Design and synthesis of pyrazolo[3,4-d]pyrimidines: Nitric oxide releasing compounds targeting hepatocellular carcinoma

Article abstract of DOI:10.1016/j.bmc.2017.03.002

A new series of pyrazolo[3,4-d]pyrimidines tethered with nitric oxide (NO) producing functionality was designed and synthesized. Sulforhodamine B (SRB) protein assay revealed that NO releasing moiety in the synthesized compounds significantly decreased the cell growth more than the des-NO analogues. Compounds 7C and 7G possessing N-para-substituted phenyl group, released the highest NO concentration of 4.6% and 4.7% respectively. Anti-proliferative activity of synthesized compounds on HepG2 cell line identified compounds 7h, 7p, 14a and 14b as the most cytotoxic compounds in the series of IC₅₀?=?3, 5, 3 and 5?μM, respectively, compared to erlotinib as a reference drug (IC₅₀?=?25?μM). Flow cytometry studies revealed that 7?h arrested the cells in G0/G1 phase of cell cycle while 7p arrested the cells in S phase. Moreover, docking study of the synthesized compounds on EGFR (PDB code: 1M17) and cytotoxicity study indicated that N-1 phenyl para substitution, pyrazole C-3 alkyl substitution and tethering the nitrate moiety through butyl group had a significant impact on the activity.

Full text of DOI:10.1016/j.bmc.2017.03.002

Accepted Manuscript
Design and Synthesis of Pyrazolo[3,4-d]Pyrimidines: Nitric Oxide Releasing
Compounds Targeting Hepatocellular Carcinoma
Yaseen A.M.M. Elshaier, Mohamed A. Shaaban, Mohammed K. Abd El Hamid,
Mostafa H. Abdelrahman, Mahrous A. Abou-Salim, Sara M. Elgazwi, Fathi
Halaweish
PII:
S0968-0896(16)31498-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.002
BMC 13598
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 December 2016
26 February 2017
3 March 2017
Please cite this article as: Elshaier, Y.A.M., Shaaban, M.A., Abd El Hamid, M.K., Abdelrahman, M.H., Abou-Salim,
M.A., Elgazwi, S.M., Halaweish, F., Design and Synthesis of Pyrazolo[3,4-d]Pyrimidines: Nitric Oxide Releasing
Compounds Targeting Hepatocellular Carcinoma, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.03.002
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Design and Synthesis of Pyrazolo[3,4-d]Pyrimidines: Nitric Oxide Releasing
Compounds Targeting Hepatocellular Carcinoma
Yaseen A. M. M. Elshaier1, Mohamed A. Shaaban*2, Mohammed K. Abd El Hamid2,
Mostafa H. Abdelrahman1, Mahrous A. Abou-Salim1,3, Sara M. Elgazwi3 and Fathi
Halaweish*3
1
Al-Azhar University, Faculty of Pharmacy, Pharmaceutical Organic Chemistry
Department, Assiut 71524, Egypt
2
Cairo University, Faculty of Pharmacy, Pharmaceutical Organic Chemistry
Department, Cairo 11562, Egypt
3South Dakota State University, Faculty of Science, Chemistry Department, Brookings,
SD 57007, USA
Abstract
A new series of pyrazolo[3,4-d]pyrimidines tethered with nitric oxide (NO) producing
functionality was designed and synthesized. Sulforhodamine B (SRB) protein assay
revealed that NO releasing moiety in the synthesized compounds significantly decreased
the cell growth more than the des-NO analogues. Compounds 7C and 7G possessing N-
para-substituted phenyl group, released the highest NO concentration of 4.6% and 4.7%
respectively. Anti-proliferative activity of synthesized compounds on HepG2 cell line
identified compounds 7h, 7p, 14a and 14b as the most cytotoxic compounds in the series
of IC50 = 3, 5, 3 and 5 µM, respectively, compared to erlotinib as a reference drug (IC50 =
25 µM). Flow cytometry studies revealed that 7h arrested the cells in G0/G1 phase of cell
cycle while 7p arrested the cells in S phase. Moreover, docking study of the synthesized
compounds on EGFR (PDB code: 1M17) and cytotoxicity study indicated that N-1
phenyl para substitution, pyrazole C-3 alkyl substitution and tethering the nitrate moiety
through butyl group had a significant impact on the activity.
Keywords
pyrazolo[3,4-d]pyrimidines - Nitric oxide - Hepatocellular carcinoma-EGFR
1. Introduction
Hepatocellular carcinoma (HCC) is the second cause of cancer-related deaths
worldwide since it has high incidence to mortality ratio (1.07).1 Currently used drugs for
-1-

treatment of liver cancer showed severe side effects and do not overcome cancer drug
resistance.2 Cancer resistance to chemotherapeutic agents is a common reason in patient
mortality and poor/early prognosis of cancer.3 Due to this problem and severe side effects
of chemotherapeutic agents, de novo design and synthesis of new chemotherapeutic
agents is a hot topic in cancer research.
Nitric oxide (NO) is an endogenous short-lived free radical obtained from
conversion of L-arginine to L-citroline,4 which is mediated by nitric oxide synthase
(NOS).5 NO producing compounds have a wide biological function such as improving
6
cardiovascular activities,4, and cerebral circulation disorders,6 peripheral ischemia
revascularization,4 limb reperfusion and salvage,4 and also potent as anti-inflammatory,4,
7-8 analgesic,8 antipyretic,8 anti-platelet,8 antibacterial9-10 and anti-cancer agents.4, 8, 11-13 In
addition, NO exerts gastrointestinal safety8 and hepatoprotective properties.14 NO is
known to reverse chemotherapy resistance in colon cancer cells, it was reported that NO
inhibited the MRP-3 efflux pump by nitrating the tyrosine and thereby led to inversion of
drug resistance.15-17 Hence, it is likely that NO can be used in conjunction with traditional
cancer drugs that have become ineffective due to such modes of efflux. Furthermore, NO
is considered as a novel potential therapeutic agent in patients with refractory cancer by
sensitizing tumor cells to chemotherapy, radiotherapy or immunotherapy.18-20 It is
noteworthy to mention that, NCX-1000 (Fig. 1) is the first hepatoprotective NO donor
drug, however, it has not met the endpoint in phase 2 clinical trials.14, 21
-2-

Epidermal growth factor receptor protein tyrosine kinase (EGFR) is highly
expressed in HCC and plays a central hepatoprotective and pro-regenerative role in the
liver.22-23 Treatment of HCC cells with EGFR-specific tyrosine kinase inhibitors such as
erlotinib (Tarceva®) (Fig.1), neutralizing antibodies cetuximab (Erbitux®) or cyclin-
dependent kinase inhibitors (CDKI) such as roscovitine (Seliciclib®) to induce cell
cycle arrest and apoptosis and increases chemo sensitivity. Hence, EGFR inhibition has
long been an attractive target for anticancer drugs,23 many efforts have been directed at
developing anticancer agents that can interfere with EGFR activity, such as monoclonal
antibodies and small-molecule inhibitors.22
O
O
NO2
O
O
O
H
O
H
H
HO
OH
H
NCX-1000
R1
N
N
HN
N
N
HN
N
O
O
R = H, Cl
R1 = H, Cl
O
O
N
N
R
Erlotinib
(I)
Figure 1: Structures of hepatoprotective14, 21 and antitumor drugs.24-25
Moreover, pyrazolo[3,4-d]pyrimidine derivatives have a considerable chemical
and pharmacological importance such as anti-inflammatory,26-27 tuberculostatic,26
antiviral,26-27 antimicrobial,26-28 and anticancer agents (Compound I, Fig. 1).26-29 They
were also found to exhibit variable degrees of anticancer activities against HCC,26, 28
colon carcinoma,28 cervical carcinoma,28 skin carcinoma27 and breast adenocarcinoma26
by inhibiting different types of enzymes such as EGFR-TK,28-29 CDK,28-29 Src and Abl
-3-

tyrosine kinase,29 glycogen synthase kinase-3,28-29 and B-Raf kinase.28 There are so many
documented cancer resistance to current chemotherapeutic agents including erlotinib.30 In
this regard, further efforts are required to develop NO based strategies for cancer
prevention and treatment.
In this context, we envisioned that a novel drug candidate of hybrid structure
from two important pharmcophores, the NO donating group and the pyrazolo[3,4-
d]pyrimidines moiety will be fruitful to hit cancer cells especially HCC cell line and to
overcome the cancer resistance to chemotherapy. To achieve this, we designed a series
of novel analogs possessing pyrazolo[3,4-d]pyrimidines linked through alkyl bridge to
an organic nitrate ester functionality.
2. Results and Discussion
2.1. Molecular docking study
A library of pyrazolo[3,4-d]pyrimidine/nitrate ester hybrids was designed and
energy minimized using MMFF94 force field calculations. The catalytic domain of
EGFR was obtained from protein data bank (PDB code: 1M17)31 and was prepared for
docking using Open Eye® software. Open Eye Omega application was used to generate
different conformations of each ligand. Docking was conducted using Fred and the data
was visualized by Veda application. This software package generates consensus scoring
which is a filtering processes to obtain virtual binding affinity, the lower consensus
score, the better binding affinity of the ligands towards the receptor. This study revealed
that compounds 7h, 7p and 14a, b showed better consensus score than the lead drug
erlotinib, Table 1. The standard erlotinib showed a hydrogen bond (1.71 Å) towards
ATP-binding site of EGFR coming from pyrimidine-N2 with Met:769:A (Fig. 2). This
docking mode similar to cocrystalized docking pose with receptor.31 However
compounds 7h, 7p and 14a,b showed a hydrophobic-hydrophobic interaction towards
the ATP binding site as well as a hydrogen bond between pyrazole-N2 and Lys:721:A
with bond length 2.02, 2.33, 2.28 and 2.40 Å, respectively (Fig. 3 and 4). Furthermore,
Compound 7a showed a hydrogen bonding towards ATP-binding site of EGFR with the
-4-

