Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line

Article abstract of DOI:10.1016/j.ejmech.2016.07.034

Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in?vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562?cell line. They can be considered as new hits for further structural optimization to obtain better activity.

Full text of DOI:10.1016/j.ejmech.2016.07.034

Accepted Manuscript
Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form
of Abl and the leukemia K-562 cell line
Samir M. El-Moghazy, Riham F. George, Essam Eldin A. Osman, Ahmed A.
Elbatrawy, Miroslava Kissova, Ambra Colombo, Emmanuele Crespan, Giovanni Maga
PII:
S0223-5234(16)30582-7
10.1016/j.ejmech.2016.07.034
EJMECH 8752
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 April 2016
Revised Date: 14 July 2016
Accepted Date: 18 July 2016
Please cite this article as: S.M. El-Moghazy, R.F. George, E.E.A. Osman, A.A. Elbatrawy, M. Kissova,
A. Colombo, E. Crespan, G. Maga, Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active
against mutant form of Abl and the leukemia K-562 cell line, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.07.034.
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ACCEPTED MANUSCRIPT
Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active
against mutant form of Abl and the leukemia K-562 cell line
a
a*
a
Samir M. El-Moghazy , Riham F. George , Essam Eldin A. Osman , Ahmed A.
b
c
c
c
Elbatrawy , Miroslava Kissova , Ambra Colombo , Emmanuele Crespan , Giovanni
c
Maga

ACCEPTED MANUSCRIPT
Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active
against mutant form of Abl and the leukemia K-562 cell line
a
a*
a
Samir M. El-Moghazy , Riham F. George , Essam Eldin A. Osman , Ahmed A.
b
c
c
c
Elbatrawy , Miroslava Kissova , Ambra Colombo , Emmanuele Crespan , Giovanni
c
Maga
aPharmaceutical chemistry department, Faculty of Pharmacy, Cairo University
1562, Egypt
, El-kasr Elaini Street, Cairo
1
b
Pharmaceutical chemistry department, Faculty of Pharmacy, Nahda University
,
Beni suif, Egypt.
c
Istituto di GeneticaMolecolare, IGM-CNR, Via Abbiategrasso 207, I-27100 Pavia, Italy.
Abstract
Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and
pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14
were synthesized and characterized by spectral and elemental analyses. They were
screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a
and 7b revealed the highest activity against both wild and mutant Abl kinases as well as
the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for
further structural optimization to obtain better activity.
Keywords: pyrazolo[3,4-d]pyrimidines, Src inhibition, Abl inhibition, K-562 cell line,
molecular modeling.
*
Corresponding author.
Riham F. George, E-mail address: rihamfgeorge@yahoo.com

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1
. Introduction
Cancer is considered as a health burden worldwide due to its clinical and economic
outcomes. It is the second leading cause of death after the cardiovascular diseases [1, 2].
The new cancer cases are expected to increase by as much as 15 million per year by
2
020, according to the World Health Organization, unless further preventive measures
are put into practice [3]. The Agency for Healthcare Research and Quality estimates that
the direct medical costs (total of all health care expenditures) for cancer in the US in 2013
were $74.8 billion [1]. Chronic myeloid leukemia (CML) is a type of cancer that starts in
certain blood-forming cells of the bone marrow. In CML, a genetic change takes place
forming an abnormal gene called Bcr-Abl, which turns the cell into a CML cell. The
leukemia cells grow and divide, building up in the bone marrow and spilling over into the
blood. In time, the cells can also settle in other parts of the body, including the spleen.
CML is a fairly slow growing leukemia, but it can also change into a fast-growing acute
leukemia that is hard to treat [1]. The future of cancer treatment depends on development
of targeted agents that specifically block key proteins involved in progression of specific
types of cancer [4]. Therefore, the first-line treatment for CML was the selective
inhibition of Bcr-Abl kinase activity by imatinib mesylate (Gleevec) I. However,
resistance to Gleevec has been documented in patients through mutations both in and
outside the Bcr-Abl kinase domain leading to interference with imatinib binding to Bcr-
Abl [5]. As a consequence, there was a growing need for developing second-generation
small molecule inhibitors able to treat Gleevec-resistant CML [6-9].
Second generation Abl inhibitors were synthesized through rational drug design
approaches to overcome resistance to I [10]. Recently, Novartis disclosed Nilotinib
(
Fig.1, II), structurally related to I, which showed promising results in preclinical studies
and was 10–25-fold more potent compared to I in cellular assays. Compound II also
inhibited the growth of cell lines expressing Bcr-Abl mutants resistant to I [11]. On the
other hand, unlike I and its derivatives, III (Fig. 1) interacted with the peptide substrate
binding site of Abl and inhibited wild type kinase 10-fold more potently than I, and it
also had activity against kinase domain mutations resistant to I [12].
Furthermore, many 4-amino-substituted pyrazolo[3,4-d]pyrimidines were synthesized
and found to be active on different oncogenic tyrosine kinases and cancer cell lines

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depending on the nature and position of substituents on the heterocyclic core [13−23].
Compound IV was an inhibitor of Abl (wild type) with K = 0.04 ꢀM [22], while
i
compound V had K = 0.025 ꢀM on Abl T315I (mutant form) (Fig. 1) [14]. Although the
i
most widely accepted mechanism for imatinib resistance is the presence of kinase domain
mutations [5], cells from patients resistant to imatinib express an activated form of the
(Src Family Kinases, SFKs) Lyn [24]. Moreover, Hck (another SFK) and Lyn are over-
expressed and activated in CML blast-crisis patients and their up-regulation correlates
with disease progression and resistance in cell lines and patients treated with imatinib
[25, 26]. Therefore, compounds that can inhibit both Src and Abl might be useful in the
treatment of patients who have relapsed on imatinib. Abl is known to share significant
sequence homology with Src and in its active conformation it bears remarkable structural
resemblance with most SFKs [27]. As a result, ATP-competitive compounds originally
developed as Src inhibitors frequently exhibited potent inhibition of Abl kinase [28, 29].
Thus, several second generation Bcr-Abl kinase inhibitors targeting both Src and Abl
kinases were synthesized in order to combat imatinib resistance, such as dasatinib VI and
bosutinib VII (Fig.2) [30, 31]. Additionally, many pyrazolo[3,4-d]pyrimidines that were
designed originally as Src inhibitors, exhibited potent inhibition of Abl kinase as PP2,
VIII [32] and compound IX (Fig. 2) that revealed Ki values 0.22 and 0.19 ꢀM against
Src and Abl enzymes, respectively [19].
It was found that most of the reported compounds carried substitutions at positions 1, 4
and 6 of the pyrazolo[3,4-d]pyrimidine core, therefore, this work aimed to explore the
effect of the substitution of a phenyl ring at position 5 in addition to the functional
moieties at positions 1 and 6 hoping to develop a better structure activity relationship.
Moreover, some pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines were prepared to study
the effect of this structure rigidification on the biological activity. Furthermore,
compounds with promising activity on the enzyme have been screened against Leukemia
K-562 cell lines. Moreover, the activity of the most active compound against both Abl
and Src kinases has been validated through molecular modeling technique.

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Fig. 1. Structures of some Abl inhibitors
Cl
Cl
O
S
N
NH
N
O
NH
H
Cl
N
NC
O
O
N
N
OH
N
N
N
Dasatinib, VI
Bosutinib, VII
Cl
NH
NH2
N
N
N
N
N
S
N
Br
N
N
PP2, VIII
IX
Fig. 2. Structures of some dual Abl-Src kinase inhibitors.

