, differentiation, migration and metabolism, by transferring the ATP
terminal phosphate to tyrosine residues of protein substrates.[36]
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II. Serine/Threonine kinases: They act on serine and threonine amino acids. Six other
groups have been identified that primarily phosphorylate serine and threonine residues.[37]
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III. Histidine Kinases: They autophosphorylate a histidine moiety in the active site and then
transfer the phosphate to an aspartate moiety.[38]
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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 diabetes, inflammation, neurological disorders and
cancer.[39] Thus, kinases represent important targets for anticancer drug development.

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Small molecules protein kinases inhibitors can be classified into three classes according to
the activity state of the kinase they recognize, and their reversibility.
Type I inhibitors: bind to the ATP 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.[40]

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Type II inhibitors: act on the inactive (unphosphorylated) conformation of enzyme, by
stabilizing the inactive conformation. Type I and II work as ATP competitive inhibitors and

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target ATP binding site.[41]
Type III inhibitors: they are noncompetitive ATP inhibitors, they bind to the allosteric site of

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the protein distant from ATP pocket. They don’t require any typical hinge binding motifs and
can bind the enzyme regardless it’s activation state.[42]

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2. Synthetic strategies AN
A plenty of synthetic methods (Fig. 1) have been outlined for the synthesis of pyrazolo[3,4-
d]pyrimidines . 5-Amino-4-cyanopyrazole I has been used in the number of reactions to attain
the synthesis of desired compounds through different routes. Reaction of I with formamide at
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180 °C for 24 h afforded the corresponding compounds (route a) [43, 44] . Two step reaction in
which compound I is reacted with N,N-dimethylphosgeniminium chloride in dichloromethane
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under reflux for 2 h gave the corresponding dimethylcarbamimidic chloride derivative , which
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was then cyclized with hydrochloric acid to obtain the 4-chloro derivative of target compound
(route b).[45] Moreover, Rashad et al.[46] reported that treatment of compounds I with a mixture
of hydrochloric acid/acetic acid (1:3) under reflux, gave pyrazolo[3,4-d]pyrimidine-4-one
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derivatives (route c). In addition, it was reported that treatment of I with triethyl orthoformate
and acetic anhydride under reflux for 24 h. afforded the corresponding pyrazolo[3,4-
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d]pyrimidine derivatives at good yield (route d).[47, 48]

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Meanwhile, concentrated sulfuric acid mediated hydrolysis of compound I gave the

corresponding 5-amino-4-pyrazolcarboxamide II derivative which underwent oxidative
cyclization with various substituted aromatic aldehydes in the presence of equimolar molecular
iodine as a mild Lewis acid and oxidant under neutral conditions in boiling acetonitrile to give a
new series of pyrazolo[3,4-d]pyrimidine derivatives (route e).[12, 49] On the other hand, a
microwave-assisted condensation employing 5-amino-4-pyrazolcarboxamide derivative II in
formamide smoothly afforded the corresponding analogous 1H-pyrazolo[3,4-d]pyrimidines

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(route f).[50] Pyrimidine derivatives can also used as a starting material for the synthesis of the
corresponding pyrazolo[3,4-d]pyrimidines. It is reported that chlorination of barbituic acid using
POCl3 introduced the corresponding tri-chloro pyrimidine derivative III[51, 52] that can be
converted to pyrazolo[3,4-d]pyrimidines via reaction with 1-benzyl-4-hydrazinylpiperidine
dihydrochloride in presence of tri-ethyl amine at 78 °C (route g).[52, 53] Soth et al.[54] carried

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out the reaction of tert-butyl carbazate (route h) with 4-chloro-5- cyano pyrimidine IV in ethyl
amine to afford the target derivative. Also, the title compounds pyrazolo[3,4-d]pyrimidine were

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reported to be prepared by reacting 4-amino-6-aryl-2-phenyl pyrimidine-5-carbonitrile V
derivatives and hydrazine hydrate in EtOH under reflux (route i).[55]

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CN
R1
NH2
N N
CN
R
I NH2
NH2
N N a NC
N

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R I X= NH2
b R N R
X= Cl
i V
HCONH2, 180 °C, 24 h NH2NH2.H2O X= R