same amino acid similar to the standard erlotinib. This hydrogen bond comes from
anilinic NH with Met:769:A (2.07 Å).
Figure 2: Visual representation for erlotinib docked with PDB: 1M17.The dashed lines
showing a hydrogen bond towards ATP-binding site of EGFR coming from pyrimidine-
N2 with Met:769:A.
-5-

Figure 3: Visual representation for 7h docked with PDB: 1M17, the dashed lines
showing the hydrogen bonding towards ATP-binding site of EGFR between pyrazolo
N-2 and Lys:721:A.
-6-

Figure 4: Visual representation for 14a docked with PDB: 1M17 showing the
hydrophobic-hydrophobic interaction towards the ATP binding site and the dashed lines
showing the hydrogen bonding between pyrazolo N-2 and Lys:721:A.
-7-

Table 1: Molecular modeling consensus scores, IC50 and NO release
(%) of pyrazolo[3,4-d]pyrimidine/nitrate ester hybrids 7a-p, 14a,b and erlotinib as a
reference drug.
IC50
NO release
(%)
Compound
Consensus score
(µM)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
14a
60
66
65
43
56
40
56
16
53
43
52
37
36
30
36
17
14
14
20
-
>100
>100
20
25
25
>100
10
3
>100
>100
10
10
25
20
10
5
3
5
25
2.3
2.1
4.6
3.1
1.5
2.6
4.7
3.7
2.6
2.2
3.8
1.3
1.3
4.1
4.2
2.2
0.7
3.6
-
14b
Erlotinib
GTN*
-
10.132
* glycerol trinitrate
2.2. Chemistry
The main precursors 2a-d were synthesized as outlined in Scheme 1 through
Michael addition of phenylhydrazine derivatives to the specific alkylidene 1.27, 29, 33-38
The generated amino cyanopyrazole 2a-d were subjected to intramolecular
condensations through refluxing in formic acid or acetic acid to afford pyrazolo[3,4-
d]pyrimidines-4-ones 3a-h. The resulting pyrazolo[3,4-d]pyrimidines-4-ones 3a-h were
refluxed in phosphorus oxychloride to give 4-chloropyrazolopyrimidine derivatives 4a-
h, Scheme 1.33, 39
-8-

Refluxing 4-chloropyrazolopyrimidine derivatives 4a-h with p-aminophenol
using triethylamine and sodium iodide as catalysts gave 5a-h. O-alkylation of anilino
derivatives 5a-h with dibromoalkane named 1,3-dibromopropane and 1,4-
dibromobutane in K2CO3/CH3CN under reflux generated the corresponding bromo-
alkyl derivatives 6a-h. The target nitrates 7a-b were prepared successfully by heating
the alkyl bromides 6a-h with AgNO3 in dry acetonitrile, Scheme 2.
Cl
R1
O
R1
N
R1
CN
R2
NH
NH2
R1
CN
CN
POCl3
R3COOH
N
NH
R3
N
N
105 oC
100-118 oC
89 - 91 %
EtOH, 78 oC
NH2
N
N
N
EtO
N
R3
N
85 - 90 %
1
R2
R2
R2
2a-d
4a-h
3a-h
3a, 4a, R1 = H, R2 = H, R3 = H
3b, 4b, R1 = H, R2 = Br, R3 = H
2a, R1 = H, R2 = H
1a, R1 = H
1b, R1 = CH3
2b, R1 = CH3, R2 = H
2c, R1 = H, R2 = Br
2d, R1 = CH3, R2 = Br
3c, 4c, R1 = CH3, R2 = H, R3 = H
3d, 4d, R1 = CH3, R2 = Br, R3 = H
3e, 4e, R1 = H, R2 = H, R3 = CH3
3f, 4f, R1 = H, R2 = Br, R3 = CH3
3g, 4g, R1 = CH3, R2 = H, R3 = CH3
3h, 4h, R1 = CH3, R2 = Br, R3 = CH3
Scheme 1. Synthesis of 4-chloropyrazolo[3,4-d]pyrimidine intermediates.
ONO2
n
Br
O
O
OH
n
HN
N
HN
HN
N
R1
R1
R1
H2N
OH
AgNO3
Br
Br
N
N
N
n
N
N
N
4
CH3CN, 80 oC
30 - 91 %
TEA, NaI
i-PrOH, 80 oC
K2CO3
CH3CN, 80 oC
17 - 64 %
N
N
N
N
R3
R3
R3
80 - 89 %
R2
R2
R2
7a-p
6a-p
5a-h
6i, 7i, R1 = H, R2 = H, R3 = CH3, n = 3
6j, 7j, R1 = H, R2 = H, R3 = CH3, n = 4
5a, R1 = H, R2 = H, R3 = H
5b, R1 = H, R2 = Br, R3 = H
5c, R1 = CH3, R2 = H, R3 = H
5d, R1 = CH3, R2 = Br, R3 = H
5e, R1 = H, R2 = H, R3 = CH3
5f, R1 = H, R2 = Br, R3 = CH3
6a, 7a, R1 = H, R2 = H, R3 = H, n = 3
6b, 7b, R1 = H, R2 = H, R3 = H, n = 4
6c, 7c, R1 = H, R2 = Br, R3 = H, n = 3
6d, 7d, R1 = H, R2 = Br, R3 = H, n = 4
6e, 7e, R1 = CH3, R2 = H, R3 = H, n = 3
6f, 7f, R1 = CH3, R2 = H, R3 = H, n = 4
6g, 7g, R1 = CH3, R2 = Br, R3 = H, n = 3
6h, 7h, R1 = CH3, R2 = Br, R3 = H, n = 4
6k, 7k, R1 = H, R2 = Br, R3 = CH3, n = 3
6l, 7l, R1 = H, R2 = Br, R3 = CH3, n = 4
6m, 7m, R1 = CH3, R2 = H, R3 = CH3, n = 3
6n, 7n, R1 = CH3, R2 = H, R3 = CH3, n = 4
6o, 7o, R1 = CH3, R2 = Br, R3 = CH3, n = 3
6p, 7p, R1 = CH3, R2 = Br, R3 = CH3, n = 4
5g, R1 = CH3, R2 = H, R3 = CH3
5h, R1 = CH3, R2 = Br, R3 = CH3
-9-

Scheme 2. Synthesis of NO releasing pyrazolo[3,4-d]pyrimidines.
In order to prepare the designed analogs which contain SMe tethering pyrazole
moiety, the departure point starts with preparing the alkylidene compound 8,40 Scheme
3. Having in hand compound 8, compounds 14a-b were obtained in high yield, Scheme
4, by following similar procedures explained before in schemes 1, and 2.
S
CN
NH2
O
N
S
Cl
N
S
NHNH2
EtOH
78 oC
S
S
CN
CN
RCOOH
POCl3
N
NH
N
+
N
N
N
80-118 oC
48 - 51 %
105 oC
N
R
N
R
59 - 66 %
Br
8
Br
9
Br
Br
11a,b
10a, 11a, R = H
10b, 11b, R = CH3
10a,b
Scheme 3. Synthesis of 3-methylthio 4-chloropyrazolo[3,4-d]pyrimidine intermediates.
O
OH
Br
OH
HN
S
HN
S
Br
Br
N
H2N
TEA, NaI
N
N
11a,b
N
N
N
R
NaOH, EtOH
78 oC
N
N
R
i-PrOH
80 oC
49 - 83 %
13a,b
84 - 91 %
Br
Br
12a,b
AgNO3 CH3CN, 80 oC
68 - 70 %
O
ONO2
HN
N
S
N
N
N
R
12a, 13a, 14a, R = H
12b, 13b, 14b, R = CH3
Br
14a,b
Scheme4. Synthesis of NO releasing 3-methylthio pyrazolo[3,4-d]pyrimidines.
-10-

