Synthesis, anticancer evaluation, and molecular docking studies of some novel 4,6-disubstituted pyrazolo[3,4-d]pyrimidines as cyclin-dependent kinase 2 (CDK2) inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.02.030

A novel series of 4,6-disubstituted pyrazolo[3,4-d]pyrimidines (7–43) bearing various anilines at C-4 position and thiophenethyl or thiopentane moieties at C-6 position have been designed and synthesized by molecular hybridization approach. All the synthesized compounds were evaluated for in vitro CDK2/cyclin E and Abl kinase inhibitory activity as well as anti-proliferative activity against K-562 (chronic myelogeneous leukemia), and MCF-7 (breast adenocarcinoma) cell lines. The structure–activity relationship (SAR) studies revealed that compounds with thiophenethyl group at C-6 with mono-substituted anilines at C-4 exhibited better CDK2 inhibitory activity compared to alkyl group (thiopentane) at C-6 and di-substituted anilines at C-4 of the scaffold. In particular, compounds having 2-chloro, 3-nitro and 4-methylthio aniline groups at C-4 displayed significant enzymatic inhibitory activity against CDK2 with single digit micromolar IC₅₀ values. The in silico molecular docking studies suggested possible binding orientation and the binding energies were in agreement with the observed SAR as well as experimental results. In addition, some of the synthesized compounds showed anti-proliferative effects against K-562 and MCF-7 cancer cell lines with IC₅₀ values in a micromolar range. Thus, the synthesized compounds could be considered as new anticancer hits for further lead optimization.

Full text of DOI:10.1016/j.bioorg.2018.02.030

Accepted Manuscript
Synthesis, anticancer evaluation, and molecular docking studies of some novel
4,6-disubstituted pyrazolo[3,4-d]pyrimidines as cyclin dependent kinase 2
(CDK2) inhibitors
Srinivasulu Cherukupalli, Balakumar Chandrasekaran, Vladimir Krystof,
Rajeshwar Reddy Aleti, Nisar Sayyad, Srinivas Reddy Merugu, Narva Deshwar
Kushwaha, Rajshekhar Karpoormath
PII:
S0045-2068(18)30057-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.02.030
YBIOO 2290
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 January 2018
26 February 2018
27 February 2018
Please cite this article as: S. Cherukupalli, B. Chandrasekaran, V. Krystof, R. Reddy Aleti, N. Sayyad, S. Reddy
Merugu, N. Deshwar Kushwaha, R. Karpoormath, Synthesis, anticancer evaluation, and molecular docking studies
of some novel 4,6-disubstituted pyrazolo[3,4-d]pyrimidines as cyclin dependent kinase 2 (CDK2) inhibitors,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.02.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, anticancer evaluation, and molecular docking studies of some novel
4,6-disubstituted pyrazolo[3,4-d]pyrimidines as cyclin dependent kinase 2
(CDK2) inhibitors
Srinivasulu Cherukupalli1, Balakumar Chandrasekaran1, Vladimir Krystof2, Rajeshwar Reddy
Aleti1, Nisar Sayyad1, Srinivas Reddy Merugu1, Narva Deshwar Kushwaha, Rajshekhar
Karpoormath 1*.
1Department of Pharmaceutical Chemistry, College of Health Sciences, University of KwaZulu-
Natal, Durban 4000, South Africa.
2Laboratory of Growth Regulators, Centre of the Region Hana for Biotechnological and
Agricultural Research, Palacky University & Institute of Experimental Botany ASCR, Slechtitelu
27, 78371 Olomouc, Czech Republic.
3Dept. of Pharmaceutical Chemistry, Discipline of Pharmaceutical Sciences, College of Health
Sciences, University of KwaZulu-Natal, Durban South Africa
*Corresponding Author
Dr. Rajshekhar Karpoormath
Tel: + 27-312607179, Fax: + 27-312607792
Email: karpoormath@ukzn.ac.za
1

Abstract
A novel series of 4,6-disubstituted pyrazolo[3,4-d]pyrimidines (7-43) bearing various anilines
at C-4 position and thiophenethyl or thiopentane moieties at C-6 position have been designed and
synthesized by molecular hybridization approach. All the synthesized compounds were evaluated
for in vitro CDK2/cyclin E and Abl kinase inhibitory activity as well as anti-proliferative activity
against K-562 (chronic myelogeneous leukemia), and MCF-7 (breast adenocarcinoma) cell lines.
The structure-activity relationship (SAR) studies revealed that compounds with thiophenethyl
group at C-6 with mono-substituted anilines at C-4 exhibited better CDK2 inhibitory activity
compared to alkyl group (thiopentane) at C-6 and di-substituted anilines at C-4 of the scaffold. In
particular, compounds having 2-chloro, 3-nitro and 4-methylthio aniline groups at C-4 displayed
significant enzymatic inhibitory activity against CDK2 with single digit micro molar IC50 values.
The in silico molecular docking studies suggested possible binding orientation and the binding
energies were in agreement with the observed SAR as well as experimental results. In addition,
some of the synthesized compounds indicated anti-proliferative effects against K-562 and MCF-7
cancer cell lines with IC50 values in a micro molar range. Thus, the synthesized compounds could
be considered as new anticancer hits for further lead optimization.
Key words: Pyrazolo[3,4-d]pyrimidine; cyclin dependent kinase inhibitor; Anti-proliferative
activity; Molecular docking; GLIDE.
1. Introduction
Cancer is an enormous global health burden, affecting almost every region and socio-economic
level. It is the second leading cause of death worldwide, accounted for 8.8 million deaths in 2015
and nearly 1 in 6 of all global deaths.1 The new cancer cases are expected to increase to 15 million
per year by 2020, according to the World Health Organization (WHO), unless further
precautionary measures are followed.2 Wide efforts are being carried out in order to discover new
treatment approaches as well as to improve prevention and molecular diagnostic systems.3,4 It is
becoming noteworthy to investigate new druggable molecular targets, identify and develop their
modulators as novel drugs for the treatment of cancer. Amongst others, protein kinases have
2

become an important group of drug targets and number of kinase inhibitors in clinical development
is rapidly increasing.5
Cyclin-dependent kinases (CDKs) are a group of serine/threonine kinases comprising 20
members, of which some are linked with regulation of cell-cycle progression by phosphorylating
proteins involved in cell division. For example, formation of active complex composed of CDK2
and cyclin E enables pRb phosphorylation, activation of transcription factor E2F which initiates
S phase of the cell cycle.6 CDK2 then also associates with cyclin A, governing continuous DNA
replication and properly programed deactivation of E2F. Deregulations of CDKs or cyclins, as
well as the loss of endogenous inhibitory proteins, result in abrogation of cell cycle control,
which is connected with development of tumors. Thus CDKs are considered important targets for
anticancer drugs.7,8 The lack of clarity as to which CDK is the most suitable drug target followed
by poor selectivity hindered the clinical development of specific CDK inhibitors.9
Initially, the importance of CDK2 as a drug target for cancer therapy was in question since
CDK2 knockdown investigations failed to block cell proliferation in a number of tumour cell
lines,10 and by mouse knockout investigations where the animals were viable.11,12 Current
investigations employing a chemical genetic method in which CDK2 countenance was
maintained, but enzymatic activity was inhibited, affords interesting sign that CDK2 is a valid
anticancer drug target.6 Up to date many CDK2 inhibitors have been developed and some of
them (including roscovitine, CYC065, dinaciclib, AT7519, milciclib) undergo clinical
evaluation.13,14 CDK2 inhibitors are also anticipated to have efficacy in combinations with other
drugs or where synthetic lethality can be recognized.15 Recent study proved that a combination
of phosphatidylinositol-3-kinase and CDK2 inhibitors induced apoptosis in malignant glioma
xenografts via synthetic-lethal interaction.16 CDK2 was proved as a therapeutic target in BRCA-
deficient cancers,17 ovarian cancer.18
Fused pyrimidines have received a great deal of attention due to their immense role as
active pharmacophores.19 The pyrazole annulated on the pyrimidine scaffold leads to
pyrazolopyrimidine, which can be looked upon as the bio-isostere of purine.20 The
pharmacological significance of purine nucleus is well established. Oxypurinol and its congeners
allopurinol and thiopurinol (Tisopurine) which contains pyrazolo[3,4-d]pyrimidine scaffold
3

inhibit xanthine oxidase enzyme and interfere with the biosynthesis of uric acid, further
allopurinol acting as a causative agent for gout.21 Ibrutinib/PCI-32765 was recently approved by
the US FDA for the treatment of mantle cell lymphoma, chronic lymphocytic leukemia and
Waldenstrom's macroglobulinemia diseases (Figure 1).22
Figure 1. Structures of active drugs containing fused pyrimidine.
Pyrazolo[3,4-d]pyrimidine is also reported to encompass biological potential as
anticancer, antiviral, antimicrobial, herbicidal, CNS agents (phosphodiesterase 9 and
benzodiazepine receptors), anti-inflammatory and cardiovascular activities.23 Figure 2 depicts
several reported analogs of pyrazolo[3,4-d]pyrimidine with anti-cancer activities related to
inhibition of various protein kinases.24-29
Based on the above mentioned facts and in continuation of our research work on
anticancer drug discovery, we envisaged to further exploit the pyrazolo[3,4-d]pyrimidine
scaffold to synthesize novel CDK2 inhibitors.30,31 Thus a series of 4,6-disubstituted pyrazolo[3,4-
d]pyrimidine was synthesized with a design strategy: a) bioisosteric replacement of purine
nucleus, b) incorporation of more lipophilic aromatic amines (C-4) and aliphatic or aromatic
groups (C-6). These derivatives were also evaluated against Abl kinase and two cancer cell lines
K-562 (chronic myelogeneous leukemia) and MCF-7 (breast adenocarcinoma). Further, in silico
molecular docking studies were performed to calculate the binding energies and orientations of
4

these compounds with respect to the active site of CDK-2 protein. The computational results
were in agreement with our experimental observations.
Figure 2. Known derivatives of pyrazolo[3,4-d]pyrimidine and their anticancer activities along
with the designed molecules. A: (Ki50 against Src, AblT315I = 0.056, 0.01 µM);24 B: (IC50
against CDK9 = 17 nM);25 C: (IC50 against CDK2 = 0.5µM);26 D: (Ki50 against Abl = 80 nM);27
E: (Ki50 against cSrc, Abl = 0.21± 0.02, 0.15±0.02 µM);28 F: (IC50 against Src = 1.2 ± 0.4 µM).29
5