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2
. Results and discussion
2
.1. Chemistry
The target compounds were synthesized as outlined in schemes 1, 2. Ethyl ethoxy
methylene cyanoacetate [33] was reacted with phenyl hydrazine according to the
1
reported procedure [34] to obtain ethyl 5-amino-1-phenyl-1H-pyrazolo-4-carboxylate 2.
The pyrazolo[3,4-d]pyrimidine core of 3 was formed from the reaction of 2 and phenyl
isothiocyanate [35]. The reaction of 3 with methyl iodide in dry acetone in the presence
1
of potassium carbonate afforded 4. H NMR spectrum showed the presence of a singlet
signal at 3.75 ppm corresponding to the methyl group. Treatment of 4 with excess
hydrazine hydrate in absolute ethanol resulted in 6-hydrazinyl derivative 5. The IR
-1
spectrum of 5 demonstrated stretching bands at 3319, 3273 and 3207cm corresponding
1
to NH and NH, respectively. Moreover, H NMR of compound 5 showed two
2
2
exchangeable signals at 5.64 and 9.68 corresponding to NH and NH, respectively, along
with the disappearance of the signal of the methyl group (Scheme 1). Condensation of 5
1
with different aldehydes in absolute ethanol afforded 6a-d. H NMR spectra of
compounds 6a-d revealed an increase in the integration of the aromatic protons indicating
the presence of additional aromatic ring. The characteristic olefinic CH singlet signal
appeared at range 7.47-9.08 ppm. Moreover, compound 6b showed the presence of OH
singlet signal at 10.08 ppm which disappeared upon deuteration. Compound 6d revealed
a singlet signal at 3.79 ppm corresponding to the methoxy group. Furthermore, the
hydrazine derivative 5 was stirred with various benzoyl chlorides in the presence of
1
pyridine at room temperature to obtain 7a-c. H NMR spectra of these compounds
revealed an increase in the integration of the aromatic protons indicating the presence of
an additional aromatic ring. Also, singlet signals appeared in the range of 9.74-13.17 ppm
1
3
for the 2NH protons which disappeared upon deuterium exchange. In addition, C NMR
spectrum of 7a and 7c supported the carbon skeleton of the obtained structure and
revealed the presence of an additional C=O signals at 171.3 and 169.2, respectively
(Scheme1). Meanwhile, the target compound 8 was obtained via reflux a solution of 5
with ethyl acetoacetate in presence of anhydrous potassium carbonate in absolute ethanol.
1
H NMR of compound 8 showed the presence of a singlet signal at 2.41 ppm due to the
1
3
methyl group, C NMR spectrum showed signals at 12.3 and 152.4 corresponding to

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CH
3
of the pyrazole and C=O of the pyrimidinone, respectively. Additionally, the mass
spectrum exhibited the molecular ion peak at m/z 384.18. The pyrazolo[4,3-
e][1,2,4]triazolo[4,3-a]pyrimidines were prepared according to scheme 2 where the
hydrazine derivative 5 was treated with different aliphatic carboxylic acids or benzoyl
1
chloride derivatives to afford 9a-c and 10a-c, respectively. H NMR spectra of 9a-c
showed the absence of NH and NH
2
signals. Compound 9b displayed a singlet signal at
2
1
.40 ppm corresponding to the methyl group while 9c revealed triplet-quartet signals at
1
.33 ppm and 2.78 ppm respectively, due to the ethyl group. On the other hand, H NMR
spectra of 10a-c showed an increase in the integration of the aromatic protons indicating
the presence of an additional aromatic moiety. Moreover, a singlet peak at 3.79 ppm
1
3
revealed the presence of methoxy group for compound 10b. The C NMR was in good
agreement with the molecular formula of compounds 10a and10b, that, the presence of
methoxy signal at 55.9 and C=O signal at 162.0 confirmed the carbon backbone of 10b.
Similarly, the reaction of 5 with chloroacetyl chloride in glacial acetic acid resulted in 8-
1
chloromethyl derivative 11. H NMR showed a singlet peak downfielded to 4.66 ppm
corresponding to methylene group. Compounds 12a,b were obtained by treatment of 11
with morpholine and 4-phenyl piperazine, respectively in dry dioxane in the presence of
1
anhydrous potassium carbonate. H NMR showed the presence of the methylene spacer
which upfielded to the range of 3.65-3.71 ppm in comparison to 4.65 ppm of the starting
1
1. Also, multiple aliphatic signals with integration of eight protons confirmed the
introduction of the new alicyclic amine. Additional phenyl ring was observed by
increased integration of the aromatic region for the compound 12b. It is noteworthy that
the reaction of 5 with different isothiocyanates in pyridine afforded 13a-c without
isolation the thiosemicarbazide intermediates. The suggested mechanism of this reaction
could be illustrated in Figure 3. Additionally, the formation of cyclic compounds could
2
be confirmed by evolution of H S during the reaction which stained the lead acetate paper
with black color.

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O
H C
O
3
O
O
H C
O
O
CH3
b
N
3
N
a
N
H N
N
2
N
S
N
H
N
1
2
3
O
N
O
N
N
c
N
N
d
g
H C
N
3
N
H N
2
S
N
N
H
5
4
e
f
O
N
Ph
N
N
N
N
N
O
N
H C
3
O
N
Ph
R2
Ph
R
Ph
N
OH
N
N
H
N
N
N
N
N
N
N
H
Ph
H
R1
8
O
Ph
7
a-c
6a-d
6a, R = R = H
7
7
7
a, R= H
1
2
b, R= OCH3
c, R= Cl
6b, R = OH, R = H
1 2
6c, R = H, R = F
1
2
6
d, R = H, R = OCH
1 2 3
Scheme 1. Reagents and reaction conditions: (a) PhNHNH , absolute ethanol, reflux, 15 h.; (b)
2
phenyl isothiocynate, pyridine, reflux, 12h.; (c) CH I, K CO , acetone, reflux, 8h.; (d) 80% NH NH ,
3
2
3
2
2
absolute ethanol, reflux 12h.; (e) app.aldehyde, absolute ethanol, reflux.; (f) app. benzoyl chloride,
pyridine, RT.; (g) ethylacetoacetate, K CO , ethanol, reflux 11h.
2
3

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O
N
N
H N
2
N
N
N
H
5
a
f
O
N
O
N
Ph
b
e
N
c
Ph
N
N
N
N
N
N
Ph
N
HN
N
O
N
Ph
O
N
R
O
Ph
S
9
a-c
a, R= H
b, R= CH3
c, R= C H
Ph
Ph
N
N
N
N
14
N
9
9
9
N
N
N
N
N
N
N
N
Ph
N
Ph
Ph
N
2
5
N
NH
R
Cl
R
13a-c
3a, R= CH3
, R= C H5
1
0a-c
11
1
1
1
0a, R= H
3b
2
1
1
0b, R= OCH3
0c, R= Cl
13c,
R= Ph
d
O
Ph
N
N
N
Ph
N
N
N
N
X
1
2a,b
2a, X= O
2b, X= NꢁPh
1
1
Scheme 2. Reagents and reaction conditions: (a) excess of aliphatic acid, reflux; (b) app. benzoyl
chloride, dry pyridine, reflux; (c) chloroacetyl chloride, acetic acid, reflux 4h.; (d) morpholine or
phenylpiperazine, KOH, dry dioxane, reflux; (e) app. isothiocyanate, pyridine, reflux; (f) C
absolute ethanol, reflux 6h.
2
S, KOH,

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Fig.3. The proposed mechanism for the formation of 13a-c.
1
H NMR spectra revealed the presence of one exchangeable signal of NH at 6.26, 6.85
1
and 9.10 ppm for 13a, 13b and 13c, respectively. H NMR spectrum for 13a showed a
singlet peak at 2.88 ppm corresponding to the methyl group. Triplet-quartet pattern
exploited the presence of ethyl group in 13b while an additional aromatic ring in 13c
could be deduced from the increase in Ar-H integration. Finally, the reaction of 5 with
carbon disulfide in absolute ethanol in the presence of potassium hydroxide resulted in 14
1
(
Scheme 2). H NMR spectrum was in good agreement with the IR spectrum, in addition,
the presence of a signal at 167.8 ppm corresponding to C=S confirmed the obtained
structure. Mass spectrum displayed the molecular ion peak consistent with its molecular
weight.