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i) N,N-Dimethylphosgeniminium EtOH, reflux
chloride, ClCH2CH2Cl, reflux, 2 h
ii) HCl

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h
t-Butyl carbazate
CN R1 X NC
Ethylamine X= R N
N
NH2 N Cl N
N N HCl, AcOH, Reflux N

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N R2
I c R 1-benzyl-4-hydrazinylpiperidine IV
R
X= OH dihydrochloride, Et3N, EtOH,
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78 °C
i) HC(OEt)3 /Ac2O g
ii) NH3
H2NCHO, mw, 200 °C, 20 min X= Cl
Cl
CN
d
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ArCHO, I2, CH3CN, reflux

N
X= OH f
NH2
N N X= OH Cl N Cl
e
R I X= OH III
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O O
NH2 NH2
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NH2 NH2
N N N N

R R
II II
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Fig.1: Synthetic approaches for pyrazolo[3,4-d]pyrimidines

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3. Anticancer activity
The biological investigations of pyrazolo[3,4-d]pyrimidines have revealed that substitution of
various groups on the scaffold imparts anticancer activity through inhibition of different
enzymes.

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3.1. p38-α mitogen-activated protein kinases (MAPK) inhibitors

p38 Kinases are members of the mitogen-activated protein kinase family that transduce
signals from various environmental stresses, growth factors, and steroid hormones. Four p38
MAP kinases, p38-α (MAPK14), -β (MAPK11), -γ (MAPK12/ ERK6), and -δ (MAPK13/

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SAPK4), have been identified. p38 is highly expressed in aggressive and invasive breast cancer.
Increased levels of activated p38 are markers of poor prognosis.[56] In 2007, Dhillon, et al.[57]
explored the role of MAPK pathways in cancer. Cancerous mutations in MAPK pathways are

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frequently affecting Ras and B-Raf in the extracellular signal-regulated kinase pathway. Stress-
activated pathways, such as Jun N-terminal kinase and p38, largely seem to counteract malignant

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transformation. The balance and integration between these signals may widely vary in different
tumors, but are important for the outcome and the sensitivity to drug therapy.[58] In 2011, Soth,

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et al.[54] designed and established 3-amino-pyrazolo[3,4-d]pyrimidines as p38-α kinase
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inhibitors. Compounds 2–6 were tested through enzyme assay for inhibition of the p38-α -
catalyzed phosphorylation of myelin basic protein. Potent inhibitors were identified from both
the amide and amine subseries. The enzymatic assay revealed that the amide functionality has a
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mild effect on potency owing to the fact that it is away from the binding site whereas amine
moiety showed high potency due to its tight binding to the active site. Compound 6 showed
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extremely high selectivity for p38-α over other kinases. That explained by the optimal fit of the
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2,4-difluorophenyl ether into the back pocket of p38-α. This pocket is specifically bigger in p38-
α than in many other kinases, due to its small gatekeeper residue (Thr106), also, the presence of
a nonplanar front pocket side chain (the branched sulfone of 5), which is particularly well-
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tolerated by p38-α versus other kinases, for example, Lck, due to a larger open space in the front
of the protein.[59] Finally, the methyl group of the sulfonic side chain of 6 fits more tightly into
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a lower hydrophobic pocket defined in part by Ala157 (Table 1).

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Table 1: IC50 of compounds (2-6) on p38-α

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p38α enzyme IC50
Cpd R HWB IC50 (µM)
(µM)

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2 0.1 --

3
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4 0.023 0.021
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5 0.038 0.126
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6 0.1 0.039
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3.2. Src kinase inhibitors

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Src kinases are a family of nine different PTKs, including c-Src, c-Yes, Fyn, Lck, Lyn, Hck,
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Frk, Blk, and c-Fgr of which Src is the prototype.[60] SrcKs can regulate a number of signaling
pathways that impact on the behavior of tumor cells, including proliferation, survival, migration,
invasion, and angiogenesis.[61] Src tyrosine kinase expression is frequently elevated in a number
of epithelial tumors including colon, breast, prostate, lung, ovary, and pancreas compared with
the adjacent normal tissues. Interestingly, Src kinase can be considered as key modulator of
cancer cell invasion and metastasis.[62-65] In 2011, Kumar, et al.[43] explored the