2. Results and discussion
2.1. Chemistry
The synthesis of novel series of pyrazolo[3,4-d]pyrimidine hybrids was achieved through an
efficient and versatile synthetic route as depicted Scheme 1. On the pyrazolopyrimidine scaffold,
chloro group was introduced at C-4 as a good leaving group, which acted as a most reactive site
for different nucleophiles, while thiophenethyl or thiopentane groups were introduced at C-6
position. With a view to prepare the target hybrid molecules (7-43), the key intermediates 4-
chloro-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidine
(6a),
4-chloro-6-(pentylthio)-1H-
pyrazolo[3,4-d]pyrimidine (6b) were synthesized from ethoxymethylene malononitrile (1) in the
sequence as illustrated in Scheme 1.
From the earlier reported literature methods compound 4 was successfully synthesized.32
Further under microwave irradiation alkylation of compound 4 was achieved by reacting with 2-
chloroethyl benzene in presence of anhydrous potassium carbonate in N,N-dimethylformamide
(DMF) to obtain 6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-ol (5a). Similarly reacting 4
with 1-bromopentane in 1M NaOH solution at 70 C resulted in 6-(pentylthio)-1H-pyrazolo[3,4-
d]pyrimidin-4-ol (5b) in moderate yield. Chlorination of 5a and 5b was easily achieved with
Vilsmeier complex (POCl3:DMF) to obtain the halogenated key intermediates 4-chloro-6-
(phenethylthio/pentylthio)-1H-pyrazolo[3,4-d]pyrimidine that are 6a and 6b. As displayed in
Scheme 1, the synthesis of the final hybrid compounds (7-43) in good yields (75-95%) was
1
13
achieved by nucleophilic substitution with various primary amines at C-4. The IR, H and C
NMR spectroscopic data of all the novel compounds were in agreement with the predicted
structures, which were further validated by the HR-MS data (Supporting information).
6

Scheme 1: Synthetic exploration for the preparation of 4,6-disubstituted pyrazolo[3,4-
d]pyrimidine analogues (7-43).
Reagents and conditions: (a) hydrazine hydrate, ethanol, 80 C, 3h, 92%; (b) Conc. H2SO4,
NH4OH, H2O, 50 C, 5h, 90%; (c) potassium ethyl xanthogenate, DMF, 120 C, 6h, 82%; (d) 2-
chloroethyl benzene, K2CO3, DMF, 70 C, microwave, 20 min, 76%; (e) 1-bromopentane,
NaOH, H2O, glacial acetic acid, 50 C, 5h, RT, overnight, 80%; (f) POCl3, DMF, 80 C, 2h,
85%; (g) R-NH2, EtOH, 80 C, 2h, 75-95%.
The 1H NMR of compound 4 exhibited very distinct singlet signals resonating at δ 13.61, 13.03,
11.86 and 8.42 ppm, which were attributed to the N-H, S-H, O-H and Ar-H protons of the
pyrazole ring. Thus, indicating the formation of the fused pyrazolopyrimidine by ring annulation
7

of 5-amino-1H-pyrazole-4-carboxamide (3) with potassium ethyl xanthogenate. For compounds
5a and 5b, the distinctive methylene signals (Ph-CH2-CH2-S- and -S-CH2-CH2-CH2-CH2-)
appeared around δ 3.43-3.39, 3.01-2.97 ppm and δ 3.17-2.87, 1.73-1.43, 1.41-1.14 respectively,
while the methyl peak for 5b was observed at δ 0.86-0.80 ppm. In particular, the disappearance
of a distinct singlet signal at around δ 13.03 ppm for mercapto (-SH) group evidently indicated
the successful alkylation of pyrimidine scaffold. Whereas the most characteristic singlet, doublet
and triplet signals at δ 7.32-7.20 ppm were attributed to the aromatic protons (C6H5-CH2-CH2-S-
) at C-6 of the pyrimidine ring. Furthermore, the absence of characteristic singlet signal at around
δ 12.22-12.28 ppm for (-OH) at C-4 of pyrimidine ring confirmed the formation of halogenated
(-Cl) key intermediates 4-chloro-6-(phenethylthio/pentylthio)-1H-pyrazolo[3,4-d]pyrimidine (6a
and 6b). These findings corroborated with their respective 13C NMR, where the methylene
carbon peaks at δ 34.61, 31.08 ppm (C6H5-CH2-CH2-) and δ 30.61-30.37, 30.16-29.69, 29.22-
28.11, 21.78-21.73 ppm (-S-CH2-CH2-CH2-CH2-) were assigned to 5a and 5b. The single
methyl peak (5b) appeared around 13.90-13.83 ppm while the prominent aromatic signals (5a
and 5b) resonated around δ 167.89-109.79 ppm.
The IR spectra of the title compounds 7-43 displayed a reasonably sturdy and
characteristic bands around 2921-3033 cm-1, 1270-1300 cm-1 accounting for N-H and C-S
stretching respectively, while the most prominent band of C-N appearing around 1180-1184 cm-1
indicated the formation of final hybrid molecules. Further, the prominent 1H NMR signals of the
title compounds 7-43 resonated around δ 13.39-10.28 ppm (exocyclic-N-H), δ 9.94-7.75 ppm
(ring-N-H) and 10.02-8.19 ppm (C-3 aromatic proton) while, the singlet or multiplet aromatic
proton peaks appeared around δ 8.47-6.84 ppm. For compounds 7-25 the methylene protons (-S-
CH2-CH2-Ph) were observed around δ 3.43-2.81 ppm while for compounds 26-43 the signals (-
S-CH2-CH2-CH2-CH2-) appeared around δ 3.10-1.20 ppm. The methyl protons for 26-43
resonated around δ 0.87-0.80 ppm. Further the methylthio (-Ph-SCH3), methyl (Br-Ph-CH3),
methoxy (Ph-OCH3) and ethynyl-H protons resonated around δ 2.47-2.44 ppm, δ 2.30-2.18 ppm,
δ 3.77-3.71 ppm and 4.18 ppm respectively. The 13C NMR spectra further confirmed the
structures of the title compounds. The characteristic carbon signals C-6, C-4 and C-3 of the
pyrimidine ring were observed around δ 167.41-166.40, 155.73-153.11 and 132.88-130.56 ppm,
while the various aromatic/heteroaromatic carbons resonated between δ 153.62ppm and 98.12
ppm. Furthermore, the prominent carbon signals observed around δ 55.66-55.61 ppm δ 21.78-
8

17.74 ppm and 30.17-15.31 ppm were attributed to the methoxy (-OCH3), methyl and
thiomethyl carbons respectively. Further, for compounds 7-25 the methylene carbon peaks (-S-
CH2-CH2-Ph) were observed around δ 35.51-31.08 ppm while for compounds 26-43 the signals
(-S-CH2-CH2-CH2-CH2-) appeared around δ 30.62-21.73 ppm and the methyl peak resonated
around δ 13.89-13.83 ppm. In addition, the HR-MS spectra of the title compounds (7-43)
displayed accurate molecular ion peaks, which were in agreement with their expected molecular
weights (supporting information).
2.2. In vitro evaluation of CDK2 and Abl kinase inhibition
All the final compounds were evaluated for CDK2/cyclin E kinase inhibition and the IC50 values
of various in vitro anticancer profiles of the final compounds are summarized in Table 1. Abl
kinase inhibition was evaluated as a counter screen, to get a preliminary information about
selectivity.
Table 1. Anticancer, CDK2 and Abl kinase inhibitory evaluation of novel 4,6-disubstituted
pyrazolo[3,4-d]pyrimidine derivatives.
Code
R
IC50 (µM)a
Abl
CDK2/Cyclin E
>12.5
7.8
K-562
19.9
MCF-7
19.2
Ph
2-ClPh
>12.5
>12.5
>25
7
>6.25
>25
>6.25
24.0
8
3-ClPh
13.3
9
4-ClPh
>25
>25
>25
>25
10
11
12
13
14
15
16
17
18
3-NO2Ph
4-NO2Ph
2-BrPh
5.1
>25
24.6
24.3
>12.5
>12.5
>25
>12.5
>12.5
>25
>6.25
>6.25
27.4
>6.25
>6.25
23.9
3-BrPh
4-BrPh
>25
>25
>50
>50
2,4-(CH3)2Ph
SCH3Ph
4-Cl-3-CF3Ph
>12.5
>25
>12.5
>25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
13.4
>25
9