Moreover, to explore the bioisosteric effect of changing the oxygen atom in p-
aminophenol by nitrogen atom using p-phenelynediamin, compounds 16 was designed.
Compound 4 was coupled with p-phenylene diamine and compound 15 was obtained in
good yield. However, cyclization reaction occurred between 1,4-dibromobutane and
anilino derivative 15 affording pyrrolidine compound 17 (Scheme 5).
H
N
Br
HN
N
X
N
N
N
NH2
16
Cl
N
HN
N
NH2
H2N
Br
N
Br
N
N
N
N
EtOH, 78 oC
N
N
K2CO3, CH3CN
80 oC
HN
N
56 %
N
15
N
N
4a
17
Scheme 5. Synthesis of compound 17
2.3. Measurement of nitric oxide release
To evaluate the thiol-induced nitric oxide generation, final compounds 7a-h and
14a,b were subjected to Griess colorimetric method.41-42 The amount of NO released
from the tested compounds, was measured relative to NO released from standard
sodium nitrite solution and calculated as percentage of NO released (Table 1). The
results of measurement of NO release revealed that compounds 7c and 7g, which
contain N-1 para-subsitituted phenyl group released the highest amount of NO among
this group (4.6 and 4.7%, respectively). The results suggested that these compounds
were able to generate nitrite at pH 1 which confirms the suggested gastrointestinal
safety of NO.43
2.4. Cytotoxicity screening studies
2.4.1. NCI 60 cell lines screening studies
-11-

Eight compounds, 6a, 6e, 6i, 6m, 7a, 7e, 7i and 7m, were selected by National
Cancer Institute (NCI) according to the protocol of the Drug Evaluation program at
National Cancer Institute,44 for in vitro anticancer screening. In vitro study using one
dose anticancer assay was conducted in full NCI 60 cell lines derived from nine tumor
subpanels, including leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate
and breast cancer cell lines. The selected compounds were added at a single
concentration (10-5 M) and the culture was incubated for 48 h. End point determinations
were made with a protein binding dye sulforhodamine B (SRB). Cytotoxicity of each
compound were reported as a mean of the percent growth of the treated cells when
compared to the untreated control cells, as depicted, in Table 2.
Cytotoxicity data (Table 2) revealed that the tested compounds showed a weak
cell growth inhibition against almost 60 cancer cell lines except compounds 6a, 7a
exhibited moderate cell growth inhibition against colon cancer cell line HCT-15 (cell
growth promotion 59.19%, inhibition 40.81%) and (cell growth promotion 57.54%,
inhibition 42.46%), respectively. Also, compounds 6e and 7e showed moderate cell
growth inhibition against non-small cell lung cancer cell line NCI-H522 (cell growth
promotion 45.63%, inhibition 54.37) and (cell growth promotion 57.65%, inhibition
42.35%), respectively. Moreover, compounds 7e and 7m record a moderate cell growth
inhibition against renal cancer cell line UO-31 (cell growth promotion 40.74%,
inhibition 59.26%) and A498 (cell growth promotion 57.57%, inhibition 42.43%),
respectively. The moderate cytotoxicity of compounds 7a, 7e, 7i, and 7m could be
attributed to the NO releasing activity of these compounds.
2.4.2. In vitro antitumor against HepG2 Cell line
All target compounds (7a-p & 14a,b) were screened for their cytotoxicity
activity against HepG2, IC50 was calculated with regard to saline control group and
potency was calculated with regard to percentage of antiproliferative activities of
erlotinib and tested compounds, as depicted, in Table 1.
Our SAR study showed that thirteen of the tested compounds have high to
moderate anti-tumor activity towards liver cell line (HepG2) with IC50 values ranges
-12-

from 3-25 µM in comparison to erlotinib as a reference drug ( IC50 = 25 µM).
Compounds with pyrazolopyrimidines linked with methyl or methylthio group at C-3 of
pyrazole ring, p-bromophenyl at pyrazole N-1, and nitrooxybutyl group (7h, 14a; IC50 =
3µM and 7p, 14b; IC50 = 5µM) have the highest activity among the tested compounds
as well as the reference drug erlotinib. Also, compounds that don`t have pyrazole C-3
methyl (7l; IC50 = 10µM) or have unsubstituted phenyl at pyrazole N-1 (7n; IC50 =
10µM) or tethered to nitrooxy function through shorter alkyl group like propyl (7g, 7o;
IC50 = 10µM) were also more active than erlotinib while compounds 7c-e, 7m and 7n
with IC50 values ranging from 20-25 µM in comparison to erlotinib. Furthermore,
because of N-1-unsubstituted phenyl and endowing the pyrazolopyrimidine core with
nitric oxide releasing moiety through short alkyl chain (propyl), compounds 7a, 7b, 7f,
7i and 7j are the least active among the series. Consequently, increasing lipophilicity at
N-1 and substitution at C-3 as well as tethering the nitroxy moiety through long alkyl
butyl chain may improve the hydrophobic hydrophobic interaction towards the ATP
binding site of EGFR and hydrogen bonding formation and thus increase the ability of
the ligands to be more active.
2.4.3. Cell Cycle Arrest
Flow cytometric analyses of compounds 7h and 7p were conducted as they are
the most active compounds among the series with IC50 = 3, 5 µM, respectively. Cell
cycle parameters were compared for untreated HepG2 cells that had been incubated for
48 h with 7h (3ꢀM) or 7p (5ꢀM), with vehicle DMSO as control. Cell cycle analysis
performed by FACS analysis revealed that compounds 7h and 7p cause accumulation of
cells in G0/G1 and S phase of the cell cycle, respectively. As shown in Figure 5,
compound 7h exhibited an increase in the fraction of cells in the G0/G1 phase (91.70%
compared to 64.21% for the control) and a decrease in the proportion of cells in the S
phase (1.88% compared to 15.90%) and the G2/M phase (4.59% compared to 19.89%).
On the other hand, compound 7p (Fig. 6) showed an increase in the fraction of cells in
the S phase (34.28% compared to 15.90% in sample control) and a decrease in the
proportion of cells in the G2/M phase (0.98% compared to 19.89%). It is concluded
-13-

from these results that 7h is a strong inhibitor of cell cycle progression at the G0/G1
phase while 7p is a strong inhibitor at the S phase.
Figure 5. Flow cytometric analysis of cell cycle parameters. HepG2 liver cancer cells
were incubated for 48 h in the presence of 3 ꢀM (IC50) of 7h.
Figure 6. Flow cytometric analysis of cell cycle parameters. HepG2 liver cancer cells
were incubated for 48 h in the presence of 5 ꢀM (IC50) of 7p.
3. Conclusion
-14-

Design and synthesis of pyrazolo[3,4-d]pyrimidine derivatives with NO
releasing properties have been achieved in an efficient manner. Molecular modeling as
well as in vitro cytotoxic studies of the target compounds against HepG2 cell lines
revealed that compounds 7h, 7p, 14a and 14b are more potent as anti-tumor agents than
the reference drug (erlotinib) through inducing G0/G1 or S cell cycle arrest. Also,
increasing lipophilicity of compounds and installing methyl or methyllthio groups on
pyrazole moiety as well as endowing with nitroxybutyl side chain played a crucial role
in cytotoxic activity. Finally, the novel synthesized NO-donating hybrids represent a
significant strategy against cancer.
4. Experimental procedures
4.1. Chemistry
13
1H and C NMR spectra were acquired on a Bruker AVANCE and Varian Unity
INOVA-400 MHz NMR spectrometer, in DMSO using TMS (ꢁ = 0 ppm) as the internal
standard for 1H NMR and DMSO-d6 (ꢁ = 39.51 ppm) for 13C NMR, with the reporting of
coupling constants in Hz and the signal multiplicities are reported as singlet (s), doublet
(d), triplet (t), quartet (q), doublet of doublets (dd), doublet of triplets (dt), multiplet (m),
or broad (br). HRMS data was obtained using Thermo Instruments MS system (LTQ
XL/LTQ Orbitrap Discovery) coupled to a Thermo Instruments HPLC system (Accela
PDA detector, Accela PDA autosampler and Pump). TLC analysis was performed using
pre-coated silica gel 60 F254 (Merck) sheets. Products were purified via column
chromatography using silica gel 150-250 ꢀm (60-120 mesh). TLC plates were visualized
by ultraviolet at 254 nm. All reagents and solvents were obtained from commercial
suppliers and used as received. All chemical reactions requiring anhydrous conditions
were performed with an oven-dried glassware.
4.1.1. Substituted 4-amino-4-cyanopyrazoles 2a-d, pyrazolo[3,4-d]pyrimidinones 3a-
c & 3e-g, 4-chloro derivatives 4a-c & 4e-g, 4-anilino derivatives 5a-e & 5g, 2-
(bis(methylthio)methylene)malononitrile
8
and 5-amino-1-(4-bromophenyl)-3-
(methylthio)-1H-pyrazole-4-carbonitrile 9 were prepared according to the reported
procedure.27, 29, 33-38
-15-