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antiproliferative and proapoptopic activities of pyrazolo[3,4-d]pyrimidines (compounds 7-9) as

Src kinase inhibitors in human ovarian adenocarcinoma (SK-Ov-3), breast carcinoma (MDA-
MB-361), and colon adenocarcinoma (HT-29)(Table 2). They concluded that pyrazolo[3,4-
d]pyrimidines are involved in the stimulation of programmed cell death and decrease the Src
phosphorylation. The SAR of the synthesized compounds revealed that the 3-phenyl group

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contributes significantly to Src kinase inhibitory activity through a hydrophobic interaction with
a large hydrophobic pocket in the ATP-binding site.[66] In addition, the variation of N1

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substitution in 3-phenylpyrazolopyrimidines with different 1,2,3-triazoles containing
hydrophobic residues can occupy and/or interact with amino acids of the cavity of carbohydrate

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binding pocket of ATP and contribute to the enhancement of Src kinase inhibitory potency. It
was found that compounds 7 and 8 exhibited modest inhibitory potency (IC50 = 6.2, 5.6 µM)

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against Src kinase. Structure–activity relationship studies suggested that the incorporation of aryl
groups at N1 position of PhPP is less tolerated (Table 2).
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Table 2: IC50 of compounds (7-9) on Src kinase:
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Cpd R Src kinase IC50 (µM)

7 H 6.2
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8 CH3(CH2)4CH2– 5.6
9 4-CH3OC6H4– 17.4
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3.3. Cyclin-dependent kinase 2 inhibitors

The cyclin-dependent kinases (CDKs) are a family of serine threonine protein kinases, which
play a crucial role in the cell cycle progression.[67, 68] In 2003, Kim, et al.[69] introduced a new
series of 1,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines (compounds 10-15) capable of
selectively inhibiting CDK2 activity. The synthesized compounds were evaluated for their

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inhibitory activity against CDK2 and EGFR enzymes (Table 3). Moreover, the synthesized
compounds were further evaluated for their cell division inhibitory activities against three human
tumor cell lines (A431, SNU638 and HCT116). In general, 4-anilino compounds 12, 13 showed
better CDK2 and cell division inhibitory activities than the corresponding 4-benzyl analogues 10,
11. The unsubstituted compounds 12, 13 at N-1 exhibited higher potency compared to the

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substituted compounds 14, 15 for the CDK2 inhibitory activity (Table 3).

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Table 3: IC50 of compounds (10-15) on CDK2 and EGFR:

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IC50 (µM) IC50 (µM)
Cpd R1 R2 R3
CDK2 EGFR A431 SNU638 HCT116
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10 H H H 29.5 93.3 26.56 20.77 4.5

11 H H (CH2)2OH 37.2 55.0 > 100 28.98 93.63
12 m-Br H H 8.1 > 100 > 100 69.22 > 100
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13 m-Br H (CH2)2OH 3.8 36.3 > 100 38.88 > 100

14 m-OCH3 CH3 H 57.5 > 100 12.94 10.49 17.19
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15 m-Br (CH3)2CH2 (CH2)2OH 29.5 > 100 2.58 4.10 5.06

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3.4. Abl-tyrosin kinase inhibitors

Bcr-Abl is regarded as highly attractive target for drug intervention since the Bcr-Abl fusion
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gene encodes a constitutively activated kinase. Drug discovery that specifically targeted the ATP
binding site of a single kinase is regarded as quite a challenging task since hundreds of protein
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kinases were known in the human genome. In 2008, Schenone et al.[45] reported the synthesis of
new 4-amino substituted pyrazolo[3,4-d]pyrimidines (compounds 16-18) along with their
activity in cell-free enzymatic assays on Src and Abl tyrosine kinases. Some compounds
emerged as good dual inhibitors of the two enzymes, showed antiproliferative effects on two
Bcr-Abl positive leukemia cell lines K-562 and KU-812, and induced apoptosis (Table 4).