4-Br-3-CF3Ph
4-Cl-3-NO2Ph
4-F-2-CH3Ph
3-Br-4-CH3Ph
2-Cl-4-FPh
3,4-(OCH3)Ph
4-(ethynyl)Ph
Ph
>25
>25
>25
>25
>6.25
>6.25
>6.25
>6.25
>12.5
>6.25
>12.5
>100
>25
>6.25
>6.25
>6.25
>6.25
>12.5
>6.25
>12.5
>100
>25
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
>12.5
>25
>12.5
>25
>12.5
15.9
>25
>12.5
>12.5
>25
>50
>50
2-ClPh
8.7
>12.5
>25
3-ClPh
>25
>12.5
>6.25
>12.5
>6.25
>12.5
>6.25
>6.25
>12.5
>6.25
>12.5
>6.25
>12.5
>12.5
>6.25
>6.25
>6.25
42
>12.5
>6.25
>12.5
>6.25
>12.5
>6.25
>6.25
>12.5
>6.25
>12.5
>6.25
>12.5
>12.5
>6.25
>6.25
>6.25
11
4-ClPh
>12.5
17.7
>25
>12.5
>25
3-NO2Ph
4-NO2Ph
>25
2-BrPh
>25
>25
3-BrPh
>25
>25
4-BrPh
>25
>25
2,4-(CH3)2Ph
SCH3Ph
>25
>25
8.8
>25
4-Cl-3-CF3Ph
4-Br-3-CF3Ph
4-Cl-3-NO2Ph
4-F-2-CH3Ph
3-Br-4-CH3Ph
2-Cl-4-FPh
3,4-(OCH3)Ph
Roscovitine
Imatinib
>25
>25
>25
>25
>25
>25
>12.5
>25
>12.5
>25
>12.5
>12.5
0.1
>12.5
>12.5
>100
0.2
>100
0.5
>10
a IC50 values were determined in triplicate in the range of 0.05 to 100 µM. IC50 value indicates concentration (µM)
that inhibits activity of the tested enzyme to 50%. For cytotoxic assays, IC50 means the concentration (μM) that
inhibits the growth of 50% of cells during a three-day cultivation
10

Some synthesized compounds displayed an activity in the single digit micro molar range
against CDK2/cyclin E. Fascinatingly, it was observed that some compounds containing
thiophenethyl group at C-6 displayed prominent anticancer activity (7, 9, 11, 14) as compared to
compounds 26-43 having thiopentane group. It was also observed that incorporation of
phenyl/substituted phenyl groups at C-4 of the pyrazolo[3,4-d]pyrimidine nucleus was essential
for CDK2 activity. From the tested thiophenethyl series the highest CDK2 inhibitory activity was
recorded for compounds 11 (IC50 = 5.1 µM) and 8 (IC50 = 7.8 µM) with 3-nitroaniline and 2-
chloroaniline group at C-4 respectively. Notable significant inhibition was also observed for
compounds bearing 3-chloroaniline (9; IC50 = 13.3 µM), 4-chloro-3-trifluoromethylaniline (18;
IC50 = 13.4 µM) and 3,4-dimethoxyaniline (24; IC50 = 15.9 µM). For the remaining compounds
of this thiophenethyl series, IC50 values could not be measured due to a solubility limit (IC50
value >12.5 or >25 µM).
Similarly, some thiopentane derivatives (series 26-43) also exhibited a reasonable activity
profile with IC50 starting from 8.7 µM. From this series, compounds with 2-chloroaniline (27)
and 4-methylthioaniline (36) showed the best activity with IC50 8.7 µM and 8.8 µM,
respectively; this was followed by 3-nitroaniline derivative (30) with IC50 17.7 µM. The
remaining compounds in this series were less soluble and their IC50 were not achieved. In
addition, all compounds (7-43) were also evaluated against Abl kinase but none of the
compounds showed any inhibition in the assayed concentration range, confirming reasonable
selectivity towards CDK2 over unrelated Abl.
2.3. Anti-proliferative activity against K-562 and MCF-7 cell lines
All the newly synthesized 4,6-disubstituted pyrazolo[3,4-d]pyrimidine analogues 7-43 were
further evaluated for their in vitro anti-proliferative activity against K-562 (chronic
myelogeneous leukemia) and MCF-7 (breast adenocarcinoma) cell lines. Several compounds are
displaying appreciable activity with measurable IC50 values against the two cell lines, such as
compounds 7 (IC50 = 19.9, 19.2 µM), 9 (IC50 = >25, 24 µM), 11 (IC50 = 24.6, 24.3 µM), and 14
(IC50 = 27.4, 23.9 µM). The remaining compounds were not active in the tested concentration
range.
11

2.4. Structure-activity relationship (SAR) studies
In general, a careful observation of the structure-activity relationship (SAR) indicated that the
CDK inhibitory activity was considerably affected by the nature of various substituents present
at C-4 (aromatic ring) and C-6 positions on pyrazolo[3,4-d]pyrimidine scaffold. From the two
series of compounds (phenethyl and pentane at C6), the phenethyl series gave most number of
active compounds (8, 9, 11, 18 and 24), while in pentane series only three molecules (27, 30 and
36) were active. Further analysis of both the series revealed that compounds 8, 9, 11, 27, 30 and
36 with mono substituted phenyl ring (2-Cl-Ph, 3-Cl-Ph, 3-NO2-Ph, and 4-SCH3-Ph groups) at
C-4 showed better activity than compounds with disubstituted phenyl groups except for
compounds 18 and 24 (4Cl-3CF3-Ph and 3,4-OCH3-Ph). Compound 11 followed by 8, 9, 27, 30
and 36 presented highest inhibitory activity.
Further, no specific activity was observed for all the compounds against Abl kinase (IC50 = >12.5
to >50.0). However, compounds 7, 11 and 14, displayed specific anti-proliferative activity
against K-562 (IC50 = 19.9, 24.6 and 27.4) and MCF-7 (IC50 = 19.2, 24.3 and 23.9), indicating
that mono-substitutions at C4 with phenethyl at C6 was favorable. In summary, compound 11
was most active, however due to low solubility profile for most of the compounds a conclusive
SAR could not be derived. Figure 3 provides a brief overview of the SAR.
Figure 3. SAR study of 4,6-disubstituted pyrazolo[3,4-d]pyrimidines as potent anticancer agents.
12

2.5. Molecular docking study
Computational and bioinformatics tools have become essential part for the design and
development of therapeutically effective novel chemical entity.33 Dinaciclib, a pyrazolo[1,5-
a]pyrimidine compound, displayed higher binding affinity towards CDKs.34 Hence, to further
understand and substantiate our observed experimental data, molecular docking simulation was
performed for the synthesized compounds with the target CDK2 protein.
To validate the docking protocols and to reproduce the reported orientation of R-
roscovitine in the predefined binding site of CDK2 (PDB ID: 2A4L), docking studies were
performed using Glide program of Schrodinger-Maestro 11.2. From the docking results, the pose
of R-roscovitine obtained revealed similar molecular interactions as reported.35 The docked
complex presented characteristic hydrogen bonding (H-bond) interactions with crucial residues
of the active-site, such as Leu83 interacted with roscovitine by forming a strong [C=O with
benzylamino NH (1.91 Å)] and a weak [(C=O with ring nitrogen (2.39 Å)] H-bond. Similarly,
the residue Asp86 interacted with OH group of roscovitine via a strong H-bond (1.67 Å),
whereas Lys89 exhibited π–cation interaction with phenyl ring of the ligand. Figure 4 represents
the reproduced 3D molecular interactions of the docked pose, validating the docking protocols.
Figure 4: Reported pose of Roscovitine (black thin tube model) and docked pose (thick tube
model) into the active-site showing similar interactions (docking validation). Magenta coloured
13

dashed lines indicate hydrogen bonding, while grey-coloured thin tube amino acids are
considered as crucial residues.
The docking experimental data with our synthesized compounds revealed that they fit
well into the binding-site and display favourable interactions with the crucial amino acid
residues. Interestingly, two nitrogen atoms of pyrazole ring of the best active compound 11 (IC50
= 5.1 µM) displayed strong H bond interactions with NH of Leu83 (2.37 Å) and C=O of Glu81
(2.02 Å) respectively, signifying these H bond interactions were crucial for the CDK-2
inhibition. The nitro group of the ligand was in close proximity to Lys89 presenting weak vdW
interaction, whereas the acidic and basic amino acid residues (Lys129, Glu131, Asn132 and
Asp145) surrounded thiophenethyl group. The exocyclic NH and the side chain sulphur
displayed no characteristic interactions with the protein (Figure 5a). In the case of moderately
active compound 27, the ring NH displayed H bond interaction with Leu83 (1.69 Å), while the
exocyclic NH presented weak H bond interaction (2.59 Å) with Asp86. The side chain was
anchored within the catalytic residues Phe80 and Asp145, whereas the pocket consisting of
Val18, Lys33, and Asp145 residues surrounded chlorophenyl ring. While in the case of the least
active compound 30, the orientation of the docked pose was observed to be different with respect
to best and the moderately active compounds (11 and 27). The nitro group showed weak H bond
interactions with the basic residues (Lys33 and Asn 132), which could have contributed to the
altered pose of the ligand. Furthermore the aliphatic side chain occupied the hydrophobic region
(Val64, Phe80, Leu134, and Ala144) resulting in H-bond interactions of the ring and exocyclic
nitrogen with different residues of the active site (Leu83 and Ile10). Figure 5 presents the
different molecular interactions of 11, 27 and 30 with the active site residues of CDK-2 protein
(PDB ID: 2A4L).
14