4.1.2. General procedure for preparation of 1-aryl-3-alkyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-ones (3a-d, 10a)
A suspension of the appropriate intermediates 2a-d or 9 (0.01 mol) in 85%
formic acid (40 mL) was heated under reflux for 10 h. The reaction mixture was cooled
and the separated precipitate was filtered, washed with water, dried and recrystallized
from formic acid to afford desired compounds 3a-d or 10a.33, 45-46
4.1.2.1 1-(4-Bromophenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (3d)
o
White solid; yield 89%; mp 344-345 C; IR (KBr, cm-1): 3437 (NH), 1686 (CO), 1584
(C=N); 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.68 (s, 3H, 6-CH3), 7.73 (d, 2H, J
= 8.4 Hz, Ar-H), 8.04 (d, 2H, J = 8.4 Hz, Ar-H), 8.17 (s, 1H, 6-H), 12.39 (s, NH,
exchangeable); Anal. Calcd. for C12H9BrN4O (305.13): C, 47.24; H, 2.97; N, 18.36.
Found: C, 47.17; H, 2.68; N, 18.11.
4.1.2.2. 1-(4-Bromophenyl)-3-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(10a)
o
White solid; yield 51%; mp 284-286 C; IR (KBr, cm-1): 3350 (NH), 1685 (CO), 1585
(C=N); 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.63 (s, 3H, SCH3), 7.75 (d, 2H, J =
8.8 Hz, Ar-H), 8.05 (d, 2H, J = 8.8 Hz, Ar-H), 8.21 (s, 1H, 6-H), 12.52 (s, 1H, NH,
exchangeable); Anal. Calcd. for C12H9BrN4OS (337.20): C, 42.74; H, 2.69; N, 16.62;
Found: C, 42.90; H, 2.74; N, 16.80.
4.1.3. General procedure for preparation of 1-Aryl-3,6-dialkyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-ones (3e-h, 10b)
A suspension of the appropriate derivatives 2a-d or 9 (0.001 mol) was dissolved
in glacial acetic acid (3 mL) then POCl3 (0.2 mL) was added quickly. The reaction
mixture was refluxed for 2 h (the reaction system was carefully observed by TLC).
After cooling the mixture, ice-water was added and the formed precipitate was filtered,
washed with water, dried and recrystallized from formic acid to give compounds 3e-h
or 10b.39
-16-

4.1.3.1.
(3h)
1-(4-Bromophenyl)-3,6-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
o
White solid; yield 91%; mp 344-346 C; IR (KBr, cm-1): 3429 (NH), 1683 (CO), 1607
(C=N); 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.27 (s, 3H, 6-CH3), 2.35 (s, 3H, 3-
CH3), 7.66 (d, 2H, J = 8.0 Hz , Ar-H), 7.96 (d, 2H, J = 8.0 Hz, Ar-H), 12.55 (s, 1H, NH);
Anal. Calcd. for C13H11BrN4O (319.16): C, 48.92; H, 3.47; N, 17.55. Found: C, 49.24; H,
3.27; N, 17.80.
4.1.3.2. 1-(4-bromophenyl)-6-methyl-3-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (10b)
o
White solid; yield 48%; mp 340-342 C; IR (KBr, cm-1): 3420 (NH), 1678 (CO), 1581
1
(C=N); H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.40 (s, 3H, 6-CH3), 2.61 (s, 3H,
SCH3), 7.74 (d, 2H, J = 8.8 Hz, Ar-H), 8.06 (d, 2H, J = 8.8 Hz, Ar-H), 12.38 (s, 1H, NH,
exchangeable); Anal. Calcd. for C13H11BrN4OS (351.22): C, 44.46; H, 3.16; N, 15.95;
Found: C, 44.69; H, 3.21; N, 16.13.
4.1.4. General procedure for preparation of 1-Aryl-4-chloro-1H-pyrazolo[3,4-
d]pyrimidines (4a-h, 11a,b)
A suspension of the appropriate derivatives 3a-h or 10a,b (0.001 mol) in
phosphorous oxychloride (8 mL) was heated under reflux for 12 h. The reaction mixture
was cooled and poured onto ice-cooled water. The formed precipitate was filtered,
washed with water, dried and recrystallized from n-hexane to afford the desired pure
compounds 4a-h or 11a,b.33
4.1.4.1. 1-(4-Bromophenyl)-4-chloro-3-methyl-1H-pyrazolo[3,4-d]pyrimidine (4d)
o
1
White solid; yield 90%; mp 170-172 C; IR (KBr, cm-1): 1574 (C=N); H NMR (400
MHz, DMSO-d6, δ = ppm) δ = 2.74 (s, 3H, 3-CH3), 7.80 (d, 2H, J = 8.8 Hz, Ar-H), 8.14
(d, 2H, J = 8.8 Hz, Ar-H), 8.95 (s, 1H, 6-H); Anal. Calcd. for C12H8BrClN4 (323.58): C,
44.54; H, 2.49; N, 17.31; Found: C, 44.70; H, 2.24; N, 17,45.
-17-

4.1.4.2.
(4h)
1-(4-Bromophenyl)-4-chloro-3,6-dimethyl-1H-pyrazolo[3,4-d]pyrimidine
o
1
White solid; yield 89%; mp 178-180 C; IR (KBr, cm-1): 1599 (C=N); H NMR (400
MHz, DMSO-d6, δ = ppm) δ = 2.71 (s, 3H, 6-CH3), 2.74 (s, 3H, 3-CH3), 7.79 (d, 2H, J =
8.8 Hz, Ar-H), 8.15 (d, 2H, J = 8.8 Hz, Ar-H); Anal. Calcd. for C13H10BrClN4 (337.60):
C, 46.25; H, 2.99; N, 16.60. Found: C, 46.50; H, 2.72; N, 16.64.
4.1.4.3. 1-(4-bromophenyl)-4-chloro-3-(methylthio)-1H-pyrazolo[3,4-d]pyrimidine
(11a)
o
1
White solid; yield 66%; mp 160-162 C; IR (KBr, cm-1): 1573 (C=N); H NMR (400
MHz, DMSO-d6, δ = ppm) δ = 2.76 (s, 3H, SCH3), 7.81 (d, 2H, J = 8.8 Hz, Ar-H), 8.16
(d, 2H, J = 8.8 Hz, Ar-H), 8.95 (s, 1H, 6-H); Anal. Calcd. for C12H8BrClN4S (355.64): C,
40.53; H, 2.27; N, 15.75. Found: C, 40.78; H, 2.25; N, 15.98.
4.1.4.4.
1-(4-bromophenyl)-4-chloro-6-methyl-3-(methylthio)-1H-pyrazolo[3,4-
d]pyrimidine (11b)
o
1
White solid; yield 59%; mp 164-165 C; IR (KBr, cm-1): 1577 (C=N); H NMR (400
MHz, DMSO-d6, δ = ppm) δ = 2.73 (s, 3H, 6-CH3), 2.74 (s, 3H, SCH3), 7.80 (d, 2H, J =
7.2 Hz, Ar-H), 8.15 (d, 2H, J = 7.2 Hz, Ar-H); Anal. Calcd. for C13H10BrClN4S (369.67):
C, 42.24; H, 2.73; N, 15.16. Found: C, 42.48; H, 2.71; N, 15.38.
4.1.5. General procedure for preparation of 4-((1-Aryl-3,6-(un)substituted-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)amino)phenols (5a-h, 12a,b)
A mixture of appropriate derivatives 4a-h or 11a-b (10 mmol), 4-aminophenol
(1.09 g, 10 mmol), sodium iodide (0.21 g, 1.4 mmol) and triethyl amine (0.84 mL, 10
mmol) in isopropyl alcohol (15 mL) was heated under reflux for 2 h. After cooling, the
reaction mixture was diluted with water, and the formed precipitate was collected by
filtration, washed with water, and crystallized from ethanol – water to afford required
compounds 5a-h, 12a,b.47
-18-