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Table 4: IC50 of compounds (16-18) on Src and Abl kinases:

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Ki (µM) IC50 (µM)
Compound R R1 R2

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Src Abl K-562 KU-812
16 SCH3 NHC4H9 CH2-CHCl-C6H4-pF 5.02 0.11 29.2 37.5
17 SC3H7 NHCH2C6H5 CH2-CHCl-C6H5 0.51 0.10 27.3 67.1

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18 SCH3 NHCH2C6H5 CH2-CHBr-C6H5 0.22 0.19 25.0 35.9

While, in 2011, Radi et al.[70] synthesized pyrazolo[3,4-d]pyrimidine derivatives (19, 20)

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having inhibitory activity in hypoxic human leukemia cells and reported the in vitro ADME
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properties and metabolic activities. These compounds were verified to act via inhibition of Bcr-
Abl kinase activity, increasing caspase-3 activity and escalating cleavage of poly-ADP-ribose-
polymerase. Molecular simulation studies featured the binding modes and structural
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requirements in the ATP pocket for dual Src/Abl inhibitors so that C-4 amino group has to forms
hydrogen bond with Met318. The synthesized compounds were found to suffer from
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pharmacokinetic issues due to polar group substitution on C-6. A beneficial equilibrium was
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exhibited by compounds with respect to different ADME properties and biological activity in
leukemia cells (Table 5).
Table 5: IC50 of compounds (19, 20) on Abl enzyme:
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IC50 (µM)
Compound R1 R2 Abl Ki (µM)
K 562 MEG-01 KU-812
19 NHCH2(o-F-Ph) F 0.02 36 15 --
20 NHCH2(o-F-Ph) Cl 0.08 9 5.5 8

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3.5. Aurora kinases and CDK1 inhibitors

Aurora kinase is a serine/threonine kinase involved in the regulation of various stages of
mitosis. Therefore, it is an important regulator in maintaining normal cell cycle process.[71]

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Aurora A is expressed from the late S phase, peaking at G2/M phase and declining at the G1
phase and is involved in centrosome maturation and separation, bipolar spindle assembly, and
mitotic entry.[72] Aurora kinases are known to be overexpressed in solid tumors and also may be

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inappropriately activated in certain cell types. Inhibition of Aurora activity was shown to slow
down cell growth and division and hence it is regarded as an attractive target in kinase-based

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drug discovery program.[73]
In 2012, Brazidec et al.[74] reported SAR of the synthesized 1,6-disubstituted-1H-

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pyrazolo[3,4-d]pyrimidines as dual inhibitors of Aurora kinases and CDK1. With the objective
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of designing biochemically potent and controlled clogP value they envisioned that R is
significant for activity since it interacts with binding site of CDK1. It was observed that
spirobicyclic, fused tricyclic, monocyclic rings were metabolically unstable and possessed high
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potency. S-Methoxymethyl (spirobicylic) displayed higher ability to inhibit AKB

phosphorylation. The pharmacokinetics study revealed that compound 22 was highly potent with
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low distribution volumes, high clearance rate, good ADME properties, neutropenic index of 21
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and was a good antitumor agent (Table 6).

Table 6: IC50 of compounds (21, 22) on Aurora kinas A, B and CDK1:
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IC50 (l µM) G2/M 100% HCT116

Compound R
AKA AKB CDK1 (l µM) IC50 (l µM)
21 (S)CH2OMe 0.004 0.005 0.022 0.03 0.01
22 (R)CH2OMe 0.016 0.006 0.15 0.065 0.06

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3.6. Ribosomal protein S6 kinase (7Akt/p70S6K) inhibitors

p70S6 kinase (p70S6K) is an important regulator of cell proliferation. p70S6 kinase has
emerged as an important regulator of cell growth, playing a positive role during progression

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through the G1 phase of the cell cycle.[75] P70S6K is phosphorylated by PDK1 on Thr-229.
Prior phosphorylation of Thr-389 is reported to be necessary before PDK1 can phosphorylate

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Thr-229. Phosphorylation of Thr-389 appears to be rate limiting and correlates with activity;
however, activation of mTOR depends on Akt activation. P70S6K is also phosphorylated on the