Figure 5. Molecular interactions of a) best active compound 11 b) moderately active compound
27 c) less active compound 30 into the binding site of CDK2/cyclin E protein. Nonpolar
hydrogens were hidden for clarity and pink dashed lines indicate H bond.
3. Conclusion
In summary, we have successfully synthesized and characterized a new series of pyrazolo[3,4-
d]pyrimidine derivatives 7-43 in good yields. The key intermediates 4-chloro-6-(phenethylthio)-
1H-pyrazolo[3,4-d]pyrimidine (6a), 4-chloro-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidine (6b)
allowed us to generate a library of condensed pyrimidines. All synthesized compounds were
evaluated for their in vitro enzymatic inhibitory activity against CDK2/cyclin E and four
15

compounds (8, 11, 27, and 36) were significantly active with IC50 values ranging from 5.1 µM to
8.8 µM. From the SAR, it was clear that the presence of thiophenethyl group at C-6 with mono-
substituted anilines at C-4 of the pyrazolo[3,4-d]pyrimidine nucleus was essential for anticancer
activity. In addition, the binding energies of the best active compounds were in agreement with
the experimental data and supported the SAR studies. However, the moderate to poor activity
observed for the majority of the compounds could be attributed to the solubility limit and this is
currently being addressed in our laboratory. Thus, these preliminary research findings can
further guide the researchers at large in developing novel pyrazolo[3,4-d]pyrimidine based
CDK2 inhibitors as potential anticancer agents.
4. Experimental Section
All the chemicals used in this research work were purchased from Sigma-Aldrich and Merck
Millipore, South Africa. All the solvents, except those of laboratory-reagent grade, were dried
and purified when necessary according to previously published methods. The progress of the
reactions was monitored by thin-layer chromatography (TLC) on pre-coated silica gel plates
procured from E. Merck and Co. (Darmstadt, Germany) using 36% ethyl acetate in n-hexane as
the mobile phase and iodine vapor as the visualizing agent. Purification of crude compounds
were performed by crystallization using appropriate solvent
and by flash column
chromatography using 100-200 mesh silica gel with Methanol (MeOH) and DCM as solvents.
The melting points of the synthesized compounds were determined using a Thermo Fisher
Scientific (IA9000, UK) digital melting point apparatus and are uncorrected. The IR spectra were
recorded on a Bruker Alpha FT-IR spectrometer (Billerica, MA, USA) using the ATR technique.
1
13
The H NMR and C NMR spectra were recorded on a Bruker AVANCE 400 and 600 MHz
(Bruker, Rheinstetten/Karlsruhe, Germany) spectrometers using CDCl3 and DMSO-d6. The
chemical shifts are reported in δ ppm units with respect to TMS as an internal standard. HR-MS
was recorded on an Autospec mass spectrometer with electron impact at 70 eV.
4.1. Synthesis of 6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-ol (5a)
To a stirred solution of 6-mercapto-1H-pyrazolo[3,4-d]pyrimidin-4-ol (compound 4, 0.3g,
0.00179 mol) in N,N-dimethyl formamide (2 mL), K2CO3 (0.247 g, 0.0017 9 mol) was added and
stirred at room temperature for 10 min. To this constantly stirred reaction mass, 2-chloroethyl
16

benzene (0.28 mL, 0.002 mol) was slowly added dropwise and heated at 80 C for 20 min. in a
microwave reactor at 150 psi. After completion of reaction (monitored on TLC), the reaction
mixture was poured in ice cold water and extracted with dichloromethane (DCM). The extracted
organic layer was dried over anhydrous sodium sulphate and concentrated under reduced
pressure to obtain crude product (dark brown viscous liquid) which was further purified by flash
silica column [MeOH/ DCM, 10:90] to afford the desired compound 5a as light brown solid.
Yield: 76%; mp 210-212 C; FTIR (ATR, cm-1) max: 3022 (NH Str.), 2920 (Ar-H Str. of Pyr.),
1671 (C=O Str.); 1H-NMR (400 MHz, DMSO-d6) δ: 13.59 (s, 1H, NH), 12.22 (s, 1H, OH), 8.03
(s, 1H, ArH), 7.31 (t, J = 2.52 Hz, 4H, ArH), 7.25-7.20 (m, 1H, ArH), 3.41 (t, J = 5.04 Hz, 2H,
13
CH2), 2.99 (t, J = 7.56 Hz, 2H, CH2) ppm; C NMR (100 MHz, DMSO-d6) δ: 158.20, 139.93,
128.73, 128.62, 128.41, 126.41, 102.89, 34.61 (CH2), 31.08 (CH2) ppm.
4.2. Synthesis of 6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-ol (5b)
To a stirred solution of 6-mercapto-1H-pyrazolo[3,4-d]pyrimidin-4-ol (compound 4, 1g, 0.005
mol) in 1M NaOH solution (12 mL), 1-bromopentane (1.48 mL, 0.011 mol) was added dropwise
and heated at 70 C for 6h and later slowly brought to RT and continued stirring for overnight.
After completion of reaction (monitored on TLC), glacial acetic acid was added dropwise to
yield the crude solid, which further washed with petroleum ether and purified by flash silica
column [MeOH/DCM, 05:95] to afford the desired compound 5b as yellow solid. Yield: 80%;
mp 201-203 C; FTIR (ATR, cm-1) max: 3180 (NH Str.), 2953 (Ar-H Str.), 1678 (C=O Str.); 1H-
NMR (400 MHz, DMSO-d6) δ: 13.54 (s, 1H, NH), 12.28 (s, 1H, OH), 7.93 (s, 1H, ArH), 3.15 (t,
J = 7.26 Hz, 2H, CH2), 1.70-1.63 (m, 2H, CH2), 1.39-1.26 (m, 4H, (CH2)2), 0.86 (t, J = 7.08 Hz,
13
3H, CH3) ppm; C NMR (100 MHz, DMSO-d6) δ: 159.49, 157.75, 135.24, 30.37 (CH2), 29.69
(CH2), 28.24 (CH2), 21.66 (CH2), 13.84 (CH3) ppm.
4.3. Synthesis of 4-chloro-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidine(6a)
Vilsmeier-Haack reagent was freshly prepared by the careful addition of POCl3 (3.75 mL, 0.041
mol) in DMF (0.85 mL, 0.011 mol) at 0 °C with constant stirring. To this reaction mixture
(maintained at 0 °C), added 6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-ol (5a, 1g, 0.003
mol) and stirred initially for 30 min and later slowly brought to RT and continued stirring for
another 30 min. Finally, the reaction mixture was allowed to reflux at 80 C for 2 h until the TLC
17

showed full consumption of starting material. The reaction mixture was then poured on ice cold
water and neutralized with 10% NaOH solution. Thus, the obtained precipitate was filtered under
suction and further purified by flash silica column [MeOH/DCM, 05:95] to afford the desired
compound 6a, as light yellow solid. Yield: 85%; mp 218-220 C; FTIR (ATR, cm-1) max: 3208
(NH Str.), 2927 (Ar-H Str.); 1H-NMR (400 MHz, DMSO-d6) δ: 14.24 (s, 1H, NH), 8.30 (s, 1H,
ArH), 7.31 (t, J = 2.48 Hz, 4H, ArH), 7.25-7.19 (m, 1H, ArH), 3.41 (t, J = 7.66 Hz, 2H, CH2),
3.01 (t, J = 7.62 Hz, 2H, CH2) ppm;13C NMR (100 MHz, DMSO-d6) δ: 167.89, 140.07, 128.73,
128.63, 128.39, 126.36, 109.79, 34.58 (CH2), 31.93 (CH2) ppm.
4.4. Synthesis of 4-chloro-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidine (6b)
Vilsmeier-Haack reagent was freshly prepared by the careful addition of POCl3 (4.29 mL, 0.047
mol) in DMF (0.97 mL, 0.012 mol) at 0 °C with constant stirring. To this reaction mixture
(maintained at 0 °C) was added 6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-ol (5b, 1g, 0.004
mol) and stirred initially for 30 min and later slowly brought to RT and continued stirring for
another 30 min. Finally, the reaction mixture was allowed to reflux at 80 C for 2 h until the TLC
showed full consumption of starting material. The reaction mixture was then poured in ice cold
water and neutralized with 10% NaOH solution. Thus, the generated precipitate was filtered
under suction and further purified by flash silica column [MeOH/DCM, 05:95] to afford the
desired compound 6b as light yellow solid. Yield: 85%; mp 194-196 C; FTIR (ATR, cm-1)max
:
1
3095 (NH Str.), 2956 (Ar-H Str.), 1602 , 1556, 1465; H-NMR (400 MHz, DMSO-d6) δ: 14.19
(s, 1H, NH), 8.28 (s, 1H, ArH), 3.16 (t, J = 7.32 Hz, 2H, CH2), 1.73-1.66 (m, 2H, CH2), 1.42-
13
1.27 (m, 4H, (CH2)2), 0.87 (t, J = 7.16 Hz, 3H, CH3) ppm; C NMR (100 MHz, DMSO-d6) δ:
168.23, 109.70, 30.42 (CH2), 30.38 (CH2), 28.11 (CH2), 21.66 (CH2), 13.84 (CH3) ppm.
4.5. General procedure for the synthesis of 6-(phenethylthio/pentylthio)-N-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amines (7-43)
To a well stirred solution of compound 6a or 6b (0.2g, 1Eq.) in absolute ethanol (10 mL), the
appropriately substituted anilines (1.1 Eq) was added and the reaction mixture was refluxed for
2-3 h until TLC showed full consumption of starting materials. The excess of solvent was
evaporated under reduced pressure to yield the crude solids, which were further purified by
recrystallization with methanol to afford the desired title compounds (7-43).
18