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2
.2. In vitro enzyme inhibitory assay
All the target compounds were evaluated for their enzyme inhibitory activity in
in-vitro assays against wild type Abl (wt). First, Abl (wt) was treated with the
compounds at a concentration of 100 µM and the % residual Abl (wt) activity of the
selected compounds was summarized in Table 1. Second, the most promising compounds
at 100 µM (7a, 7b, 10a and 10b) have been further tested against the kinases Src and
PI3K and their % residual activity was presented in Table 2. Moreover, the most active
compounds 7a, 7b and 10b were also screened against the mutant Abl tyrosine kinase
(T315I), and their IC50 are represented in Table 3.
Table 1.% Residual Abl (wt) activity of compounds at 100 µM.
Compd.
% activity Abl (wt) at 100
µM
% activity Abl (wt) at 100
Compd. ID
ID
4
5
µM
71.0 ± 8.0
a
84.0 ± 9.0
46.0 ± 6.0
47.0 ± 7.0
9b
9c
7
2
0.0 ± 7.0
8.0 ± 3.0
6
a
10a
6
6
b
c
96.0 ± 10.0
36.0 ± 4.0
77.0 ± 8.0
6.3 ± 0.5
10b
10c
11
17.0 ± 2.0
68.0 ± 7.0
68.0 ± 7.0
>100.0
6d
7
a
13a
7
7
8
9
b
c
23.0 ± 2.0
26.0 ± 3.0
98.0 ± 8.0
39.0 ± 4.0
13b
13c
14
>100.0
33.0 ± 5.0
66.0 ± 8.0
< 1.0
a
Dasatinib
a
values are the mean of two independent replicates ±S.D.
Table 2. % Residual Src and PI3K activity of selected compounds at 100 µM
Compd. ID % activity Src at 100 µM % activity PI3K at 100 µM
a
7
7
1
a
b
0a
0b
7 ± 1
100 ± 11
80 ± 10
82 ± 6
20 ± 4
50 ± 4
35 ± 3
1
90 ± 3
a
values are the mean of two independent replicates ±S.D.

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Table 3: The IC50 (µM) of selected compounds toward Src, Abl (wt) and T315I Abl and
their (cytotoxic concentration) CC against K-562 cell line.
50
c
CC (µM) ±SD
5
0
Compd.
ID
b
b
b
IC (µM) ±SD
IC (µM) ±SD
IC (µM) ±SD
Leukemia K-562
Cell line
5
0
50
50
(
n.d
n.d
Src)
(Abl wt)
(Abl T315I)
a
a
a
2.3700 ± 0.0800
4.0600 ± 0.1400
0.0700 ± 0.0020
5
n.d
n.d
a
a
6
c
110.0000 ± 10.0000
20.9000 ± 0.9000
n.d
4
.7000 ± 0.7000
7
a
13.8000 ± 0.8000
12.2000 ± 0.5000
7b
24.0000 ± 2.0000
26.1000 ± 0.5000
65.6000 ± 0.6000
40.0000 ± 5.0000
19.0000 ± 1.0000
0.0500 ± 0.0010
0.8600 ± 0.0200
a
a
n.d
7
c
n.d
a
a
1
0a
0b
3c
n.d
n.d
0.9200 ± 0.0300
1.0600 ± 0.0500
6
7.0000 ± 8.0000
1
20.0000 ± 2.0000
a
a
n.d
4.9200 ± 0.1600
24.5000 ± 0.7500
0.2500 ± 0.0200
1
150.0000 ± 10.0000
0.5000 ± 0.2000
0.0040 ± 0.0002
n.d
d
0.1800 ± 0.0500
a
PP2
n.d
a
n.d
Dasatinib
0.0800 ± 0.0200
a
n.d = not determined
values are the mean of two independent replicates ±S.D.
values are the mean of tetrareplicates ±S.D
b
c
d
PP2, pyrazolopyrimidine Src/Abl inhibitor [19]
From the obtained results (Tables 1, 2 and 3) it was observed that among the
tested compounds, only compounds7a, 7b and 10b are the most active against the wild
type Abl. Compounds 7a and 7b also displayed comparable activity against Src, but not
against PI3K. Interestingly, p-methoxybenzohydrazide 7b and its unsubstituted analog 7a
revealed also better activity against mutant Abl (T315I) with IC equal to 12.2 and 13.8
5
0
µM, respectively. Compound 10b, the cyclized form of 7b, also maintained the same
activity against the mutant as observed against the wild type (IC50 of 20 µM and 19.1
µM, respectively). On the other hand, the cyclization of 7b into 10b resulted in decreased
the activity against Src kinase with IC50 values of 24 and 67 µM, respectively. Thus,
compounds 7a, 7b and 10b seem to be resilient towards the resistance induced by the
gatekeeper mutation T315I. Their chemical scaffolds could be further exploited to
develop dual Src-Abl inhibitors, also active against the clinically relevant Abl mutant
T315I.

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2
.3. In vitro cytotoxic activity against Leukemia K-562 cell line
Eight compounds that showed promising IC50 values against Abl enzyme (wt)
were tested for their cytotoxic activity against the leukemia cell line K-562. This
screening was performed at Vacsera, Cairo, Egypt and the results are summarized in
Table 3. The tested compounds revealed remarkable activity with cytotoxic concentration
CC50 ranging from 0.05 to 4.92 ꢀM. Moreover, from the obtained results, structure
activity relationships revealed that acylation of compound 5 with unsubstituted or 4-
methoxybenzoyl chloride resulted in the most active derivatives 7a and 7b, respectively.
On the other hand, the 4-chloro derivative 7c revealed a lower activity. Thus, the
presence of an electron donating group is more favorable than an electron withdrawing
moiety. On the other hand, conversion of the hydrazine derivative 5 into 4-
fluorobenzohydrazone 6c resulted in decrease in the activity. Furthermore, 8-aryl-1H-
pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidine-4(5H)-ones 10a,b exhibited higher
activity than the phenylamino congener 13c. Moreover, cyclization of 7a,b into 10a,b
resulted in decrease in the cytotoxic activity. In general, the CC values exerted towards
5
0
the K-562 cell line were lower than the corresponding IC50 values towards the single
recombinant Src or Abl enzymes (Table 3). However, the effects of the compounds at the
cellular level may be amplified through signal transduction cascades activated in
response to the simultaneous inhibition of both Src and Abl activity, resulting in a
synergic effect. In addition, differences between in vitro enzymatic assays and cellular
viability measurements may also account for these discrepancies. For example, the
absolute enzyme concentrations are surely different between in vitro and cellular tests.
2
.4. Molecular modeling
Molecular docking technique was used to interpret the observed promising
activity of compound 7b on both Abl types. Therefore, docking of 7b into the three
dimensional X-ray crystal structure of the wild type human Bcr-Abl kinase (PDB code:
4
WA9) and at the crystal structure of human Abl T315I gatekeeper mutant form (PDB
code: 4TWP) [36] both in complex with axitinib were carried out using Accelry’s
Discovery Studio software package [37]. The selection of these X-ray resolved
complexes for the docking study was based on providing a comparative perspective for