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pseudo substrate or auto inhibitory domain at Ser-411, Ser-418, Thr-421, and Ser-424, but the
kinases responsible for phosphorylating these sites are debated.[76]
In 2012, Rice et al.[77] evaluated the role of pyrazolopyrimidines as dual Akt/p70S6K

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inhibitors. Compounds having bromine at C-3 position e.g. 23 exhibited high inhibitory activity
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against Akt and in PC-3 cell lines. Further, Akt activity was enhanced by the presence of larger
lipophilic groups at position-5 which resulted in a potent, metabolically stable potential
candidate exhibiting good pharmacokinetic profile (Table 7).
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Table 7: IC50 of compound (23) on p70S6K and AKT1:

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H
R2 N
R3
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R1 N
C

N
N
N
AC

N
H
IC50 (nM) IC50 (nM)
Compound R1 R2 R3 GSK3β-
p70S6K Akt1 PC-3
p

23 -Br -COCH2CH9 2 1 30/55 194

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3.7. EGFR Tyrosine Kinase Inhibitors

EGFR (epidermal growth factor receptor) is a receptor tyrosine kinase that exists on the cell
surface and is activated by binding with specific ligands, including epidermal growth factor and

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transforming growth factor α (TGFα). EGFR signaling pathway is one of the most important
pathways that regulate growth, survival, proliferation, and differentiation in mammalian cells.
Overexpression or constant activation of EGFR results in uncontrolled cell division which has

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been associated with a number of cancers, including lung cancer, anal cancers and glioblastoma
multiform.[78] In 1997, Traxler et al.[79] optimized a class of the pyrazolo[3,4-d]pyrimidines

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producing a series of 4-(phenylamino)-1H-pyrazolo[3,4-d]-pyrimidines (24 and 25) as highly
potent inhibitors of the EGF-R tyrosine kinase. Compounds were tested for inhibition of the

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tyrosine kinase activity of a recombinant, intracellular domain of the EGF-R (ICD) using
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angiotensin II as the phosphate-acceptor substrate. Selectivity was assayed against a panel of
tyrosine (c-Src and v-Abl) and serine/threonine kinases (PKC-R and CDK1). The amino group in
position 6 of the pyrimidine ring can be removed without loss of activity. Introduction of an
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anilino, or especially the m-chloroanilino moiety in the position 4 of the pyrimidine ring led to
compounds with improved activity (26 and 27). In exploiting the size of the large hydrophobic
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pocket, literature reported that replacement of the anilino moiety in position 3 of the pyrazole
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ring by a larger substituted benzyl amino moiety (28 and 29) or by a substituted phenyl ring
directly attached to the pyrazole ring (30 and 31) also led to highly potent inhibitors in the Nano
molar range (Table 8).
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Table 8: IC50 of compounds (24-31) on EGFR compared to other kinases:

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AC

IC50 (µM)
Compound R1
EGF-R v-Abl c-Src PKC-Rα CDK1
24 NH2 0.22 1.90 >10 >100 --
25 H 0.16 0.41 6 24 --

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IC50 (µM)
Compound R1 R2 X EGF-
v-Abl c-Src PKC-Rα CDK1

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R
26 H H NH 0.058 1.96 75 6.9 --
27 m-Cl Cl NH 0.033 1.98 5.3 5.9 --

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28 H Cl NHCH2 0.007 6.60 >10 10.0 7.40
29 m-OCH3 Cl NHCH2 0.008 3.50 >10 >100 3.10

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IC50 (µM)
D

Compound R1 R2
EGF-R v-Abl c-Src PKC-Rα CDK1
30 H Cl 0.019 3.76 >10 >10 2.80
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31 m-OH Cl 0.026 0.18 0.002 >10 0.2

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3.8. Glycogen Synthase Kinase-3 (GSK-3) Inhibitors

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Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine protein kinase which exists in

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two isoforms GSK-3α and GSK-3β.[80] GSK-3 has recently been the subject of much research
because it has been implicated in a number of diseases, including type 2 diabetes, Alzheimer’s
disease (AD), bipolar disorders, inflammation, and cancer.[81] Several recent studies have shed
new light on the activity of GSK-3 in cancer and provide insight into the molecular mechanisms
by which it regulates tumor cell proliferation and survival of multiple human malignancies. In
fact, GSK-3β is a critical regulator of nuclear factor (NF) κB nuclear activity, which suggest that