4.1.5.1.
4-((1-(4-Bromophenyl)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenol (5f)
o
White solid; yield 80%; mp 299-300 C; IR (KBr, cm-1): 3422-3189 (br, OH & NH) and
1
1580 (C=N); H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.52 (s, 3H, CH3), 6.82 (d,
2H, J = 8.0 Hz, Ar-H); 7.57 (d, 2H, J = 8.0 Hz, Ar-H); 7.74 (d, 2H, J = 8.0 Hz, Ar-H),
8.22 (d, 2H, J = 8.0 Hz, Ar-H); 8.49 (s, 1H, 3-H); 9.39 (bs, 1H, OH, exchangeable); 9.91
(s, 1H, NH, exchangeable); Anal. Calcd. for C18H14BrN5O (396.24): C, 54.56; H, 3.56; N,
17.67; Found: C, 54.40; H, 3.28; N, 17.80.
4.1.5.2.
4-((1-(4-Bromophenyl)-3,6-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenol (5h)
o
White solid; yield 89%; mp 226-227 C; IR (KBr, cm-1): 3431-3275 (br, OH & NH) and
1581 (C=N); 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.45 (s, 3H, 3-CH3), 2.67 (s,
3H, 6-CH3), 6.79 (d, 2H, J = 8.0 Hz, Ar-H); 7.42 (d, 2H, J = 8.0 Hz, Ar-H); 7.71 (d, 2H,
J = 8.4 Hz, Ar-H), 8.21 (d, 2H, J = 8.4 Hz, Ar-H); 8.56 (s, 1H, NH, exchangeable); 9.38
(s, 1H, OH, exchangeable). Anal. Calcd. for C19H16BrN5O (410.27): C, 55.62; H, 3.93; N,
17.07; Found: C, 55.95; H, 3.72; N, 16.81.
4.1.5.3.
4-((1-(4-bromophenyl)-3-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenol (12a)
o
Light brown solid; yield 84%; mp 206-207 C; IR (KBr, cm-1): 3367 (NH), 3228 (OH)
and 1620 (C=N); 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.73 (s, 3H, SCH3), 6.79
(d, 2H, J = 8.8 Hz, Ar-H); 7.41 (d, 2H, J = 8.8 Hz, Ar-H); 7.75 (d, 2H, J = 8.8 Hz, Ar-H),
8.21 (d, 2H, J = 8.8 Hz, Ar-H); 8.42 (s, 1H, 6-H); 8.45 (s, 1H, NH, exchangeable); 9.41
(s, 1H, OH, exchangeable); Anal. Calcd. for C18H14BrN5OS (428.31): C, 50.48; H, 3.29;
N, 16.35; Found: C, 50.64; H, 3.32; N, 16.52.
4.1.5.4.
4-((1-(4-bromophenyl)-6-methyl-3-(methylthio)-1H-pyrazolo[3,4-
d]pyrimidin-4-yl)amino)phenol (12b)
o
White solid; yield 91%; mp 216-218 C; IR (KBr, cm-1): 3344 (OH & NH) and 1612
1
(C=N); H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.49 (s, 3H, 6-CH3), 2.71 (s, 3H,
-19-

SCH3), 6.79 (d, 2H, J = 8.8 Hz, Ar-H); 7.46 (d, 2H, J = 8.4 Hz, Ar-H); 7.75 (d, 2H, J =
8.4 Hz, Ar-H), 8.21 (d, 2H, J = 8.8 Hz, Ar-H), 8.31 (s, 1H, NH, exchangeable), 9.38 (s,
1H, OH, exchangeable); Anal. Calcd. for C19H16BrN5OS (442.33): C, 51.59; H, 3.65; N,
15.83; Found: C, 51.82; H, 3.69; N, 16.04.
4.1.6. General procedure for preparation of 1-Aryl-N-(4-(bromoalkoxy)phenyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6a-p)
To a solution of the appropriate compounds 5a-h (0.001 mol) in acetonitrile (15
mL), dihaloalkane (0.010 mol), and anhydrous potassium carbonate (0.276 g, 0.002
mol) were added and the reaction mixture was heated under reflux for 10 h. After
removal of acetonitrile under vacuo, dichloromethane and water were added to the
reaction mixture and the organic layer was washed with water, dried over Na2SO4 and
the solvent was evaporated under reduced pressure to give a crude product which was
purified by flash chromatography on silica gel (AcOEt : n-hexane; 1:9) .48-50
4.1.6.1.
N-(4-(3-Bromopropoxy)phenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (6a)
o
1
White solid; yield 50%; mp 158-159 C; IR (KBr, cm-1): 3437 (NH), 1583 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.26 (p, 2H, J = 6.3 Hz, OCH2CH2CH2Br),
3.69 (t, 2H, J = 6.5 Hz, OCH2CH2CH2Br), 4.10 (t, 2H, J = 6.0 Hz, OCH2CH2CH2Br),
7.02 (d, 2H, J = 8.0 Hz, Ar-H), 7.36 (t, 1H, J = 8.0 Hz, Ar-H), 7.56 (t, 2H, J = 7.8 Hz,
Ar-H), 7.70 (d, 2H, J = 8.0 Hz, Ar-H), 8.20 (d, 2H, J = 8.0 Hz, Ar-H), 8.47 (s, 1H, 6-H),
10.11 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6, δ = ppm) δ = 31.3, 31.9, 65.5, 114.7,
120.8, 126.3, 129.2, 133.7, 138.8, 142.0, 153.0, 155.0, 156.2; Anal Calcd. for
C20H18BrN5O (424.29): C, 56.62; H, 4.28; N, 16.51. Found: C,56.32; H,4.33; N,16.50.
4.1.6.2.
N-(4-(4-Bromobutoxy)phenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (6b)
o
1
White solid; yield 64%; mp 164-165 C; IR (KBr, cm-1): 3426 (NH), 1582 (C=N). H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.91–1.80 (m, 2H, OCH2CH2CH2CH2Br);
2.05–1.93(m, 2H, OCH2CH2CH2CH2Br); 3.63 (t, 2H, J = 6.6 Hz, OCH2CH2CH2CH2Br);
-20-

4.03 (t, 2H, J = 6.3 Hz, OCH2CH2CH2CH2Br); 6.00 (d, 2H, J = 8.0 Hz, Ar-H); 7.36 (t,
1H, J = 7.6 Hz, Ar-H), 7.57 (t, 2H, J = 8.0 Hz, Ar-H), 7.68 (d, 2H, J = 8.0 Hz, Ar-H),
8.20 (d, 2H, J = 8.0 Hz, Ar-H); 8.47 (s, 1H, 6-H); 8.50 (s, 1H, 3-H); 10.10 (s, 1H, NH);
Anal Calcd. for C21H20BrN5O (438.32): C, 57.54; H, 4.60; N, 15.98, Found: C, 57.88; H,
4.67; N, 15.76.
4.1.6.3.
1-(4-Bromophenyl)-N-(4-(3-bromopropoxy)phenyl)-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6c)
o
1
White solid; yield 50%; mp 198-199 C; IR (KBr, cm-1): 3436 (NH), 1588 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.25-2.29 (m, 2H, OCH2CH2CH2Br), 3.69 (t,
2H, J = 6.45 Hz, OCH2CH2CH2Br), 4.11 (t, 2H, J = 6.0 Hz, OCH2CH2CH2Br), 7.02 (d,
2H, J = 9.0 Hz, Ar-H), 7.68 (d, 2H, J = 8.4 Hz, Ar-H), 7.76 (d, 2H, J = 8.4 Hz, Ar-H),
8.23 (d, 2H, J = 9.0 Hz, Ar-H), 8.49 (s, 2H, 6-H & 3-H), 10.13 (s, NH, exchangeable);
Anal Calcd. for C20H17Br2N5O (503.19): C, 47.74; H, 3.41; N, 13.92; Found: C, 47.49; H,
3.47; N, 13.93.
4.1.6.4.
N-(4-(4-Bromobutoxy)phenyl)-1-(4-bromophenyl)-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6d)
o
1
White solid; yield 48%; mp 202-203 C; IR (KBr, cm-1): 3434 (NH), 1583 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.83-1.88 (m, 2H, OCH2CH2CH2CH2Br),
1.97-1.99 (m, 2H, OCH2CH2CH2CH2Br), 3.63 (t, 2H, J = 6.6 Hz, OCH2CH2CH2Br), 4.04
(t, 2H, J = 6.0 Hz, OCH2CH2CH2Br), 7.00 (d, 2H, J = 8.1 Hz, Ar-H), 7.67 (d, 2H, J = 8.4
Hz, Ar-H), 7.77 (d, 2H, J = 8.4 Hz, Ar-H), 8.22 (d, 2H, J = 8.1 Hz, Ar-H), 8.49 (s, 2H, 6-
H & 3-H), 10.12 (s, NH, D2O exchangeable); Anal Calcd. for C21H19Br2N5O (517.22): C,
48.77; H, 3.70; N, 13.54; Found: C, 49.03; H, 3.84; N, 13.32.
4.1.6.5.
N-(4-(3-Bromopropoxy)phenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6e)
o
1
White solid; yield 36%; mp 110-111 C; IR (KBr, cm-1): 3410 (NH), 1603 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.26 (p, 2H, J = 6.3 Hz, OCH2CH2CH2Br),
2.76 (s, 3H, CH3), 3.69 (t, 2H, J = 6.5 Hz, OCH2CH2CH2Br), 4.10 (t, 2H, J = 6.0 Hz, 2H,
-21-