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the conformational changes taking place in the enzyme upon ligand binding. Recently,
axitinib was shown to bind the wild form of Abl kinase in the inactive state known as
“DFG out” in which the catalytic Asp381 is oriented away from the ATP binding site
while Phe382 is oriented towards the binding site [38]. Regarding the mutant form,
axitinib was shown to bind to the active state known as “DFG in” where the Asp381 is
oriented towards the ATP binding site while Phe382 is oriented inwards. The validity of
the docking protocol was ensured by re-docking of the co-crystallized axitinib into the
active site of both forms. The coordinates of the best scoring docking pose of axitinib was
compared with its coordinates in the co-crystallized PDB files based on the binding mode
and root mean square deviation (rmsd). The re-docked ligand showed an rmsd of 0.938
Aº with CDOCKER interaction energy of -55.3216 at the wild type Bcr-Abl kinase and
an rmsd of 0.713 Aº with CDOCKER interaction energy of -58.2649 at the mutant form
(
Figure 4, 5). In both, axitinib was forming two hydrogen bonds with the amino acids of
the hinge region Glu316 and Met318. Two π-π interactions were formed with Phe317 and
additional hydrogen bonds with Tyr253 and Lys271 were also reported. In the wild form
of Abl kinase, the methoxy substituted phenyl in 7b was extending towards the hinge
region where its methoxy group was engaged in a hydrogen bond with the crucial Met318
(
Figure 6). Multiple pi interactions between Phe382 and both pyrazolopyrimidine and its
-phenyl moiety was reported while the 5-phenyl moiety was forming a cation-pi
interaction with Lys271.
1

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Fig. 4. Superimposition of the co-crystallized 3D structure of axitinib (green) and its top
docking pose (red) in the active site of the wild type Bcr-Abl kinase (PDB: 4WA9)
Fig. 5. Superimposition of the co-crystallized 3D structure of axitinib (green) and its top
docking pose (red) in the active site of mutant Bcr-Abl kinase (PDB: 4TWP)

ACCEPTED MANUSCRIPT
Fig. 6. Overlay of axitinib (green) and compound 7b (magenta) in the active site of the
wild type Bcr-Abl kinase (PDB: 4WA9)
An interesting feature in the top docking pose of 7b at the active site of T315I mutant, as
compared to the wild form, was the flipping of the molecule in a fashion that allowed a
good alignment with that of axitinib binding fashion (Figure 7). The pyrazole ring was
engaged in one hydrogen bond with Met318. The pyrazole and the 1-phenyl ring were
forming pi interactions with Phe317. Since Phe382 in the T315I mutant form was
oriented away from the ATP binding site, its involvement in pi interactions with 7b as in
the wild enzyme is not expected. This change in the relative position of Phe382 may also
result in providing a larger room to accommodate the molecule in a binding mode similar
to that of axitinib. It is worth mentioning that neither Thr315 in the wild nor Ile315 in the
mutant forms were involved in any interactions with compound 7b.

ACCEPTED MANUSCRIPT
Fig. 7. Overlay of the best docking pose of compound 7b (magenta) and axitinib (green)
in the active site of T315I mutant Bcr-Abl kinase (PDB:4TWP)
The three dimensional X-ray crystal structure of the kinase domain of c-Src in complex
with a substituted pyrazolopyrimidine (PDB code: 4O2P) was used for molecular
docking of compounds 7a and 7b [39]. The selection of this x-ray resolved complex was
based on the structural similarity between the compounds 7a and 7b with the co-
crystalized ligand. The co-crystallized ligand interacted with the kinase domain of c-src
through pi interactions with Tyr 340 and Lys 295 and hydrogen bonds with Met 341 and
the gate keeper Thr 338 (Fig.8).

ACCEPTED MANUSCRIPT
Fig. 8. The cocrystallized pyrazolopyrimidine in c-Src (PDB code: 4O2P)
The best docking poses of compounds 7a and 7b in the kinase domain of c-Src revealed a
hydrogen bond between Lys 295 and the carbonyl moiety in the core of compounds 7a
and 7b. Lys 295 was also involved in a cation-pi interaction with the phenyl ring. Tyr 340
in the hinge region interacted with the phenyl ring in 7a or the methoxy substituted
phenyl in 7b through a pi-pi interaction. The electron donating nature of the methoxy
group in 7b is hypothized to decrease the aromatic interaction with the electron rich
phenyl ring of Tyr 340. Met 341 was in close proximity to the hydrazide oxygen in both
compounds. The target compounds 7a and 7b were not involved in interaction with the
THR338 (Fig. 9, 10). It is worthy to mention that the pyrazolopyrimidine core in 7a and
7
b was flipped relative to the native co-crystallized ligand.

ACCEPTED MANUSCRIPT
Fig. 9. The best docking pose of compound 7a in the active site of the kinase domain of
c-Src(PDB: 4O2P)
Fig. 10. The best docking pose of compound 7b in the active site of the kinase domain of
c-Src (PDB: 4O2P)

ACCEPTED MANUSCRIPT
3
. Conclusion
Some novel pyrazolo[3,4-d]pyrimidines and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]
pyrimidines were synthesized through facile procedures. They revealed mild to moderate
activity against both Src and Abl (wt) kinases. Compounds 7a, 7b and 10b were the most
active compared to the other derivatives against both enzymes and the mutant Abl type
T135I, and the leukemia K-562 cell line. However, one point of interest in the present
study, is the identification of chemical scaffolds (compounds 7a, 7b and 10b), which
were unaffected by the T315I mutation and can be further exploited in order to develop
specific inhibitors for this clinically relevant mutant.
4
. Experimental
4
.1. Chemistry
Melting points were determined by open capillary tube method using
Electrothermal 9100 melting point apparatus and are uncorrected. Elemental
microanalyses were performed at the Regional Center for Mycology and Biotechnology,
Al-Azhar University. The infrared spectra (IR) were recorded as potassium bromide discs
on Schimadzu FT-IR 8400S and Bruker FT-IR 12243136 spectrophotometers at Faculty
of Pharmacy, Cairo University and Central Research Lab, Nahda University,
1
respectively. H NMR spectra were recorded on Bruker spectrophotometer at 400 MHz.
Chemical shift values (δ) are given in parts per million (ppm) downfield from
1
3
tetramethylsilane (TMS) as internal reference. C NMR spectra were recorded using
Bruker, 100 MHz NMR spectrometer at Microanalytical Unit, Faculty of Pharmacy,
Cairo University.Mass spectra were performed on Fennigan MAT, SSQ 7000 mass
spectrophotometer at 70eV.Reactions were followed up by thin layer chromatography
(TLC), using Silica gel/TLC cards DC-Alufolien-Kiesel gel with fluorescent indicator
UV254 using chloroform or hexane : ethyl acetate 6 : 4 as the eluting system and the
spots were visualized using VilberLourmet ultraviolet lamp at ג=254nm.
Compounds 1 [33], 2 [34], 3 [35] were prepared according to reported procedures.

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4
.1.1.6-(Methylthio)–1,5-diphenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 4
A mixture of 3 (3.20 g, 10 mmol) and anhydrous potassium carbonate (1.65 g, 12 mmol)
in dry acetone (50 mL) was stirred at room temperature for 1 h., then methyl iodide (7.10
g, 50 mmol) was added dropwise and the mixture was heated under reflux for 8 h. The
solvent and the excess methyl iodide were evaporated under reduced pressure and the
remaining residue was poured onto ice cold water. The separated product was crystallized
°
from acetone to obtain white crystalline solid. Yield %: 76 (2.43 g), m.p.: 232-234 C. IR
-1
1
υmax, cm : 3053 (Ar-CH), 2954 (aliph-CH), 1701 (C=O). H NMR (DMSO-d , 400
6
MHz): 3.75 (s, 3H, -SCH
3
), 7.14 (d, 1H, J = 7.56 Hz, Ar-H), 7.29 (d, 1H, J = 7.88 Hz,
Ar-H), 7.41- 7.54 (m, 3H, Ar-H), 7.81 (t, 1H, J = 7.40 Hz, Ar-H), 8.01 (t, 2H, J = 7.92
1
3
6
Hz, Ar-H), 8.64 (d, 2H, J = 8.00, Ar-H), 8.69 (s, 1H, H3pyrazole). C NMR (DMSO- d ,
1
1
00 MHz): 43.0 (S-CH ), 103.7 (C4pyrazole), 121.3, 125.3, 125.7, 127.0, 127.7, 128.3,
3
29.5, 129.6, 129.7, 137.1, 137.2, 139.1, 146.3, 151.8, 157.0 (Ar-C), 158.4 (C=O).
14 4
Anal.Calcd. for C18H N OS (334.09): C, 64.65; H, 4.22; N, 16.75. Found: C, 64.81; H,
4
4
.29; N, 17.01.
.1.2. 6-Hydrazinyl-1,5-diphenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 5
To a solution of compound 4 (3.34 g, 10 mmol) in ethanol (50 mL), hydrazine hydrate
80%) (0.1 g, 20 mmol) was added and the reaction mixture was refluxed for 12 h. The
reaction mixture was cooled and the formed precipitate was filtered, washed with ethanol
(
°
-1
to obtain V as a white powder. Yield 71% (2.25 g), m.p.: 201-203 C. IR υmax, cm : 3319,
1
3
5
7
2
1
273 (NH ), 3207 (NH), 3082 (Ar-H), 1699 (C=O). H NMR (DMSO-d , 400 MHz):
2
6
.64 (s, 2H, NH , exchanged with D O), 7.14 (t, 1H, J = 7.34 Hz, Ar-H), 7.34 (t, 1H, J =
2
2
.38 Hz, Ar-H), 7.40 (t, 2H, J = 7.86 Hz, Ar-H), 7.52 (t, 2H, J = 7.90 Hz, Ar-H), 7.79 (d,
H, J = 7.76 Hz, Ar-H), 8.09 (d, 2H, J = 7.76 Hz, Ar-H), 8.18 (s, 1H, H3pyrazole), 9.68 (s,
+
H, NH, exchanged with D
2
O). MS m/z (%): 318.13 (M , 2.66), 77.05 (100.00).
Anal.Calcd. for C H N O (318.12): C, 64.14; H, 4.43; N, 26.40. Found: C, 64.31; H,
1
7
14
6
4
.49; N, 26.71.