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inhibition of GSK-3β could be effective in the treatment of a wide variety of tumors with
constitutively active NFκB.[82]
In 2004, Thomson and his colleagues identified a novel lead pyrazolo-pyrimidine structure
as GSK-3 inhibitor (32 and 33) (Fig.2) and proceeded to optimize this family of compounds
through synthesizing of hydrazones of the general structure different aromatic or hetero aromatic

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N1 substituents and usually a 4-pyridyl group on the hydrazone moiety.[83]
Compound 32 is a potent GSK-3 inhibitor with a pIC50 of 8.2 and low Nano molar efficacy

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for the stimulation of glycogen synthesis in vitro. In light of SAR studies and structural analysis
of the GSK-3 binding pocket, the authors incorporated a benzimidazole group at N1 and

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obtained derivatives 33. Derivative 33, with a 4-fluoro-phenyl substituent, showed similar
activity to 32 and improved cellular efficacy in stimulating glycogen synthesis in rat skeletal

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muscle L6 cell line.[83] AN
M
D
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32 33
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Fig.2: Structure of compounds 32 and 33.

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3.9. Inhibitors of urokinase plasminogen activator (uPA):

Urokinase plasminogen activator (uPA) is a serine protease that functions in the conversion
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of the circulating plasminogen to the active, broad-spectrum serine protease, plasmin. uPA is
secreted as an inactive single-chain proenzyme by many different cell types and exists in a
soluble or cell associated form by binding to a specific membrane uPA receptor (uPAR).[84, 85]
Elevated expression levels of urokinase is implicated in a large number of malignancies e.g.
cancers of breast[86], lung[87], cervix[88], prostate,[89] thyroid carcinoma,[90] ovarian,[91]
stomach, kidney, bladder, and brain.[92, 93]

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In 2014, a new pyrazolo[3,4-d]pyrimidine derivatives (compounds 34-37) were synthesized

and evaluated in vitro for their anticancer activities as inhibitors of urokinase plasminogen
activator (uPA). Moreover, their anticancer activities against the MCF-7 breast cancer, HepG2
liver cancer, and A549 lung carcinoma cell lines is evaluated [94] (Table 9).
Table 9: IC50 of compound (34-37) on uPA:

PT
RI
SC
Compound R R1 X % Inhibition of uPA

U
MCF-7 HepG2 A549
AN
34 H CH3 O 77±4.28 63±4.65 ---
35 H H O 60±2.84 56±1.98 ---
36 H SH S 7±0.94 5±0.46 ---
M

37 p-ClC6H4 SH NH 9±0.47 6±0.45 ---

D
TE

3.10. Xanthine oxidase inhibitors

EP

In 2008, Gupta, et al.[47] reported a series of pyrazolo[3,4-d]pyrimidines (38-41),

structurally related with allopurinol[95], a well-known xanthine oxidase inhibitor, clinically used
in the therapy of gout, which also have been reported as potent inhibitors of xanthine oxidase and
C

hence the growth of several human tumor cell lines. Their biological activity was pertained to
AC

xanthine oxidase inhibition which affected the growth of three tumor cell lines (MCF-7, NCI-
H460, and SF-268). The results revealed that some compounds possessed good xanthine oxidase
inhibitory activity but no effect on the growth of the three tumor cell lines. The SAR studies
highlighted that the lipophilic region adjacent to the active site of xanthine oxidase could
complex with the aromatic systems. That complex is formed with lipophilic region when the
pyrazole ring is substituted with aryl groups such as the pyrimidine ring which further increases

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the activity in the presence of an electron withdrawing group. The higher potency of compound
41 compared to compound 40 is owing to the amide bond[47] (Table 10).
Table 10: IC50 of compounds (38-41) on xanthine oxidase:

PT
RI
SC
XO inhibitory activity Inhibition of MCF-7
Compound R
IC50 (µM) cell lines GI50 (µM)
38 CN 0.4 --