OCH2CH2CH2Br), 6.99 (d, 2H, J = 8.0 Hz, Ar-H), 7.31 (t, 1H, J = 7.3 Hz, Ar-H), 7.51-
7.56 (m, 4H, Ar-H), 8.18 (d, 2H, J = 8.0 Hz, Ar-H), 8.36 (s, 1H, 6-H), 8.73 (s, 1H, NH);
13C NMR (101 MHz, DMSO-d6, δ = ppm) δ = 14.8, 31.3, 31.9, 65.5, 101.1, 114.3, 120.5,
125.6, 125.8, 129.1, 131.5, 138.7, 142.4, 154.1, 155.4, 155.8, 155.9; DEPT-13C NMR
(DMSO-d6 ꢁ ppm) 14.8, 31.3 (inverted), 31.9 (inverted), 65.5 (inverted), 114.3, 120.5,
125.6, 125.8, 129.1, 155.9. HRESI-MS m/z calcd for C21H20BrN5O: 438.0924 [M+H]+,
440.0904 [M+H+2]+, found: 438.0914 [M+H]+.
4.1.6.6.
N-(4-(4-Bromobutoxy)phenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6f)
o
1
White solid; yield 53%; mp126-127 C; IR (KBr, cm-1): 3434 (NH), 1611 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.81-1.86 (m, 2H, OCH2CH2CH2CH2Br),
1.94-2.01 (m, 2H, OCH2CH2CH2CH2Br), 2.75 (s, 3H, CH3), 3.61 (t, 2H, J = 6.6 Hz,
OCH2CH2CH2CH2Br), 4.01 (t, 2H, J = 6.3 Hz, OCH2CH2CH2CH2Br), 6.96 (d, 2H, J =
8.0 Hz, Ar-H), 7.31 (t, 1H, J = 8.0 Hz, Ar-H), 7.50-7.55 (m, 4H, Ar-H), 8.18 (d, 2H, J =
8.0 Hz, Ar-H), 8.36 (s, 1H, 6-H), 8.71 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6, δ =
ppm) δ = 15.3, 27.9, 29.6, 35.3, 67.2, 101.6, 114.7, 120.9, 125.9, 126.2, 129.5, 131.7,
139.2, 142.8, 154.5, 156.1, 156.3, 156.4; HRESI-MS m/z calcd for C22H23BrN5O:
452.1080 [M+H]+, found: 452.1067.
4.1.6.7.
1-(4-Bromophenyl)-N-(4-(3-bromopropoxy)phenyl)-3-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6g)
o
1
White solid; yield 23%; mp 176-177 C; IR (KBr, cm-1): 3420 (NH), 1608 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.24-2.30 (m, 2H, OCH2CH2CH2Br), 2.75 (s,
3H, CH3), 3.70 (t, 2H, J = 6.4 Hz, OCH2CH2CH2Br), 4.11 (t, 2H, J = 5.8 Hz,
OCH2CH2CH2Br), 7.00 (d, 2H, J = 8.8 Hz, 2H, Ar-H), 7.54 (d, 2H, J = 8.4 Hz, Ar-H),
7.73 (d, 2H, J = 8.4 Hz, Ar-H), 8.21 (d, 2H, J = 8.8 Hz, Ar-H), 8.38 (s, 1H, 6-H), 8.79 (s,
NH); Anal Calcd. for C21H19Br2N5O (517.22): C, 48.77; H, 3.70; N, 13.54; Found: C,
48.94; H, 3.36; N, 13.72.
4.1.6.8.
N-(4-(4-Bromobutoxy)phenyl)-1-(4-bromophenyl)-3-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6h)
-22-

o
1
White solid; yield 38%; mp 174-175 C; IR (KBr, cm-1): 3435 (NH), 1609 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.82-1.89 (m, 2H, -OCH2CH2CH2CH2Br),
1.96-2.03 (m, 2H, -OCH2CH2CH2CH2Br), 2.75 (s, 3H, CH3), 3.63 (t, 2H, J = 6.6 Hz,
OCH2CH2CH2Br), 4.02 (t, 2H, J = 6.2 Hz, OCH2CH2CH2Br), 6.97 (d, 2H, J = 8.8 Hz,
Ar-H), 7.52 (d, 2H, J = 8.4 Hz, Ar-H), 7.72 (d, 2H, J = 8.4 Hz, Ar-H), 8.21 (d, 2H, J =
8.8 Hz, Ar-H), 8.38 (s, 1H, 6-H), 8.78 (s, 1H, NH); Anal Calcd. for C22H21Br2N5O
(531.24): C, 49.74; H, 3.98; N, 13.18; Found: C, 50.00; H, 4.11; N, 12.88.
4.1.6.9.
N-(4-(3-Bromopropoxy)phenyl)-6-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6i)
o
1
White solid; yield 17%; mp 209-210 C; IR (KBr, cm-1): 3432 (NH), 1583 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.23-2.29 (m, 2H, OCH2CH2CH2Br), 2.53 (s,
3H, CH3), 3.69 (t, 2H, J = 6.0 Hz, OCH2CH2CH2Br), 4.10 (t, 2H, J = 5.9 Hz,
OCH2CH2CH2Br), 7.01 (d, 2H, J = 8.0 Hz, C6H4), 7.34 (t, 1H, J=7.4 Hz, C6H5), 7.56 (t,
2H, J = 8.0 Hz, C6H5), 7.72 (s, 2H, Ar-H), 8.21 (d, 2H, J = 8.0 Hz, Ar-H), 8.40 (s, 1H,
C3-H); 9.97 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6, δ = ppm) δ = 26.3, 31.3, 31.9,
65.5, 109.5, 114.7, 120.7, 126.1, 126.6, 129.1, 129.6, 133.6, 134.1, 138.9, 139.4, 154.2,
165.5; Anal Calcd. for C21H20BrN5O (438.32): C, 57.54; H, 4.60; N, 15.98; Found: C,
57.33; H, 4.47; N, 15.81.
4.1.6.10.
N-(4-(4-Bromobutoxy)phenyl)-6-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6j)
o
1
White solid; yield 39%; mp 196-197 C; IR (KBr, cm-1): 3436 (NH), 1581 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.83-1.88 (m, 2H, OCH2CH2CH2CH2Br),
1.95-2.02 (m, 2H, OCH2CH2CH2CH2Br), 2.53 (s, 3H, CH3), 3.62 (t, 2H, J = 6.6 Hz,
OCH2CH2CH2CH2Br), 4.02 (t, 2H, J = 6.2 Hz, OCH2CH2CH2CH2Br), 6.99 (d, 2H, J =
8.0 Hz, Ar-H), 7.34 (t, 1H, J = 8.0 Hz, Ar-H), 7.55 (t, 2H, J = 7.9 Hz, Ar-H), 7.70 (s, 2H,
13
Ar-H), 8.21 (d, 2H, J = 8.0 Hz, Ar-H), 8.40 (s, 1H, 3-H), 9.95 (s, 1H, NH); C NMR
(101 MHz, DMSO-d6, δ = ppm) δ = 26.3, 27.4, 29.1, 34.9, 66.8, 114.6, 120.7, 126.1,
129.1, 133.6, 138.9, 143.1, 143.4, 149.6, 151.9, 152.5, 155.5, 156.8, 165.9; Anal Calcd.
-23-

for C22H22BrN5O (452.35): C, 58.41; H, 4.90; N, 15.48; Found: C, 58.70; H, 4.61; N,
15.22.
4.1.6.11.
1-(4-bromophenyl)-N-(4-(3-bromopropoxy)phenyl)-6-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6k)
o
1
White solid; yield 37%; mp 202-203 C; IR (KBr, cm-1): 3429 (NH), 1581 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.24-2.30 (m, , 2H, OCH2CH2CH2Br), 2.54 (s,
3H, CH3), 3.70 (t, 2H, J = 6.4 Hz, OCH2CH2CH2Br), 4.11 (t, 2H, J = 6.0 Hz,
OCH2CH2CH2Br), 7.02 (d, 2H, J = 8.4 Hz, Ar-H), 7.75 (dd, 4H, J = 8.2 Hz, Ar-H), 8.23
(d, 2H, J = 8.4 Hz, Ar-H), 8.40 (s, 1H, 3-H), 10.03 (s, 1H, NH); Anal Calcd. for
C21H19Br2N5O (517.22): C, 48.77; H, 3.70; N, 13.54; Found: C, 49.10; H, 4.01; N, 13.61.
4.1.6.12.
N-(4-(4-Bromobutoxy)phenyl)-1-(4-bromophenyl)-6-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6l)
o
1
White solid; yield 38%; mp 174-175 C; IR (KBr, cm-1): 3431 (NH), 1580 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.82-1.89 (m, 2H, OCH2CH2CH2CH2Br),
1.96-2.03 (m, 2H, OCH2CH2CH2CH2Br), 2.54 (s, 3H, CH3), 3.63 (t, 2H, J = 6.6 Hz,
OCH2CH2CH2Br), 4.03 (t, 2H, J = 6.2 Hz, OCH2CH2CH2Br), 7.00 (d, 2H, J = 8.8 Hz,
Ar-H), 7.76 (dd, 4H, J = 8.4 Hz, Ar-H), 8.23 (d, 2H, J = 8.8 Hz, Ar-H), 8.50 (s, 1H, 3-H),
10.01 (s, 1H, NH, exchangeable); Anal Calcd. for C22H21BrN5O (531.24): C, 49.74; H,
3.98; N, 13.18; Found: C, 51.02; H, 4.02; N, 13.48.
4.1.6.13.
N-(4-(3-Bromopropoxy)phenyl)-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6m)
o
1
White solid; yield 35%; mp 138-139 C; IR (KBr, cm-1): 3445 (NH), 1614 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.23-2.30 (m, 2H, OCH2CH2CH2Br), 2.46 (s,
3H, 6-CH3), 2.71 (s, 3H, 3-CH3), 3.69 (t, 2H, J = 6.6 Hz, OCH2CH2CH2Br), 4.10 (t, 2H, J
= 6.0 Hz, OCH2CH2CH2Br), 6.98 (d, 2H, J = 8.0 Hz, Ar-H), 7.30 (t, 1H, J = 8.0 Hz, Ar-
H), 7.52 (t, 2H, J = 8.0 Hz, Ar-H), 7.60 (d, 2H, J = 9.0 Hz, Ar-H), 8.19 (d, 2H, J = 8.0
13
Hz, Ar-H), 8.57 (s, 1H, NH); C NMR (101 MHz, DMSO-d6, δ = ppm) δ = 14.8, 26.2,
31.3, 31.9, 65.5, 99.3, 114.3, 120.5, 125.0, 125.6, 129.0, 131.7, 138.9, 142.2, 155.2,
-24-