4.5.1. 6-(phenethylthio)-N-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (7)
White solid; yield: 90%; mp 226-228 C; FTIR (ATR, cm-1)max: 3363, 3119, 2918, 1621, 1567,
1
1491, 1470, 1301, 1183; H-NMR (400 MHz, DMSO-d6) δ: 13.44 (s, 1H, NH), 10.05 (s, 1H,
ArH), 8.17 (s, 1H, NH), 7.77 (d, J = 7.92 Hz, 2H, ArH), 7.35-7.19 (m, 7H, ArH), 7.10 (t, J =
13
7.34 Hz, 1H, ArH), 3.36 (t, J = 5.14 Hz, 2H, CH2), 2.97 (t, J = 5.14, 2H, CH2) ppm; C NMR
(100 MHz, DMSO-d6) δ: 167.40, 155.51, 153.48, 140.38, 138.90, 128.69, 128.52, 128.31,
126.20, 123.59, 121.34, 98.41, 35.30 (CH2), 31.31 (CH2) ppm; HRMS (ESI) for C19H16N5S,
[M+H]+ calcd: 346.1126, found: 346.1126.
4.5.2. N-(2-chlorophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (8)
White solid; yield: 92%; mp 268-270 C; FTIR (ATR, cm-1)max: 3055, 2918, 2718, 1626, 1560,
1379, 1269, 1184; 1H-NMR (400 MHz, DMSO-d6): δ 10.47 (s, 1H, NH), 7.98 (s, 1H, NH), 7.62-
7.58 (m, 2H, ArH), 7.41-7.08 (m, 8H, ArH), 3.20 (t, J = 7.68 Hz, 2H, CH2), 2.83 (t, J = 7.56 Hz,
2H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 166.95, 155.03, 140.21, 134.86, 130.46,
129.95, 129.39, 128.49, 128.29, 127.81, 126.20, 98.12, 35.33 (CH2), 31.37 (CH2) ppm.
4.5.3. N-(3-chlorophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (9)
White solid; yield: 85%; mp 216-218 C; FTIR (ATR, cm-1)max: 3311, 3210, 3132, 3023, 2917,
1635, 1558, 1473, 1388, 1309, 1176; 1H-NMR (400 MHz, DMSO-d6) δ: 10.46 (s, 1H, NH), 8.35
(s, 1H, NH), 8.08 (s, 1H, ArH), 7.73 (dd, J = 8.08, 1.04 Hz, 1H, ArH), 7.36-7.12 (m, 8H, ArH),
3.39 (t, J = 7.62 Hz, 2H, CH2), 2.98 (t, J = 7.62 Hz, 2H, CH2) ppm; 13C NMR (100 MHz,
DMSO-d6) δ: 167.27, 155.19, 153.30, 140.47, 140.24, 132.99, 132.48, 130.28, 128.57, 128.32,
126.25, 123.05, 120.55, 119.31, 35.06 (CH2), 31.52 (CH2) ppm; HRMS (ESI) for C19H15N5SCl,
[M+H]+ calcd: 380.0733, found: 380.0737.
4.5.4. N-(4-chlorophenyl)-6-(3-phenylpropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10)
White solid; yield: 85%; mp 286-288 C; FTIR (ATR, cm-1)max: 3052, 2911, 2725, 1627, 1566,
1487, 1383, 1276, 1184, 1080, 1015; 1H-NMR (400 MHz, DMSO-d6) δ: 10.81 (s, 1H, NH), 8.49
(s, 1H, NH), 7.84 (d, J = 8.80 Hz, 2H, ArH), 7.35-7.18 (m, 8H, ArH), 3.37 (t, J = 7.74 Hz, 2H,
13
CH2), 2.95 (t, J = 7.74 Hz, 2H, CH2) ppm; C NMR (100 MHz, DMSO-d6):δ 166.99, 154.47,
19

153.53, 140.20, 137.55, 132.23, 128.56, 128.51, 128.31, 127.63, 126.26, 123.16, 98.76, 35.21
(CH2), 31.38 (CH2) ppm.
4.5.5. N-(3-nitrophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (11)
Yellow solid; yield: 81%; mp 249-251C; FTIR (ATR, cm-1)max: 3378, 3100, 3030, 2828, 1629,
1
1566, 1525, 1478, 1429, 1349, 1250, 1109; H-NMR (400 MHz, DMSO-d6) δ: 10.63 (s, 1H,
NH), 8.91 (t, J = 2.12 Hz, 1H, ArH), 8.34 (s, 1H, NH), 8.22 (dd, J = 8.06, 1.62 Hz, 1H, ArH),
7.91 (dd, J = 8.14, 1.98 Hz, 1H, ArH), 7.61 (t, J = 8.18 Hz, 1H, ArH), 7.28-7.17 (m, 6H, ArH),
3.41 (t, J = 7.62 Hz, 2H, CH2), 2.97 (t, J = 7.60 Hz, 2H, CH2) ppm; 13C NMR (100 MHz,
DMSO-d6) δ: 167.38, 155.45, 153.12, 147.90, 140.35, 140.23, 132.53, 130.00, 128.47, 128.29,
126.46, 126.22, 117.51, 114.85, 98.72, 35.03 (CH2), 31.44 (CH2) ppm.
4.5.6. N-(4-nitrophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (12)
White solid; yield: 88%; mp 265-267 C; FTIR (ATR, cm-1)max: 3033, 2902, 2820, 2752, 1627,
1570, 1515, 1338, 1274, 1183, 1110; 1H-NMR (400 MHz, DMSO-d6) δ: 11.06 (s, 1H, NH), 9.94
(s, 1H, NH), 8.54 (s, 1H, ArH), 8.17 (s, 4H, ArH), 7.29-7.19 (m, 5H, ArH), 3.41 (t, J = 7.70 Hz,
2H, CH2), 3.00 (t, J = 7.68 Hz, 2H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.34,
155.39, 153.01, 145.64, 141.83, 140.27, 132.88, 128.51, 128.32, 126.29, 124.72, 120.14, 99.19,
35.08 (CH2), 31.39 (CH2) ppm; HRMS (ESI) for C19H15N6O2S, [M+H]+ calcd: 391.0974, found:
391.0977.
4.5.7. N-(2-bromophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (13)
White solid; yield: 85%; mp 266-268 C; FTIR (ATR, cm-1)max: 3052, 2921, 2709, 1625, 1585,
1
1564, 1547, 1507, 1415, 1380, 1268, 1185; H-NMR (400 MHz, DMSO-d6) δ: 10.57 (s, 1H,
NH), 7.96 (s, 1H, NH), 7.76 (dd, J = 8.0, 1.0 Hz, 1H, ArH), 7.57 (dd, J = 7.80, 1.52 Hz, 1H,
ArH), 7.44 (dd, J = 7.62, 1.16 Hz, 1H, ArH), 7.32-7.24 (m, 4H, ArH), 7.18 (t, J = 7.28 Hz, 1H,
ArH), 7.08 (d, J = 7.28 Hz, 2H, ArH), 3.19 (t, J = 7.72 Hz, 2H, CH2), 2.82 (t, J = 7.68 Hz, 2H,
13
CH2) ppm; C NMR (100 MHz, DMSO-d6) δ: 166.85, 155.15, 140.18, 136.35, 133.12, 131.87,
129.79, 128.88, 128.50, 128.29, 126.20, 35.32 (CH2), 31.39 (CH2) ppm.
4.5.8. N-(3-bromophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (14)
20