ACCEPTED MANUSCRIPT
4
.1.3. General procedure for preparation of 6a-d
A mixture of equimolar amounts of compound 5 and the appropriate aromatic aldehyde
10 mmol) in absolute ethanol (50 mL) was refluxed for 4-7 h. The separated solid was
filtered while hot and washed with ethanol to give the corresponding hydrazone product.
(
4
.1.3.1.
6-[2-(Benzylidene)hydrazinyl]-1,5-diphenyl-1,5-dihydro-4H-pyrazolo[3,4-d]
pyrimidin-4-one 6a
°
-1
The reaction time was 7 h, yield 65% (2.64 g), m.p.: 160-164 C. IR υmax, cm : 3421
1
(
NH), 3059 (Ar-H), 1724 (C=O). H NMR (DMSO-d
6
, 400 MHz): 7.30 (t, 1H, J = 7.36
Hz, Ar-H), 7.44-7.59 (m, 14H, 12Ar-H, NH exchanged with D O, and –CH=N), 8.02 (d,
2
1
3
2
H, J = 7.92 Hz, Ar-H), 8.46 (s, 1H, H3pyrazole). C NMR (DMSO, 100 MHz): 103.2
(C4pyrazole), 121.1, 125.3, 126.9, 128.5, 129.3, 129.5, 130.1, 130.4, 131.9, 132.9, 137.1,
+
1
39.1, 151.4 (Ar-C), 151.6 (C=O), 152.4 (C=N). MS m/z (%): 406.21 (M , 4.13), 404.21
18 6
(100.00). Anal.Calcd. for C24H N O (406.44): C, 70.92; H, 4.46; N, 20.68. Found: C,
7
1.08; H, 4.52; N, 20.89.
4
.1.3.2. 6-[2-(2-Hydroxybenzylidene)hydrazinyl]-1,5-diphenyl-1,5-dihydro-4H-pyrazolo
[
3,4-d]pyrimidin-4-one 6b
°
-1
The reaction time was 5h, yield 72% (3.04 g), m.p.: 186-187 C. IR υmax, cm : 3213 (br-
1
OH), 3196 (NH), 3062 (Ar-CH), 1687 (C=O). H NMR (DMSO-d , 400 MHz): 6.73 (t,
6
1
H, J = 7.48 Hz, Ar-H), 6.85 (d, 2H, J = 8.68 Hz, Ar-H), 7.02 (m, 1H, Ar-H), 7.13-7.24
(m, 3H, Ar-H), 7.32-7.50 (m, 2H, Ar-H), 7.56 (t, 1H, J = 7.86 Hz, Ar-H), 7.65-7.69 (m,
2
H, Ar-H), 8.07-8.09 (m, 3H, N=CH + 2 Ar-H), 8.21 (s, 1H, H3pyrazol), 9.18 (s,1H, NH,
1
3
2 2 6
exchanged with D O), 10.08 (s, 1H, OH, exchanged with D O). C NMR (DMSO- d ,
1
1
00 MHz): 103.5 (C4pyrazole), 116.3, 119.8, 121.2, 122.3, 123.8, 125.5, 127.4, 128.9,
29.5, 131.2, 136.4, 136.7, 151.9, 153.7 (Ar-C), 152.4 (C=O), 155.8 (C=N). Anal.Calcd.
for C H N O (422.44): C, 68.24; H, 4.29; N, 19.89. Found: C, 68.43; H, 4.35; N,
2
4
18
6
2
2
0.04.
4
.1.3.3.
6-
[2-(4-Fluorobenzylidene)hydrazinyl]-1,5-diphenyl-1,5-dihydro-4H-pyrazolo
[
3,4-d]pyrimidin-4-one 6c

ACCEPTED MANUSCRIPT
°
-1
The reaction time was 7h, yield 61% (2.59 g), m.p.: 222-223 C. IR υmax, cm : 3332 (NH),
1
3
059 (Ar-CH), 1712 (C=O). H NMR (DMSO-d , 400 MHz): 6.81 (d, 1H, J = 9.44 Hz,
6
Ar-H), 7.14-7.28 (m, 2H, Ar-H), 7.30-7.49 (m, 4H, Ar-H), 7.53-7.57 (m, 3H, CH=N +
Ar-H), 7.64-7.69 (m, 2H, Ar-H), 8.07 (d, 2H, J = 7.88 Hz, Ar-H), 8.18 (dd, 1H, J = 5.95,
1
3
6
.08 Hz, Ar-H), 8.21 (s, 1H, H3pyrazole), 9.20 (s, 1H, NH exchanged with D O). C NMR
2
6
(DMSO- d , 100 MHz): 104.2 (C4pyrazole), 116.1, 116.3, 121.0, 121.5, 121.6, 124.5, 127.1,
1
28.8, 129.5, 129.6, 130.1, 130.2, 138.9, 148.0, 150.0 (Ar-C), 152.7 (C=O), 154.8 (C=N).
+
MS m/z (%): 424.18 (M , 11.63), 186.08 (100.00). Anal.Calcd. for C H FN O
2
4
17
6
(424.43): C, 67.92; H, 4.04; N, 19.80. Found: C, 68.13; H, 4.12; N, 19.97.
4
.1.3.4. 6-[2-(4-Methoxybenzylidene)hydrazinyl]-1,5-diphenyl-1,5-dihydro-4H-pyrazolo
[
3,4-d]pyrimidin-4-one 6d
°
-1
The reaction time was 4h, yield 81% (3.53 g), m.p.: 214-215 C. IR υ
cm : 3321 (NH),
max,
1
3
3
035 (Ar-CH), 2953 (aliph-CH), 1660 (C=O). H NMR (DMSO-d
6
, 400 MHz): 3.79 (s,
H, OCH ), 7.01 (d, 2H, J = 8.88 Hz, Ar-H), 7.29 (t, 1H, J = 7.38 Hz, Ar-H), 7.43-7.47
3
(m, 5H, 4 Ar-H, and NH), 7.58 (s, 1H, N=CH), 7.60 (br s, 5H, Ar-H), 8.01 (d, 2H, J =
1
3
7
1
1
4
4
.88 Hz, Ar-H), 8.43 (s, 1H, H3pyrazole). C NMR (DMSO- d , 100 MHz): 55.9 (OCH ),
6 3
02.0 (C4pyrazole), 114.9, 121.1, 122.3, 124.3, 126.7, 127.0, 128.9, 129.5, 130.4, 136.6,
38.3, 139.1, 151.1, 151.7, 162.1 (Ar C), 157.4 (C=O), 161.0 (C=N). MS m/z (%):
+
36.39 (M , 1.18), 44.03 (100.00). Anal.Calcd. for C H N O (436.47): C, 68.80; H,
2
5
20
6
2
.62; N, 19.25. Found: C, 68.97; H, 4.73; N, 19.39.
4
.1.4. General procedure for preparation of 7a-c.
A solution of compound 5 (3.18 g, 10 mmol) and the appropriate benzoyl chloride (10
mmol) in pyridine (20 mL) was stirred at room temperature for 3-6 h. The reaction
mixture was poured onto cold water and neutralized with acetic acid. The obtained solid
was filtered, washed several times with water and crystallized from methanol.
4
.1.4.1. N’-(4-Oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)benzo-
hydrazide 7a