155.2, 165.1; Anal Calcd. for C22H22BrN5O (452.35): C, 58.41; H, 4.90; N, 15.48; Found:
C, 58.75; H, 5.21; N, 15.36.
4.1.6.14.
N-(4-(4-Bromobutoxy)phenyl)-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6n)
o
1
White solid; yield 36%; mp 138-139 C; IR (KBr, cm-1): 3446 (NH), 1617 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.82-1.89 (m, 2H, OCH2CH2CH2CH2Br),
1.95-2.02 (m, 2H, OCH2CH2CH2CH2Br), 2.47 (s, 3H, 6-CH3), 2.69 (s, 3H, 3-CH3), 3.62
(t, 2H, J = 6.6 Hz, OCH2CH2CH2Br), 4.03 (t, 2H, J = 6.2 Hz, OCH2CH2CH2Br), 6.98 (d,
2H, J = 8.0 Hz, Ar-H), 7.31 (t, 1H, J = 8.0 Hz, Ar-H), 7.51-7.59 (m, 4H, Ar-H), 8.16 (d,
2H, J = 8.0 Hz, Ar-H), 8.79 (bs, 1H, NH);13C NMR (101 MHz, DMSO-d6, δ = ppm) δ =
15.3, 28.0, 29.6, 33.6, 67.4, 101.0, 110.2, 115.2, 121.4, 121.6, 124.1, 126.4, 129.2, 129.3,
131.1, 138.0, 155.0, 155.1; Anal Calcd. for C23H24BrN5O (466.37): C, 59.23; H, 5.19; N,
15.02; Found: C, 59.26; H, 5.27; N, 15.17.
4.1.6.15.
1-(4-Bromophenyl)-N-(4-(3-bromopropoxy)phenyl)-3,6-dimethyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6o)
o
1
White solid; yield 32%; mp 138-139 C; IR (KBr, cm-1): 3443 (NH), 1608 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.24-2.30 (m, 2H, OCH2CH2CH2Br), 2.47 (s,
3H, 6-CH3), 2.71 (s, 3H, 3-CH3), 3.70 (t, 2H, J = 6.6 Hz, OCH2CH2CH2Br), 4.10 (t, 2H, J
= 5.8 Hz, OCH2CH2CH2Br), 6.99 (d, 2H, J = 8.4 Hz, Ar-H), 7.59 (d, 2H, J = 8.8 Hz, Ar-
H), 7.72 (d, 2H, J = 8.8 Hz, Ar-H), 8.21 (d, 2H, J = 8.4 Hz, Ar-H), 8.62 (s, 1H, NH);
Anal Calcd. for C22H21Br2N5O (531.24): C, 49.74; H, 3.98; N, 13.18; Found: C, 49.86; H,
4.05; N, 13.32.
4.1.6.16.
N-(4-(4-Bromobutoxy)phenyl)-1-(4-bromophenyl)-3,6-dimethyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (6p)
o
1
White solid; yield 30%; mp 148-147 C; IR (KBr, cm-1): 3438 (NH), 1614 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.82-1.89 (m, 2H, OCH2CH2CH2CH2Br),
1.96-2.03 (m, 2H, OCH2CH2CH2CH2Br), 2.46 (s, 3H, 6-CH3), 2.70 (s, 3H, 3-CH3), 3.63
(t, 2H, J = 6.6 Hz, OCH2CH2CH2CH2Br), 4.02 (t, 2H, J = 6.2 Hz, OCH2CH2CH2CH2Br),
-25-

6.97 (d, 2H, J = 8.4 Hz, Ar-H), 7.57 (d, 2H, J = 8.8 Hz, Ar-H), 7.72 (d, 2H, J = 8.8 Hz,
Ar-H), 8.21 (d, 2H, J = 8.4 Hz, Ar-H), 8.61 (s, 1H, NH); Anal Calcd. for C23H23Br2N5O
(545.27): C, 50.66; H, 4.25; N, 12.84; Found: C, 50.36; H, 4.19; N, 13.06.
4.1.7. General procedure for preparation of N-(4-(4-bromobutoxy)phenyl)-1-(4-
bromophenyl)-3-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (13a,b)
To a solution of the appropriate compounds 12a,b (0.001 mol) in absolute
ethanol (15 mL), 1,4-dibromobutane (1.079 g, 0.005 mol), and sodium hydroxide (0.040
g, 0.001 mol) were added and the reaction mixture was heated under reflux for 15 min.
then another 0.001 mol of sodium hydroxide was added and the reflux was continued to
another 30 min. until the starting material was consumed. The reaction mixture was
cooled, filtered and washed with ethanol (3x5 mL) and n-hexane (3x5 mL). The isolated
product was then ercrystallized from acetonitrile and further purification was achieved
by flash chromatography on silica gel (benzene).48-50
4.1.7.1.
N-(4-(4-bromobutoxy)phenyl)-1-(4-bromophenyl)-3-(methylthio)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (13a)
o
1
White solid; yield 83%; mp 154-155 C; IR (KBr, cm-1): 3387 (NH), 1612 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.84-1.87 (m, 2H, OCH2CH2CH2CH2Br),
1.97-2.01 (m, 2H, OCH2CH2CH2CH2Br), 2.75 (s, 3H, SCH3), 3.63 (t, 2H, J = 6.6 Hz,
OCH2CH2CH2CH2Br), 4.03 (t, 2H, J = 6.4 Hz, OCH2CH2CH2CH2Br), 6.97 (d, 2H, J =
8.8 Hz, Ar-H), 7.55 (d, 2H, J = 9.2 Hz, Ar-H), 7.75 (d, 2H, J = 9.2 Hz, Ar-H), 8.20 (d,
2H, J = 8.8 Hz, Ar-H), 8.44 (s, 1H, 6-H), 8.55 (s, 1H, NH); Anal Calcd. for
C22H21Br2N5OS (563.31): C, 46.91; H, 3.76; N, 12.43; Found: C, 47.08; H, 3.80; N,
12.67.
4.1.7.2. N-(4-(4-bromobutoxy)phenyl)-1-(4-bromophenyl)-6-methyl-3-(methylthio)-
1H-pyrazolo[3,4-d]pyrimidin-4-amine (13b)
o
1
White solid; yield 49%; mp 148-149 C; IR (KBr, cm-1): 3336 (NH), 1620 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.82-1.89 (m, 2H, OCH2CH2CH2CH2Br),
1.94-2.02 (m, 2H, OCH2CH2CH2CH2Br), 2.51 (s, 3H, 6-CH3), 2.73 (s, 3H, SCH3), 3.63
-26-