U
39 NO2 2.2 --
40 CF3 0.18 --
AN
41 CONHCH2COOCH3 0.08 >150
M

3.11. Generation of reactive oxygen species

D

In 2011, Rashad et al.[96] reported the synthesis and anticancer effects of some novel
pyrazolo[3,4-d]pyrimidine derivatives (42-43) by generating reactive oxygen species (ROS) in
TE

human breast adenocarcinoma cells. Reactive oxygen species (ROS) are chemically reactive
molecules containing oxygen. ROS are formed as a natural byproduct of the normal metabolism
EP

of oxygen and have important roles in cell signaling and homeostasis.[97] Cancer cells exhibit
greater ROS stress than normal cells do. At low levels, ROS facilitates cancer cell survival since
cell-cycle progression driven by growth factors and receptor tyrosine kinases (RTK) require ROS
C

for activation.[98] On the other hand, a high level of ROS can suppress tumor growth through
AC

the sustained activation of cell-cycle inhibitors.[99] In fact, the antitumor effect of these novel
pyrazolo[3,4-d]pyrimidine compounds is partly through augmenting ROS stress by production of
H2O2.[100] In general, the acyclic nucleoside of pyrazolo [3,4-d]pyrimidine derivative 42
revealed the highest anticancer activity among the other tested compounds [96] (Table 11).

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Table 11: Biological activity of compounds (42-43):

PT
SOD CAT GSH-Px GSH H2O2
Cpd/
R R1 U/mg U/mg U/mg nmol/m nmol/mg
(µg/ml)

RI
protein protein protein g protein protein
42/2 µg/ml 125.00 ± 3.60 ± 5.60 ± 20.11 ± 35.00 ±
14.00 0.30 0.60 2.25 3.70

SC
42/5 µg/ml 150.25 ± 2.90 ± 4.25 ± 19.16 ± 55.00 ±
16.50 0.35 0.50 2.20 5.50
I CH2CH3
42/10 µg/ml 360.00 ± 1.76 ± 3.00 ± 17.00 ± 60.50 ±
37.50 0.19 0.37 1.80 7.00

U
42/20 µg/ml 385.00 ± 1.55 ± 2.20 ± 14.80 ± 80.00 ±
39.00 0.20 0.24 1.80 8.60
AN
43/2 µg/ml 105.00 ± 3.90 ± 6.00 ± 23. 60 ± 30.75 ±
12.20 0.42 0.75 2.90 3.86
43/5 µg/ml 135.70 ± 3.40 ± 5.65 ± 20.95 ± 45.77 ±
M

16.12 0.37 0.66 2.80 5.90

Br CH 2CH2OCH3
43/10 µg/ml 310.25 ± 3.00 ± 4.00 ± 18.75 ± 58.50 ±
33.00 0.35 0.50 2.00 6.00
D

43/20 µg/ml 310.00 ± 2.10 ± 2.90 ± 15.60 ± 66.80 ±

35.60 0.26 0.42 1.66 7.50
TE

3.12. Antiproliferative activity

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In 2004, Schenone, et al.[101] introduced a new class of 1-aryl-4-amino-1H-pyrazolo[3,4-

d]pyrimidine derivatives (44-48) which were synthesized and biologically evaluated for their
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inhibitory activity against tumor cell line A431 responding to epidermal growth factor (EGF).
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The growth inhibitory activity of the compounds studied was compared with the activity of the
selective EGFR inhibitor AG1478.[102, 103] at the concentration of 50 nM. From the data
obtained, it was found that, large substituents at C4 exhibited better inhibition of the A431 cells
growth. Introduction of an aromatic moiety at C4, such as a benzyl amino group, was favorable
for activity (45, 112%), while increasing the size of this group into a phenylethyl amino side
chain produced a drop in activity (46, 49%). Compound 47, bearing a cyclopropylamino moiety
at C4 and an unsaturated aryl chain (i.e., styryl moiety) at N1 was characterized by an inhibitory

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profile much more interesting with respect to the corresponding phenyl chloroethyl derivative 44
(Table 12).
Table 12: Antiproliferative activity of compounds (44-48) on A431 cell line:

PT
HN Cl
O
N

RI
O N
AG1478

SC
% Inhibition on
Compound R R1 EC50
A431 cell line
44 NHcyclopropyl CH2CHClPh 53 69

U
45 NHCH2Ph CH2CHClPh 112 4.3
46 NH(CH2)2Ph CH2CHClPh 49 70
AN
47 NHcyclopropyl CH=CHPh 146 1.4
48 NH(CH2)2Ph CH=CHPh 46 78
M
D

3.13. Antitumor activities against HL-60 (human leukaemia cancer

TE

cell)
In 2011, Song and his coworkers [104] illustrated the synthesis of novel fluorinated
EP

pyrazolo[3,4-d]pyrimidine derivatives containing 1,3,4-thiadiazole (49-52). The antitumor

activities of the synthesized compounds evaluated against HL-60 by an MTT assay.[105]
C

Compounds 49, 50, 51 and 52 exhibited good anticancer activity against HL-60, especially
AC

compounds 51 and 52 which exhibited even higher activity than doxorubicin (IC50 = 0.55
µmol/L). Studying the SAR of , these compounds revealed that better anticancer activity is
optimized when H atom in position 1 of the pyrazole ring is substituted with a phenyl group[104]
(Table 13).

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Table 13: IC50 of compounds (44-48) on HL-60 cell line:

PT
RI
Compound R R1 IC50 (µmol/L)
49 H CF3 1.03

SC
50 H 2-FC6H4 3.16
51 C6H5 CF3 0.08
52 C6H5 4-CF3C6H4 0.21

U
AN
3.14. Antitumor activities against MCF-7 (breast adenocarcinoma):
M

In 2014, Abdellatif et al.[106] synthesized a new series of pyrazolo[3,4-d]pyrimidin-4-ones and

screened in vitro for antitumor activity. The series comprises the derived 5,6-disubstituted
D

pyrazolo[3,4-d]pyrimidin-4-one pharmacophore that is structurally related to Erlotinib[107] and

Lapatinib.[108] In that study, the substitution pattern at the 5,6-disubstituted pyrazolo[3,4-
TE

d]pyrimidin-4-one pharmacophore was manipulated so as to create different electronic

environments that might affect the lipophilicity and hence the activity of target molecules. The
EP

rationale for the design of target compounds was based upon some structural modifications on
the general features of anilinoquinazoline-containing compounds (Erlotinib and Lapatinib).
These modifications comprise a replacement of the benzene moiety in the quinazoline skeleton
C

by a pyrazolo moiety as the pyrazolo moiety is naturally found in the body’s purine bases and
AC

this is expected to enhance cytotoxic activity. Prompted by these claims, they present a new
series of compounds containing 5,6-disubstituted pyrazolo[3,4-d]pyrimidin-4-ones core as
anticancer agents. The design strategy was directed toward designing a variety of compounds
with diverse chemical properties hypothesizing that the potency of these compounds might be
increased by adding alternative binding groups such as a methyl group at position 6, and

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aroylhydrazone, phenylamino, amide, thioamide, thiosemicarbazide and substituted aryl at

position 5 of the pyrazolo[3,4-d]pyrimidine ring. (Table 14)
Table 14: IC50 of compounds (53-56) on MCF-7cell line

Fragment replacemnt

PT
O
F
R
HN O

RI
HN Cl
O N
O N N
N O N
O
O N N N
S N

SC
Erlotinib O O
Lapatinib
Isosteric replacement
lipophilic group insertion

U
AN
Compound R IC50 on MCF-7
M

53 H 17
54 F 12
D

55 Cl 12
56 NO2 11
TE
EP

4. Conclusion
In this review we have compiled and discussed specifically the anticancer potential of
C

pyrazolo[3,4-d]pyrimidine derivatives through inhibition of different kinase enzymes, as well as

AC

their synthetic strategies. Number of studies has explored their SAR, as well as conformation and
orientation requirements for kinase binding site through modeling studies. This could provide
insight to a medicinal chemist for a comprehensive and target oriented information for
development of clinically viable pyrazolo[3,4-d]pyrimidine -based anticancer drugs.

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