ACCEPTED MANUSCRIPT
°
-1
The reaction time was 6h, yield 56% (2.36 g), m.p.: 179-181 C. IR υmax, cm : 3318 (NH),
1
3
070 (Ar-CH), 1685 (br 2 C=O). H NMR (DMSO-d , 400 MHz): 7.21 (d, 1H, J = 7.48
6
Hz, Ar-H), 7.35-7.52 (m, 8H, Ar-H), 7.58-7.63 (m, 2H, Ar-H), 7.74 (t, 2H, J = 7.60 Hz,
Ar-H), 7.96 (d, 2H, J = 7.48 Hz, Ar-H), 8.47 (s, 1H, H3pyrazole), 11.23 (s, 1H, NH,
1
3
exchanged with D O), 12.96 (s, 1H, NH, exchanged with D O). C NMR (DMSO-d ,
2
2
6
1
1
00 MHz): 105.7 (C4pyrazole), 121.3, 122.0, 124.4, 125.3, 126.9, 127.8, 128.8, 129.9,
31.2, 132.1, 132.6, 133.2, 137.3, 137.8, 148.7 (Ar-C), 167.8 (C=Opyrimidinone), 171.3
+
(
C=O). MS m/z (%): 422.21 (M , 12.56), 404.19 (100.00). Anal.Calcd. for C H N O
2
4
18
6
2
(422.15): C, 68.24; H, 4.29; N, 19.89. Found: C, 68.37; H, 4.36; N, 20.02.
4
.1.4.2. 4-Methoxy-N’-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)benzohydrazide 7b
°
-1
The reaction time was 3h, yield 78% (3.52 g), m.p.: 197-200 C. IR υmax, cm : 3255 and
1
3
4
209 (2NH), 3062 (Ar-CH), 2935 (aliph-CH), 1685 (br 2 C=O). H NMR (DMSO-d
6
,
00 MHz): 4.04 (s, 3H, OCH ), 7.20 (t, 1H, J = 7.36 Hz, Ar-H), 7.34 (t, 1H, J = 7.26 Hz,
3
Ar-H), 7.42 (t, 2H, J = 7.78 Hz, Ar-H) 7.49 (t, 2H, J = 7.72 Hz, Ar-H), 7.59-7.71 (m, 4H,
Ar-H), 8.01 (d, 2H, J = 7.96 Hz, Ar-H), 8.06 (d, 2H, J = 7.96 Hz, Ar-H), 8.21 (s, 1H,
H3pyrazole), 9.74 (s, 1H, NH, exchanged with D O), 11.20 (s, 1H, NH, exchanged with
2
1
3
D
2
O). C NMR (DMSO-d
6 3
, 100 MHz): 55.1 (OCH ), 104.4 (C4pyrazole), 110.7, 113.5,
1
1
1
18.5, 119.6, 123.2, 126.5, 127.4, 129.1, 130.2, 131.8, 143.1, 149.0, 150.7, 160.9 (Ar-C),
20 6 3
63.7 (C=O), 171.0 (C=O). Anal.Calcd. for C25H N O (452.16): C, 66.36; H, 4.46; N,
8.57. Found: C, 66.62; H, 4.50; N, 18.74.
4
.1.4.3. 4-Chloro-N’-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)benzohydrazide 7c
°
-1
The reaction time was 4h, yield 59% (2.69 g), m.p.: 215-217 C. IR υmax, cm : 3421 and
1
3
7
387 (2 NH), 3100 (Ar-CH), 1786 and 1720 (2 C=O). H NMR (DMSO-d , 400 MHz):
6
.33 (t, 1H, J = 8.44 Hz, Ar-H), 7.46 (t, 1H, J = 7.90 Hz, Ar-H), 7.51-7.66 (m, 4H, Ar-
H), 7.72 (d, 2H, J = 8.56 Hz, Ar-H), 7.95 (d, 2H, J = 8.48 Hz, Ar-H), 8.01 (d, 2H, J =
7
2
.80 Hz, Ar-H), 8.16 (d, 2H, J = 8.60 Hz, Ar-H), 8.47 (s, 1H, H3pyrazole), 13.17 (s, 2H,
1
3
2 6
NH, exchanged with D O). C NMR (DMSO-d , 100 MHz): 105.4 (C4pyrazole), 123.4,

ACCEPTED MANUSCRIPT
1
1
3
6
26.4, 129.1, 130.6, 131.4, 131.7, 131.8, 132.3, 132.7, 133.5, 133.8, 134.9, 139.3, 140.5,
+
41.2, 152.8 (Ar-C), 154.5 (C=Opyrimidinone), 169.2 (C=O). MS m/z (%): 456.14 (M ,
+
.63), 458.86 (M + 2, 1.11), 40.15 (100.00). Anal.Calcd. for C H ClN O (456.11): C,
2
4
17
6
2
3.09; H, 3.75; N, 18.39. Found: C, 63.31; H, 3.73; N, 18.52.
4
.1.5.
6-(5-Hydroxy-3-methyl-1H-pyrazol-1-yl)-1,5-diphenyl-1,5-dihydro-4H-pyrazolo
[
3,4-d]pyrimidin-4-one 8
A mixture of compound 5 (3.18g, 10 mmol), ethyl acetoacetate (1.3g, 10 mmol) and
anhydrous potassium carbonate (2.07g, 15 mmol) in ethanol (30 mL) was heated under
reflux for 11h. The reaction mixture was cooled and the precipitated solid was collected
by filteration, washed with water, dried and crystallized from ethanol to give white
°
-1
crystalline solid. Yield %: 53 (2.01 g), m.p.: 149-152 C. IR υmax, cm : 3394 (br –OH),
1
3
094, 3051( Ar-CH), 2981, 2928 (aliph-CH), 1709 (C=O). H NMR (DMSO-d , 400
6
3
MHz): 2.41 (s, 3H, CH ), 7.29 (t, 1H, J = 7.36 Hz, Ar-H), 7.45 (t, 2H, J = 7.80 Hz, Ar-
H), 7.63-7.74 (m, 7H, 6 Ar-H, and OH exchanged with D O), 7.98 (d, 2H, J = 7.96 Hz,
2
1
3
Ar-H), 8.40 (s, 1H, H3pyrazole). C NMR (DMSO-d
6
, 100 MHz): 12.3 (CH
3
), 103.1
(
(
C4pyrazole), 121.1, 126.8, 127.9, 129.5, 130.2, 132.2, 137.0, 139.1, 150.2, 151.2, 151.4
+
Ar-C), 152.4 (C=O). MS m/z (%): 384.18 (M , 3.59), 137.08 (100.00). Anal.Calcd. for
(384.13): C, 65.62; H, 4.20; N, 21.86. Found: C, 65.71; H, 4.25; N, 21.99.
21 16 6 2
C H N O
4
.1.6. General procedure for preparation of 9a-c.
A mixture of compound 5 (3.18 g, 10 mmol) and different aliphatic carboxylic acid (30
mL) was heated under reflux for the appropriate time 6-10 h. After evaporation of the
excess acid under reduced pressure, the remaining solution was poured onto ice water.
The obtained solid was filtered, washed with water, dried, and crystallized from ethanol.
4
.1.6.1. 1,5-Diphenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidin-4(5H)-one 9a
°
-1
The reaction time was 10 h, yield 67% (2.19 g), m.p.: 118-119 C. IR υmax, cm : 3065,
1
3
047 (Ar-CH), 1724 (C=O). H NMR (DMSO-d
6
, 400 MHz): 7.36 (t, 1H, J = 7.40 Hz,
Ar-H), 7.52-7.58 (m, 3H, Ar-H), 7.68 (t, 2H, J = 7.82 Hz, Ar-H), 7.95 (d, 2H, J = 7.72
Hz, Ar-H), 8.10 (d, 2H, J = 7.80 Hz, Ar-H) 8.45 (s, 1H, H3pyrazole), 9.36 (s, 1H, H3triazole).