White solid; yield: 81%; mp 265-267 C; FTIR (ATR, cm-1)max: 3053, 2914, 2724, 1625, 1558,
1
1507, 1472, 1379, 1273, 1184; H-NMR (400 MHz, DMSO-d6) δ: 10.61 (s, 1H, NH), 8.42 (s,
1H, NH), 8.23 (s, 1H, ArH), 7.80 (t, J = 2.66 Hz, 1H, ArH), 7.29-7.20 (m, 8H, ArH), 3.39 (t, J =
13
7.58 Hz, 2H, CH2), 2.98 (t, J = 7.56 Hz, 2H, CH2) ppm; C NMR (100 MHz, DMSO-d6) δ:
167.16, 153.39, 140.52, 140.17, 132.46, 130.56, 128.59, 128.33, 126.26, 126.09, 123.55, 121.41,
119.85, 98.75, 35.02 (CH2), 31.56 (CH2) ppm; HRMS (ESI) for C19H15N5SBr, [M+H]+ calcd:
424.0224, found: 424.0232.
4.5.9. N-(4-bromophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (15)
White solid; yield: 78%; mp 284-286 C; FTIR (ATR, cm-1)max: 3100, 3025, 2917, 1613, 1579,
1
1495, 1473, 1304, 1225, 1180; H-NMR (400 MHz, DMSO-d6) δ: 10.84 (s, 1H, NH), 8.51 (s,
1H, NH), 7.79 (d, J = 8.76 Hz, 2H, ArH), 7.46 (d, J = 8.80 Hz, 2H, ArH), 7.30-7.18 (m, 6H,
ArH), 3.37 (t, J = 5.16 Hz, 2H, CH2), 2.95 (t, J = 7.74 Hz, 2H, CH2) ppm; 13C NMR (100 MHz,
DMSO-d6) δ: 166.98, 153.50, 140.20, 137.97, 132.22, 131.47, 128.51, 128.33, 126.27, 123.52,
115.74, 98.81, 35.21 (CH2), 31.38 (CH2) ppm.
4.6.0. N-(2,4-dimethylphenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (16)
White solid; yield: 76%; mp 238-240 C; FTIR (ATR, cm-1)max: 3100, 3025, 2917, 1613, 1579,
1495, 1473, 1335, 1304, 1225, 1180; 1H-NMR (400 MHz, DMSO-d6) δ:13.29 (s, 1H, NH), 9.66
(s, 1H, NH), 7.29-7.04 (m, 9H, ArH), 3.22 (s, 2H, CH2), 2.88 (s, 2H, CH2), 2.31 (s, 3H, CH3),
2.14 (s, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.40, 155.73, 140.50, 133.89,
132.55, 131.23, 128.51, 128.25, 127.04, 126.13, 35.51 (CH2), 31.28 (CH2), 20.64 (CH3), 17.74
(CH3) ppm; HRMS (ESI) for C21H20N5S, [M+H]+ calcd: 374.1447, found: 374.1439.
4.6.1. N-(4-(methylthio)phenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (17)
White solid; yield: 90%; mp 262-264 C; FTIR (ATR, cm-1)max: 3054, 2921, 2809, 2752, 1625,
1
1583, 1586, 1488, 1417, 1385, 1274, 1243, 1184; H-NMR (400 MHz, DMSO-d6) δ: 10.50 (s,
1H, NH), 8.36 (s, 1H, NH), 7.74 (d, J = 8.52 Hz, 2H, ArH), 7.31-7.19 (m, 8H, ArH), 3.37 (t, J =
13
7.80 Hz, 2H, CH2), 2.96 (t, J = 7.76 Hz, 2H, CH2), 2.44 (s, 3H, SCH3); C NMR (100 MHz,
DMSO-d6) δ: 167.07, 154.78, 153.49, 140.28, 135.91, 132.20, 128.51, 128.31, 126.70, 126.24,
21

122.23, 98.58, 35.31 (CH2), 31.31 (CH2), 15.31 (CH3) ppm; HRMS (ESI) for C20H18N5S2,
[M+H]+ calcd: 392.1000, found: 392.1004.
4.6.2. N-(4-chloro-3-(trifluoromethyl)phenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (18)
White solid; yield: 89%; mp 273-275 C; FTIR (ATR, cm-1)max: 3033, 2920, 2808, 2752, 1628,
1566, 1507, 1498, 1399, 1315, 1274, 1171, 1153, 1107, 1037; 1H-NMR (400 MHz, DMSO-d6) δ:
10.93 (s, 1H, NH), 8.49 (d, J = 2.52 Hz, 1H, ArH), 8.47 (s, 1H, NH), 8.18 (dd, J = 8.82, 2.50 Hz,
1H, ArH), 7.61 (d, J = 8.80 Hz, 1H, ArH), 7.29-7.20 (m, 6H, ArH), 3.37 (t, J = 7.68 Hz, 2H,
13
CH2), 2.96 (t, J = 7.66 Hz, 2H, CH2) ppm; C NMR (100 MHz, DMSO-d6) δ: 167.21, 155.12,
153.17, 140.21, 138.63, 132.61, 131.89, 126.76, 126.46, 125.52, 124.15, 123.86, 121.44, 119.62,
119.57, 98.83, 35.03 (CH2), 31.40 (CH2) ppm; HRMS (ESI) for C20H15N5F3SCl, [M+H]+ calcd:
449.0689, found: 448.0611.
4.6.3. N-(4-bromo-3-(trifluoromethyl)phenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (19)
White solid; yield: 86%; mp 273-275 C; FTIR (ATR, cm-1)max: 3033, 2905, 2809, 2752, 1628,
1562, 1507, 1473, 1381, 1315, 1273, 1182, 1131, 1097; 1H-NMR (400 MHz, DMSO-d6) δ: 10.71
(s, 1H, NH), 8.48 (s, 1H, NH), 8.37 (s, 1H, ArH), 8.08 (t, J =6.48 Hz, 1H, ArH), 7.76 (t, J = 7.44
Hz, 1H, ArH), 7.29-7.20 (m, 6H, ArH), 3.37 (t, J =7.66 Hz, 2H, CH2), 2.96 (t, J =7.54 Hz, 2H,
13
CH2) ppm; C NMR (100 MHz, DMSO-d6) δ: 167.27, 153.11, 140.23, 139.12, 135.27, 132.61,
128.50, 128.32, 128.26, 126.26, 125.49, 124.25, 121.53, 119.82, 35.04 (CH2), 31.38 (CH2) ppm;
HRMS (ESI) for C20H15N5F3SBr, [M+H]+ calcd: 493.0184, found: 492.0105.
4.6.4. N-(4-chloro-3-nitrophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20)
White solid; yield: 75%; mp 265-267 C; FTIR (ATR, cm-1)max: 3023, 2899, 2827, 2759, 1628,
1557, 1531, 1494, 1432, 1376, 1338, 1275, 1182, 1048; 1H-NMR (400 MHz, DMSO-d6) δ: 10.95
(s, 1H, NH), 8.75 (s, 1H, ArH), 8.43 (s, 1H, NH), 8.16 (d, J = 8.84 Hz, 1H, ArH), 7.68 (dd, J =
8.82, 1.66 Hz, 1H, ArH), 7.29-7.18 (m, 6H, ArH), 3.40 (t, J = 7.58 Hz, 2H, CH2), 2.97 (t, J =
7.56 Hz, 2H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.28, 155.29, 153.02, 147.02,
22

140.21, 139.17, 132.71, 131.82, 128.50, 128.30, 126.25, 125.57, 118.35, 117.18, 98.82, 34.98
(CH2), 31.45 (CH2) ppm.
4.6.5. N-(4-fluoro-2-methylphenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (21)
White solid; yield: 80 %; mp 268-270 C; FTIR (ATR, cm-1)max: 3103, 3026, 3000, 2934, 1593,
1
1580, 1492, 1313, 1303, 1210, 1180, 1144; H-NMR (400 MHz, DMSO-d6) δ: 13.34 (s, 1H,
NH), 9.72 (s, 1H, NH), 7.39 (q, J = 4.76 Hz, 1H, ArH), 7.29-7.04 (m, 8H, ArH), 3.20 (s, 2H,
13
CH2), 2.87 (d, J = 6.92 Hz, 2H, CH2), 2.19 (s, 3H, CH3) ppm; C NMR (100 MHz, DMSO-d6)
δ: 167.41, 161.66, 155.71, 140.44, 132.79, 132.48, 128.47, 128.24, 126.15, 117.12, 116.89,
113.21, 113.00, 35.50 (CH2), 31.28 (CH2), 17.90 (CH3) ppm; HRMS (ESI) for C20H17N5FS,
[M+H]+ calcd: 378.1191, found: 378.1189.
4.6.6.
N-(3-bromo-4-methylphenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(22)
White solid; yield: 85%; mp 279-281 C; FTIR (ATR, cm-1)max: 3056, 2923, 2745, 1626, 1583,
1
1550, 1487, 1383, 1270, 1184, 1145, 1038; H-NMR (400 MHz, DMSO-d6) δ: 10.67 (s, 1H,
NH), 8.43 (s, 1H, NH), 8.19 (d, J = 1.84 Hz, 1H, ArH), 7.68 (dd, J = 8.24, 2.00 Hz, 1H, ArH),
7.28-7.18 (m, 7H, ArH), 3.38 (t, J = 7.66 Hz, 2H, CH2), 2.96 (t, J = 7.62 Hz, 2H, CH2), 2.30 (s,
3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.06, 154.62, 153.45, 140.16, 137.83,
130.85, 128.58, 128.30, 126.25, 124.55, 123.64, 120.54, 119.29, 35.07 (CH2), 31.56 (CH2),
21.78 (CH3) ppm; HRMS (ESI) for C20H18N5SBr, [M+H]+ calcd: 439.0466, found: 438.0388.
4.6.7. N-(2-chloro-4-fluorophenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (23)
White solid; yield: 83 %; mp 282-284 C; FTIR (ATR, cm-1)max: 3037, 2907, 2724, 1629, 1569,
1488, 1380, 1269, 1256, 1196, 1180; 1H-NMR (400 MHz, DMSO-d6) δ: 10.28 (s, 1H, NH), 8.08
(s, 1H, NH), 7.65-7.57 (m, 2H, ArH), 7.28-7.17 (m, 5H, ArH), 7.09 (d, J = 10.24 Hz, 2H, ArH),
3.18 (t, J = 7.76 Hz, 2H, CH2), 2.82 (d, J = 5.80 Hz, 2H, CH2) ppm; 13C NMR (100 MHz,
DMSO-d6) δ: 155.10, 140.17, 131.55, 130.92, 130.82, 128.41, 128.26, 126.21, 117.24, 116.98,
115.09, 114.87, 35.36 (CH2), 31.32 (CH2) ppm.
4.6.8. N-(3,4-dimethoxyphenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (24)
23