(t, 2H, J = 6.6 Hz, OCH2CH2CH2CH2Br), 4.03 (t, 2H, J = 6.2 Hz, OCH2CH2CH2CH2Br),
6.97 (d, 2H, J = 8.8 Hz, Ar-H), 7.60 (d, 2H, J = 9.2 Hz, Ar-H), 7.76 (d, 2H, J = 8.8 Hz,
Ar-H), 8.21 (d, 2H, J = 9.2 Hz, Ar-H), 8.41 (s, 1H, NH, exchangeable ); Anal Calcd. for
C23H23Br2N5OS (577.33): C, 47.85; H, 4.02; N, 12.13; Found: C, 48.01; H, 4.08; N,
12.40.
4.1.8. General procedure for preparation of 4-(1-aryl-1H-pyrazolo[3,4-d]pyrimidin-
4-yl)amino)phenoxy)alkyl nitrate (7a-p, 14a,b)
A solution of the appropriate bromoalkyl derivatives 6a-p or 13a,b (0.001 mol)
in acetonitrile (5 mL) was treated portion wise with a solution of AgNO3 (0.339 g,
0.002 mol) in acetonitrile (5 mL) and the resulting mixture was heated under reflux for
8 h. the reaction mixture was then filtered, evaporated to dryness to yield the crude
product which was purified by flash chromatography on silica gel (n-hexane/AcOEt 9:1
for 7a-p and benzene for 14a,b).20, 50-51
4.1.8.1.
3-(4-((1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)phenoxy)propyl
nitrate (7a)
o
1
White solid; yield 50%; mp 176-177 C; IR (KBr, cm-1): 3431(NH), 1638 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.16 (p, 2H, J = 6.3 Hz, OCH2CH2CH2ONO2),
4.09 (t, 2H, J = 6.1 Hz, OCH2CH2CH2ONO2), 4.71 (t, 2H, J = 6.4 Hz,
OCH2CH2CH2ONO2), 7.00 (d, 2H, J = 8.0 Hz, Ar-H), 7.36 (t, 1H, J = 7.4 Hz, Ar-H),
7.56 (t, 2H, J = 7.8 Hz, Ar-H), 7.70 (d, 2H, J = 8.0 Hz, Ar-H), 8.20 (d, 2H, J = 8.0 Hz,
13
Ar-H), 8.47 (s, 1H, 6-H), 8.50 (s, 1H, 3-H), 10.11 (s, 1H, NH); C NMR (101 MHz,
DMSO-d6, δ = ppm) δ = 26.3, 64.2, 71.0, 102.5, 115.1, 121.2, 126.7, 129.6, 132.3, 134.2,
139.2, 145.7, 153.5, 156.4, 156.6, 158.5; HRESI-MS m/z calcd for [M+H]+ C20H19N6O4:
407.1462, found: 407.1448.
4.1.8.2.
4-(4-((1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)phenoxy)butyl
nitrate (7b)
o
1
Yellow solid; yield 81%; mp 171-172 C; IR (KBr, cm-1): 3432 (NH), 1620 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.80–1.87 (m, 4H, OCH2CH2CH2CH2ONO2),
-27-

4.03 (t, 2H, J = 6.0 Hz, OCH2CH2CH2CH2ONO2), 4.61 (t, 2H, J = 6.0 Hz,
OCH2CH2CH2CH2ONO2), 6.99 (d, 2H, J = 8.0 Hz, Ar-H); 7.37 (t, 1H, J = 8.0 Hz, Ar-H),
7.56 (t, 2H, J = 8.0 Hz, Ar-H), 7.68 (s, 2H, Ar-H), 8.20 (d, 2H, J = 8.0 Hz, Ar-H); 8.47
(s, 1H, 6-H), 8.50 (s, 1H, 3-H); 10.11 (s, 1H, NH); HRESI-MS m/z calcd for [M+H]+
C21H21N6O4: 421.1619, found: 421.1604.
4.1.8.3.
3-(4-((1-(4-Bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenoxy)propyl nitrate (7c)
o
1
White solid; yield 68%; mp 200-201 C; IR (KBr, cm-1): 3435 (NH), 1632 (C=N); H
NMR (300 MHz, DMSO-d6, δ = ppm) δ = 2.14-2.18 (m, 2H, OCH2CH2CH2ONO2), 4.09
(t, 2H, J = 6.0 Hz, OCH2CH2CH2ONO2), 4.71 (t, 2H, J = 6.3 Hz, OCH2CH2CH2ONO2),
7.00 (d, 2H, J = 9.0 Hz, Ar-H), 7.68 (d, 2H, J = 9.0 Hz, Ar-H), 7.75 (d, 2H, J = 9.0 Hz,
Ar-H), 8.22 (d, 2H, J = 9.0 Hz, Ar-H), 8.48 (s, 2H, 6-H & 3-H), 10.12 (s, 1H, NH,
exchangeable); Anal Calcd. For C20H17BrN6O4 (485.29): C, 49.50; H, 3.53; N, 17.32;
Found: C, 49.17; H, 3.56; N, 17.26;
4.1.8.4.
4-(4-((1-(4-Bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenoxy)butyl nitrate (7d)
o
1
White solid; yield 30%; mp 176-177 C; IR (KBr, cm-1): 3427 (NH), 1622 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.82-1.86 (m, 4H, OCH2CH2CH2CH2ONO2),
4.03 (t, 2H, J = 5.6 Hz, OCH2CH2CH2ONO2), 4.61 (t, 2H, J = 5.6 Hz,
OCH2CH2CH2ONO2), 7.00 (d, 2H, J = 8.0 Hz, Ar-H), 7.56-7.73 (m, 2H, Ar-H), 7.77 (d,
2H, J = 8.0 Hz, Ar-H), 8.23 (d, 2H, J = 8.0 Hz, Ar-H), 8.49 (s, 1H, 6-H), 8.55 (s, 1H, 3-
H), 10.16 (s, 1H, NH); Anal Calcd. for C21H19BrN6O4 (499.32): C, 50.51; H, 3.84; N,
16.83; Found: C, 50.38; H, 3.54; N, 17.08.
4.1.8.5.
3-(4-((3-Methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenoxy)propyl nitrate (7e)
o
Yellow solid; yield 76%; mp 130-131 C; IR (KBr, cm-1): 3418 (NH), 1618 (C=N); 1H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.16 (p, 2H, J = 6.3 Hz, OCH2CH2CH2ONO2),
2.76 (s, 3H, 3-CH3), 4.09 (t, 2H, J = 6.1 Hz, OCH2CH2CH2ONO2), 4.71 (t, 2H, J = 6.4
-28-

Hz, OCH2CH2CH2ONO2), 6.98 (d, 2H, J = 8.0 Hz, Ar-H), 7.31 (t, 1H, J = 7.4 Hz, Ar-H),
7.51-7.57 (m, 4H, Ar-H), 8.18 (d, 2H, J = 8.0 Hz, Ar-H), 8.36 (s, 1H, 6-H), 8.73 (s, 1H,
NH); 13C NMR (101 MHz, DMSO-d6, δ = ppm) δ = 14.8, 26.3, 64.2, 71.0, 101.1, 114.3,
120.5, 125.6, 125.8, 129.1, 131.5, 138.7, 142.4, 154.1, 155.3, 155.8, 155.9; HRESI-MS
m/z calcd for [M+H]+ C21H21N6O4: 421.1619, found: 421.1613.
4.1.8.6.
4-(4-((3-Methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenoxy)butyl nitrate (7f)
o
1
Yellow solid; yield 81%; mp 144-145 C; IR (KBr, cm-1): 3398 (NH), 1626 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.79-1.87 (m, 4H, OCH2CH2CH2CH2ONO2),
2.75 (s, 3H, 3-CH3), 4.02 (t, 2H, J = 6.0 Hz, OCH2CH2CH2CH2ONO2), 4.60 (t, 2H, J =
6.0 Hz, OCH2CH2CH2CH2ONO2), 6.96 (d, 2H, J = 8.0 Hz, Ar-H), 7.31 (t, 1H, J = 8.0
Hz, Ar-H), 7.51-7.55 (m, 4H, Ar-H), 8.18 (d, 2H, J = 8.0 Hz, Ar-H), 8.36 (s, 1H, 6-H),
13
8.72 (s, 1H, NH); C NMR (101 MHz, DMSO-d6, δ = ppm) δ = 14.8, 23.0, 25.0, 67.0,
73.6, 101.1, 114.2, 120.5, 125.6, 125.8, 129.1, 131.3, 138.7, 142.4, 154.1, 155.6, 155.8,
156.0; HRESI-MS m/z calcd for [M+H]+ C22H23N6O4: 435.1775, found: 435.1767.
4.1.8.7.
3-(4-((1-(4-Bromophenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenoxy)propyl nitrate (7g)
o
1
Orange solid; yield 50%; mp 202-203 C; IR (KBr, cm-1): 3443 (NH), 1622 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.15-2.18 (m, 2H, OCH2CH2CH2ONO2), 2.76
(s, 3H, 3-CH3), 4.09 (t, 2H, J = 6.2 Hz, OCH2CH2CH2ONO2), 4.72 (t, 2H, J = 6.4 Hz,
OCH2CH2CH2ONO2), 6.99 (d, 2H, J = 8.8 Hz, Ar-H), 7.54 (d, 2H, J = 8.8 Hz, Ar-H),
7.73 (d, 2H, J = 8.4 Hz, Ar-H), 8.21 (d, 2H, J = 8.4 Hz, Ar-H), 8.39 (s, 1H, 6-H), 8.80 (s,
1H, NH); Anal Calcd. for C21H19BrN6O4 (499.32): C, 50.51; H, 3.84; N, 16.83; Found: C,
50.23; H, 3.88; N, 16.75.
4.1.8.8.
4-(4-((1-(4-Bromophenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)amino)phenoxy)butyl nitrate (7h)
o
1
Yellow solid; yield 91%; mp 194-195 C; IR (KBr, cm-1): 3404 (NH), 1618 (C=N); H
NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.79-1.90 (m, 4H, OCH2CH2CH2CH2ONO2),
-29-