ACCEPTED MANUSCRIPT
1
3
C NMR (DMSO-d
6
, 100 MHz): 103.0 (C4pyrazole), 121.5, 124.0, 127.3, 129.3, 129.7,
+
1
30.1, 133.0, 137.0, 138.7, 142.3 (Ar-C), 152.4 (C=O). MS m/z (%): 328.15 (M , 17.67),
7.05 (100.00). Anal.Calcd. for C H N O (328.11): C, 65.85; H, 3.68; N, 25.60. Found:
7
1
8
12
6
C, 65.97; H, 3.72; N, 25.89.
4
4
.1.6.2.
8-Methyl-1,5-diphenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidin-
(5H)-one 9b
°
-1
The reaction time was 6 h, yield 81% (2.77 g), m.p.: 124-125 C. IR υmax, cm : 3055 (Ar-
1
CH), 2982, 2928 (aliph-CH), 1712 (C=O). H NMR (DMSO-d
6
, 400 MHz): 2.41 (s, 3H,
CH ) 7.29 (t, 1H, J = 7.38 Hz, Ar-H), 7.45 (t, 2H, J = 7.88 Hz, Ar-H), 7.63-7.70 (m, 3H,
3
Ar-H), 7.73 (d, 2H, J = 7.52 Hz, Ar-H), 7.98 (d, 2H, J =7.92 Hz, Ar-H), 8.40 (s, 1H,
1
3
H3pyrazole). C NMR (DMSO-d , 100 MHz): 12.3 (CH ), 103.2 (C4pyrazole), 121.1, 121.2,
6
3
1
1
22.3, 126.8, 127.9, 128.9, 129.5, 130.2, 130.4, 132.2, 136.6, 137.0, 139.1, 150.2, 151.2,
+
51.4 (Ar-C), 152.4 (C=O). MS m/z (%): 342.14 (M , 17.32), 77.07 (100.00).
Anal.Calcd. for C H N O (342.12): C, 66.66; H, 4.12; N, 24.55. Found: C, 66.85; H,
1
9
14
6
4
.17; N, 24.72.
4
.1.6.3. 8-Ethyl-1,5-diphenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidin-4(5H)-
one 9c
°
-1
The reaction time was 7 h, yield 86% (3.06 g), m.p.: 133-134 C. IR υmax, cm : 3067,
1
3
1
055 (Ar-CH), 2978, 2944, 2920 (aliph-CH), 1712 (C=O). H NMR (CDCl
3
, 400 MHz):
.33 (t, 3H, J = 7.48 Hz, CH ), 2.78 (q, 2H, J = 7.48 Hz, -CH -), 7.21 (t, 1H, J = 7.38 Hz,
3
2
Ar-H), 7.35 (t, 2H, J = 7.82 Hz, Ar-H), 7.48 (d, 2H, J = 7.72 Hz, Ar-H), 7.60-7.65 (m,
1
3
3
H, Ar-H), 8.01 (d, 2H, J =7.96, Ar-H), 8.31 (s, 1H, H3pyrazole). C NMR (CDCl
3
, 100
MHz): 10.4 (CH ), 19.7 (CH ), 103.2 (C4pyrazole), 120.8, 126.1, 127.1, 128.8, 130.1,
3
2
1
3
4
30.3, 131.4, 136.5, 138.9, 149.3, 151.8, 151.9 (Ar-C), 154.5 (C=O). MS m/z (%):
+
56.17 (M , 12.05), 77.05 (100.00). Anal.Calcd. for C H N O (356.14): C, 67.40; H,
2
0
16
6
.53; N, 23.58. Found: C, 67.62; H, 4.61; N, 23.70.
4
.1.7. General procedure for preparation of 10a-c.

ACCEPTED MANUSCRIPT
A mixture of compound 5 (3.18 g, 10 mmol) and the appropriate benzoyl chloride (12
mmol) in dry pyridine (10 mL) was heated under reflux for 24 h. The reaction mixture
was cooled and the formed precipitate was filtered, washed with water then ethanol, dried
and crystallized from ethanol.
4
4
.1.7.1.
1,5-Diphenyl-8-phenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidin-
(5H)-one 10a
°
-1
Yield 59% (2.38 g), m.p.: 218-220 C. IR υmax, cm : 3059, 3017 (Ar-CH), 1721 (C=O).
1
6
H NMR (DMSO-d , 400 MHz): 7.30 (t, 1H, J = 7.20 Hz, Ar-H), 7.44-7.59 (m, 12H, Ar-
1
3
H), 8.01 (d, 2H, J = 8.40 Hz, Ar-H), 8.46 (s, 1H, H3pyrazole). C NMR (DMSO-d , 100
6
MHz): 103.2 (C4pyrazole), 121.1, 125.3, 126.9, 128.5, 129.3, 129.5, 130.1, 130.4, 131.9,
1
32.9, 137.1, 139.1, 150.7, 151.5, 151.6 (Ar-C), 152.4 (C=O). Anal.Calcd. for
C H N O (404.15): C, 71.28; H, 3.99; N, 20.78. Found: C, 71.49; H, 4.06; N, 20.96.
24
16
6
4
.1.7.2.
8-(4-Methoxyphenyl)-1,5-diphenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]
pyrimidin-4(5H)-one 10b
°
-1
Yield 79% (3.42 g), m.p.: 229-231 C. IR υmax, cm : 3082, 3063 (Ar-CH), 2965, 2926
1
(
aliph-CH), 1720 (C=O). H NMR (DMSO-d , 400 MHz): 3.79 (s, 3H, OCH ), 7.00 (d,
6
3
2
H, J = 8.40 Hz, Ar-H), 7.29 (t, 1H, J = 7.16 Hz, Ar-H), 7.42-7.46 (m, 4H, Ar-H), 7.60
1
3
(
s, 5H, Ar-H) 8.00 (d, 2H, J = 7.92 Hz, Ar-H), 8.43 (s, 1H, H3pyrazole). C NMR (DMSO-
d
6 3
, 100 MHz): 55.9 (OCH ), 103.2 (C4pyrazole), 114.8, 117.2, 121.0, 126.8, 128.5, 129.5,
1
30.1, 130.4, 131.0, 133.1, 137.1, 139.1, 150.7, 151.3, 151.6 (Ar-C), 162.0 (C=O). MS
+
m/z (%): 434.22 (M , 3.83), 77.06 (100.00). Anal.Calcd. for C H N O (434.17): C,
2
5
18
6
2
6
9.11; H, 4.18; N, 19.34. Found: C, 69.34; H, 4.25; N, 19.49.
4
.1.7.3.
8-(4-Chlorophenyl)-1,5-diphenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-
a]pyrimidin-4(5H)-one 10c
Yield 75% (3.28 g), m.p.: 229-231 C. IR υmax, cm : 3060 (Ar-CH), 1723 (C=O). H
NMR (DMSO-d , 400 MHz): 7.30 (t, 1H, J = 7.40 Hz, Ar-H), 7.46 (t, 1H, J = 7.80 Hz,
°
-1
1
6
Ar-H), 7.51 (d, 1H, J = 8.80 Hz, Ar-H), 7.56-7.60 (m, 3H, Ar-H), 7.71 (d, 2H, J = 8.40
Hz, Ar-H), 7.94 (d, 2H, J = 8.00 Hz, Ar-H), 8.01 (d, 2H, J = 8.00 Hz, Ar-H), 8.16 (d, 2H,