Light yellow solid; yield: 78%; mp 265-267 C; FTIR (ATR, cm-1)max: 3063, 2930, 2716, 1629,
1584, 1505, 1457, 1388, 1259, 1232, 1204, 1184, 1129; 1H-NMR (400 MHz, DMSO-d6) δ: 10.97
(s, 1H, NH), 8.63 (s, 1H, NH), 7.39-7.14 (m, 8H, ArH), 6.85 (d, J = 8.40 Hz, 1H, ArH), 3.73 (s,
3H, OCH3), 3.71 (s, 3H, OCH3), 3.38 (t, J = 7.80 Hz, 2H, CH2), 2.93 (t, J = 7.74 Hz, 2H, CH2)
ppm; 13C NMR (100 MHz, DMSO-d6) δ: 166.40, 148.58, 140.05, 131.11, 128.52, 128.30,
126.29, 111.69, 55.66 (O-CH3), 55.51 (O-CH3), 35.19 (CH2), 31.46 (CH2) ppm.
4.6.9. N-(3-ethynylphenyl)-6-(phenethylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25)
Light yellow solid; yield: 78%; mp 254-256 C; FTIR (ATR, cm-1)max: 3230, 3065, 2914, 2750,
1
1627, 1571, 1543, 1509, 1381, 1257, 1167, 1051; H-NMR (400 MHz, DMSO-d6) δ: 10.82 (s,
1H, NH), 8.51 (s, 1H, NH), 8.03 (s, 1H, ArH), 7.86 (d, J = 4.54 Hz, 1H, ArH), 7.35-7.18 (m, 8H,
ArH), 4.18 (s, 1H, Ethynyl-H), 3.39 (t, J = 7.54 Hz, 2H, CH2), 2.97 (t, J = 7.52 Hz, 2H, CH2)
ppm; 13C NMR (100 MHz, DMSO-d6) δ: 166.99, 153.62, 140.13, 138.92, 129.13, 127.04,
126.27, 124.41, 122.09, 122.04, 98.80, 83.30, 80.75, 35.04 (CH2), 31.56 (CH2) ppm.
4.7.0. 6-(pentylthio)-N-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (26)
White solid; yield: 86%; mp 202-204 C; FTIR (ATR, cm-1)max: 3079, 2924, 1624, 1568, 1540,
1
1507, 1496, 1309, 1185; H-NMR (400 MHz, DMSO-d6) δ: 13.39 (s, 1H, NH), 10.02 (s, 1H,
ArH), 8.15 (s, 1H, NH), 7.78 (d, J=7.88 Hz, 2H, ArH), 7.38 (t, J = 7.92 Hz, 2H, ArH), 7.12 (t, J
= 7.32 Hz, 1H, ArH), 3.09 (t, J = 7.38 Hz, 2H, CH2), 1.66 (m, 2H, CH2), 1.25-1.38 (m, 4H,
(CH2)2), 0.85 (t, J = 7.16 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.73, 155.51,
153.39, 138.96, 128.64, 123.53, 121.22, 98.34, 30.57 (CH2), 29.97 (CH2), 28.97 (CH2), 21.75
(CH2), 13.85 (CH3) ppm; HRMS (ESI) for C16H18N5S, [M+H]+ calcd: 312.1284, found:
312.1283.
4.7.1. N-(2-chlorophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (27)
White solid; yield: 75%; mp 231-233 C; FTIR (ATR, cm-1)max: 3054, 2924, 2719, 1624, 1571,
1
1383, 1273, 1185, 1057; H-NMR (400 MHz, DMSO-d6) δ: 10.48 (s, 1H, NH), 7.99 (S, 1H,
NH), 7.57-7.61 (m, 2H, ArH), 7.35-7.44 (m, 2H, ArH), 2.90 (t, J = 7.44 Hz, 2H, CH2), 1.46-1.53
13
(m, 2H, CH2), 1.14-1.22 (m, 4H, (CH2)2), 0.82 (t, J = 6.86 Hz, 3H, CH3) ppm; C NMR (100
24

MHz, DMSO-d6) δ: 167.35, 154.87, 134.96, 132.05, 129.84, 129.43, 128.19, 127.70, 97.95,
30.50 (CH2), 29.99 (CH2), 29.11 (CH2), 21.74(CH2), 13.87 (CH3) ppm.
4.7.2. N-(3-chlorophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (28)
White solid; yield: 92%; mp 224-226 C; FTIR (ATR, cm-1)max: 3099, 2928, 1623, 1558, 1541,
1473, 1386, 1304, 1180; 1H-NMR (400 MHz, DMSO-d6) δ: 10.35 (s, 1H, NH), 8.30 (s, 1H, NH),
8.12 (s, 1H, ArH), 7.70 (d, J = 8.28 Hz, 1H, ArH), 7.39 (t, J = 8.10 Hz, 1H, ArH), 7.15 (dd, J =
7.90, 1.70 Hz, 1H, ArH), 3.12 (t, J = 7.32 Hz, 2H, CH2), 1.63-1.70 (m, 2H, CH2), 1.21-1.41 (m,
13
4H (CH2)2), 0.84 (t, J = 7.16 Hz, 3H, CH3) ppm; C NMR (100 MHz, DMSO-d6) δ: 167.64,
155.27, 153.21, 140.53, 132.96, 132.43, 130.28, 123.00, 120.47, 119.19, 98.62, 30.51 (CH2),
30.14 (CH2), 28.75 (CH2), 21.76 (CH2), 13.87 (CH3) ppm.
4.7.3. N-(4-chlorophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (29)
White solid; yield: 88%; mp 253-255 C; FTIR (ATR, cm-1)max: 3056, 2923, 2717, 1625, 1567,
1541, 1384, 1267, 1183, 1080, 1013; 1H-NMR (400 MHz, DMSO-d6) δ: 10.58 (s, 1H, NH), 8.39
(s, 1H, NH), 7.85 (d, J = 8.88 Hz, 2H, ArH), 7.43 (d, J = 8.88 Hz, 2H, ArH), 3.09 (t, J = 7.38 Hz,
2H, CH2), 1.61-1.68 (m, 2H, CH2), 1.22-1.37 (m, 4H, (CH2)2), 0.84 (t, J = 7.16 Hz, 3H, CH3)
ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.45, 153.38, 137.76, 132.27, 128.51, 127.40,
122.95, 98.60, 30.58 (CH2), 30.12 (CH2), 28.88 (CH2), 21.76 (CH2), 13.87 (CH3) ppm.
4.7.4. N-(3-nitrophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30)
Brown solid; yield: 84%; mp 210-212 C; FTIR (ATR, cm-1)max: 3080, 2928, 2757, 1625, 1558,
1522, 1348, 1299, 1175, 1072; 1H-NMR (400 MHz, DMSO-d6) δ: 10.70 (s, 1H, NH), 8.96 (t, J =
2.14 Hz, 1H, ArH), 8.37 (s, 1H, ArH), 8.20-8.23 (m, 1H, ArH), 7.92-7.94 (m, 1H, ArH), 7.66 (t,
J = 8.22 Hz, 1H, ArH), 3.15 (t, J = 7.30 Hz, 2H, CH2), 1.62-1.69 (m, 2H, CH2), 1.21-1.38 (m,
13
4H, (CH2)2), 0.82 (t, J = 7.20 Hz, 3H, CH3) ppm; C NMR (100 MHz, DMSO-d6) δ: 167.72,
155.34, 153.14, 147.93, 140.40, 132.55, 130.00, 126.49, 117.56, 114.85, 98.72, 30.49 (CH2),
30.16 (CH2), 28.57 (CH2), 21.74 (CH2), 13.86 (CH3) ppm.
4.7.5. N-(4-nitrophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (31)
25