ACCEPTED MANUSCRIPT
1
3
J = 8.40 Hz, Ar-H), 8.47 (s, 1H, H3pyrazole). C NMR (DMSO-d
6
, 100 MHz): 103.2
(C4pyrazole), 121.1, 125.3, 126.9, 128.5, 129.3, 129.5, 129.6, 130.1, 130.4, 131.9, 132.9,
+
1
37.2, 139.1, 150.8, 151.6 (Ar-C), 152.4 (C=O). MS m/z (%): 438.14 (M , 11.04), 440.16
+
(
M + 2, 4.69), 77.06 (100.00). Anal.Calcd. for C24
H
15ClN
6
O (438.10): C, 65.68; H, 3.45;
N, 19.15. Found: C, 65.84; H, 3.41; N, 19.34.
4
4
.1.8. 8-Chloromethyl-1,5-diphenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidin-
(5H)-one 11
A mixture of 5 (3.18 g, 10 mmol) and chloroacetyl chloride (1.38 g, 12 mmol) in acetic
acid (15 mL) was heated under reflux for 4 h. The reaction mixture was cooled, poured
onto water (50 mL) and the solid was filtered, dried and crystallized from acetic acid.
-1
Yield: 55.1% (2.10 g), m.p: 254-256 °C. IR υmax, cm : 3098, 3063, 3046, (Ar-CH), 2972
1
(
aliph-CH), 1717 (C=O). H NMR (CDCl , 400 MHz): 4.66 (s, 2H, -CH -), 7.25 (t, 1H, J
3
2
=
7.22 Hz, Ar-H), 7.38 (t, 2H, J = 7.78 Hz, Ar-H), 7.60-7.68 (m, 5H, Ar-H), 8.01 (d, 2H,
1
3
J = 8.08 Hz, Ar-H), 8.33 (s, 1H, H3pyrazole). C NMR (DMSO-d , 100 MHz): 48.5
6
(
CH
2
), 103.3 (C4pyrazole), 121.3, 127.0, 127.9, 129.6, 130.3, 130.8, 131.7, 137.2, 139.0,
1
2
49.7 (Ar-C), 151.4 (C=O). Anal.Calcd. for C H ClN O (370.08): C, 60.56; H, 3.48; N,
1
9
13
6
2.30. Found: C, 60.67; H, 3.51; N, 22.48.
4
.1.9. General procedure for preparation of 12a,b.
A mixture of 11 (3.76 g, 10 mmol), the appropriate alicyclic amine (20 mmol) and
anhydrous potassium hydroxide (0.1 g) in dioxane (50 mL) was refluxed for 24 h. The
reaction mixture was cooled and poured onto ice water. The formed precipitate was
filtered, washed with water, dried and crystallized from ethanol to afford 12a,b.
4
.1.9.1.
8-Morpholinomethyl-1,5-diphenyl-1H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]
pyrimidin-4(5H)-one 12a
-1
Yield: 65% (2.77 g), m.p: 261-263 °C. IR υmax, cm : 3094, 3051 (Ar-CH), 2955, 2928,
1
2
855 (aliph-CH), 1717 (C=O). H NMR (CDCl
3
, 400 MHz): 2.52-2,54 (m, 4H, H2,6
morpholine), 3.59-3.61 (m, 4H, H3,5 morpholine), 3.65 (s, 2H, -CH -), 7.27 (t, 2H, J = 6.82 Hz,
2
Ar-H), 7.41 (t, 2H, J = 7.86 Hz, Ar-H), 7.62-7.69 (m, 4H, Ar-H), 8.06 (d, 2H, J = 7.92

ACCEPTED MANUSCRIPT
1
3
Hz, Ar-H), 8.38 (s, 1H, H3pyrazole). C NMR (DMSO-d
6 2
, 100 MHz): 52.5 (CH ), 52.8 (N-
CH2 morpholinyl), 66.4 (O-CH2 morpholinyl), 103.2 (C4pyrazole), 121.2, 126.8, 127.7, 127.9,
1
1
29.6, 130.0, 130.2, 130.4, 132.2, 132.7, 137.0, 139.1, 150.2, 150.5, 151.2, 151.5 (Ar-C),
+
52.4 (C=O). MS m/z (%): 427.17 (M , 1.56), 40.14 (100.00). Anal.Calcd. for
C H N O (427.18): C, 64.63; H, 4.95; N, 22.94. Found: C, 64.89; H, 5.04; N, 23.17.
23
21
7
2
4
.1.9.2. 1,5-Diphenyl-8-(4-phenylpiperazinyl)methyl-1H-pyrazolo [4,3-e][1,2,4]triazolo
[
4,3-a]pyrimidin-4(5H)-one 12b
-1
Yield: 73 % (3.66 g), m.p > 300 °C. IR υmax, cm : 3060 (Ar-CH), 2926, 2854, 2826
1
(
aliph-CH), 1715 (C=O). H NMR (CDCl , 400 MHz): 2.69 (br s, 4H, H2,6 piperazine), 3.10
3
2
(br s , 4H, H3,5 piperazine), 3.71 (s, 2H, -CH -), 6.89-6.94 (m, 3H, Ar-H), 7.41-7.51 (m, 6H,
1
3
Ar-H), 7.63-7.69 (m, 4H, Ar-H), 8.06-8.08 (m, 2H, Ar-H), 8.39 (s, 1H, H3pyrazole). C
NMR (CDCl , 100 MHz): 49.7 (piperazine-C), 52.0 (CH ), 52.8 (piperazine-C), 103.4
3
2
(
C4pyrazole), 116.1, 116.7, 117.3, 117.2, 120.1, 120.6, 121.1, 126.3, 126.7, 126.9, 127.2,
28.9, 129.1, 129.3, 129.4, 130.3, 131.5, 136.7, 149.4, 150.1 (Ar-C), 152.0 (C=O).
Anal.Calcd. for C29 O (502.22): C, 69.31; H, 5.21; N, 22.30. Found: C, 69.46; H,
1
26 8
H N
5
.27; N, 22.47.
4
.1.10. General procedure for preparation of 13a-c
A mixture of compound 5 (3.18 g, 10 mmol) and the substituted isothiocyante (15 mmol)
in pyridine (10 mL) was refluxed for 12-24 h. The solution was cooled, poured onto ice
water and acidified with acetic acid. The formed precipitate was filtered, washed several
times with water and crystallized from ethanol to afford the desired compounds.
4
.1.10.1. 8-Methylamino-1,5-diphenyl-1H-pyrazolo[4,3e][1,2,4]triazolo[4,3-a]pyrimidin
-4(5H)-one 13a
-1
The reaction time was 12 h, yield: 60 % (2.14 g), m.p: 184-186 °C. IR υmax, cm : 3321
1
(
NH), 3066, 3034 (Ar-CH), 2924, 2853 (aliph-CH), 1701 (C=O). H NMR (DMSO-d ,
6
4
00 MHz): 2.88 (s, 3H, -CH
3 2
), 6.26 (s, 1H, NH, exchanged with D O), 7.08 (t, 1H, J =
7
.20 Hz, Ar-H), 7.23 (t, 2H, J = 7.20 Hz, Ar-H), 7.45-7.54 (m, 5H, Ar-H), 7.88 (d, 2H, J
1
3
=
6 3
7.60 Hz, Ar-H), 8.09 (s, 1H, H3pyrazole). C NMR (DMSO-d , 100 MHz): 34.0 (CH ),