Yellow solid; yield: 93%; mp 268-270 C; FTIR (ATR, cm-1)max: 3108, 2923, 1626, 1568,
1508, 1336, 1297, 1250, 1182, 1111; 1H-NMR (400 MHz, DMSO-d6) δ: 10.66 (s, 1H, NH), 8.33
(s, 1H, NH), 8.27 (d, J = 9.24 Hz, 2H, ArH), 8.13 (d, J = 9.24 Hz, 2H, ArH), 3.14 (t, J = 7.32 Hz,
2H, CH2), 1.66-1.73 (m, 2H, CH2), 1.25-1.43 (m, 4H, (CH2)2), 0.85 (t, J = 7.18 Hz, 3H, CH3)
ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.77, 155.64, 152.77, 145.70, 141.75, 132.57,
124.79, 119.87, 99.00, 30.60 (CH2), 30.12 (CH2), 28.73 (CH2), 21.77 (CH2), 13.86 (CH3) ppm.
4.7.6. N-(2-bromophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (32)
White solid; yield: 81%; mp 255-257 C; FTIR (ATR, cm-1)max: 3052, 2924, 2725, 1626, 1570,
1542, 1507, 1379, 1275, 1184, 1044; 1H-NMR (400 MHz, DMSO-d6) δ: 10.53 (s, 1H, NH), 7.96
(s, 1H, NH), 7.75-7.78 (m, 1H, ArH), 7.53-7.56 (m, 1H, ArH), 7.45-7.49 (m, 1H, ArH), 7.29-
7.33 (m, 1H, ArH), 2.90 (t, J = 7.42 Hz, 2H, CH2), 1.45-1.53 (m, 2H, CH2), 1.14-1.22 (m, 4H,
(CH2)2, 0.82 (t, J = 6.98 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.15, 155.06,
136.37, 133.01, 131.81, 129.82, 128.80, 128.40, 121.69, 97.97, 30.49 (CH2), 30.06 (CH2), 29.15
(CH2), 21.75 (CH2), 13.88 (CH3) ppm.
4.7.7. N-(3-bromophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (33)
Brown solid; yield: 76%; mp 232-234 C; FTIR (ATR, cm-1)max: 3099, 2927, 1622, 1557, 1472,
1298, 1178, 1063; 1H-NMR (400 MHz, DMSO-d6) δ: 10.41 (s, 1H, NH), 8.32 (s, 1H, NH), 8.26
(t, J = 1.78 Hz, 1H, ArH), 7.75 (d, J = 8.04 Hz, 1H, ArH), 7.27-7.35 (m, 2H, ArH), 3.12 (t, J =
7.28 Hz, 2H, CH2), 1.63-1.71 (m, 2H, CH2), 1.23-1.41 (m, 4H, (CH2)2), 0.84 (t, J = 7.22 Hz, 3H,
CH2) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.59, 155.19, 153.18, 140.63, 132.41, 130.56,
125.88, 123.32, 121.39, 119.57, 98.61, 30.49 (CH2), 30.13 (CH2), 28.70 (CH2), 21.75 (CH2),
13.86 (CH3) ppm.
4.7.8. N-(4-bromophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (34)
White solid; yield: 90%; mp 256-258 C; FTIR (ATR, cm-1)max: 3056, 2924, 2718, 1626, 1559,
1
1474, 1383, 1269, 1183, 1065; H-NMR (400 MHz, DMSO-d6) δ: 10.53 (s, 1H, NH), 8.36 (s,
1H, NH), 7.80 (d, J = 8.64 Hz, 2H, ArH), 7.55 (d, J = 8.80 Hz, 2H, ArH), 3.09 (t, J = 7.36 Hz,
2H, CH2), 1.61-1.68 (m, 2H, CH2), 1.22-1.37 (m, 4H, (CH2)2), 0.85 (t, J = 7.08 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) δ: 167.48, 153.34, 138.22, 132.27, 131.41, 123.25, 98.61, 30.59
26

(CH2), 30.12 (CH2), 28.89 (CH2), 21.76 (CH2), 13.89 (CH3) ppm; HRMS (ESI) for C16H17N5SBr,
[M+H]+ calcd: 390.0383, found: 390.0388.
4.7.9. N-(2,4-dimethylphenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (35)
Brown solid; yield: 90%; mp 256-258 C; FTIR (ATR, cm-1)max: 3099, 2922, 1614, 1578, 1473,
1315, 1223, 1180; 1H-NMR (400 MHz, DMSO-d6) δ: 13.24 (s, 1H, NH), 9.62 (s, 1H, ArH), 7.17
(t, J = 9.64 Hz, 2H, ArH), 7.06 (d, J = 7.68 Hz, 1H, ArH), 2.95 (s, 2H, CH2), 2.31 (s, 3H, CH3),
2.13 (s, 3H, CH3), 1.56 (s, 2H, CH2), 1.25 (s, 4H, (CH2)2), 0.84 (t, J = 6.68 Hz, 3H, CH3) ppm;
13C NMR (100 MHz, DMSO-d6) δ: 167.72, 155.74, 133.93, 132.51, 131.16, 127.51, 127.00,
30.58 (CH2), 29.80 (CH2), 29.10 (CH3), 21.76 (CH2), 20.62 (CH3), 17.72 (CH2), 13.90 (CH3)
ppm; HRMS (ESI) for C18H22N5S, [M+H]+ calcd: 340.1601, found: 340.1596.
4.8.0. N-(4-(methylthio)phenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (36)
Brown solid; yield: 77%; mp 242-244 C; FTIR (ATR, cm-1)max: 3064, 2923, 2724, 1624, 1558,
1520, 1507, 1386, 1185, 1083; 1H-NMR (400 MHz, DMSO-d6) δ: 10.65 (s, 1H, NH), 8.42 (d, J =
9.84, 1H, NH), 7.75 (d, J = 8.60 Hz, 2H, ArH), 7.29 (d, J = 8.64 Hz, 2H, ArH), 3.09 (t, J = 7.42
Hz, 2H, CH2), 2.47 (s, 3H, SCH3), 1.60-1.68 (m, 2H, CH2), 1.21-1.36 (m, 4H, (CH2)2), 0.84 (t, J
13
= 7.12 Hz, 3H, CH3) ppm; C NMR (100 MHz, DMSO-d6) δ: 167.25, 153.57, 135.82, 131.96,
126.74, 122.41, 98.61, 30.61 (CH2), 30.17 (CH2), 28.98 (CH2), 21.77 (CH2), 15.38 (CH3), 13.90
(CH3) ppm.
4.8.1.
N-(4-chloro-3-(trifluoromethyl)phenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (37)
White solid; yield: 83%; mp 258-260 C; FTIR (ATR, cm-1)max: 3101, 2927, 1628, 1567, 1478,
1437, 1385, 1321, 1256, 1176, 1132; 1H-NMR (400 MHz, DMSO-d6) δ: 10.60 (s, 1H, NH), 8.51
(d, J = 2.52 Hz, 1H, ArH), 8.32 (s, 1H, ArH), 8.10 (dd, J = 8.80, 2.52 Hz, 1H, ArH), 7.71 (d, J =
8.80 Hz, 1H, ArH), 3.10 (t, J = 7.34 Hz, 2H, CH2), 1.61-1.68 (m, 2H, CH2), 1.21-1.38 (m, 4H,
(CH2)2), 0.84 (t, J = 7.18 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.67, 155.41,
152.99, 138.72, 132.49, 131.94, 126.45, 125.33, 123.73, 119.47, 98.66, 30.48 (CH2), 30.01
(CH2), 28.57 (CH2), 21.73 (CH2), 13.83 (CH3) ppm.
27

4.8.2.
N-(4-bromo-3-(trifluoromethyl)phenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (38)
Brown solid; yield: 79%; mp 257-259 C; FTIR (ATR, cm-1)max: 3099, 2926, 1633, 1559, 1541,
1474, 1436, 1257, 1145, 1097; 1H-NMR (400 MHz, DMSO-d6) δ: 10.62 (s, 1H, NH), 8.51 (d, J =
2.52 Hz, 1H, ArH), 8.34 (s, 1H, NH), 8.03 (dd, J = 8.74, 2.50 Hz, 1H, ArH), 7.86 (d, J = 8.72
Hz, 1H, ArH), 3.10 (t, J = 7.32 Hz, 2H, CH2), 1.61-1.68 (m, 2H, CH2), 1.22-1.38 (m, 4H,
(CH2)2), 0.84 (t, J = 7.14 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.64, 155.37,
152.98, 139.16, 135.28, 132.50, 128.54, 125.39, 124.29, 119.73, 111.31, 98.70, 30.48 (CH2),
30.00 (CH2), 28.56 (CH2), 21.73 (CH2), 13.83 (CH3) ppm; HRMS (ESI) for C17H16N5F3SBr,
[M+H]+ calcd: 458.0258, found: 458.0262.
4.8.3. N-(4-chloro-3-nitrophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (39)
Yellow solid; yield: 91%; mp 251-253 C; FTIR (ATR, cm-1)max: 3098, 2925, 1626, 1558,
1540, 1473, 1339, 1297, 1265, 1182; 1H-NMR (400 MHz, DMSO-d6) δ: 10.73 (s, 1H, NH), 8.78
(d, J = 2.52 Hz, 1H, ArH), 8.33 (s, 1H, ArH), 8.08 (dd, J = 8.92, 2.56 Hz, 1H, ArH), 7.76 (d, J =
8.88 Hz, 1H, ArH), 3.12 (t, J = 7.26 Hz, 2H, CH2), 1.61-1.68 (m, 2H, CH2), 1.20-1.39 (m, 4H,
(CH2)2), 0.84 (t, J = 7.14 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.70, 155.45,
152.90, 147.12, 139.23, 132.54, 131.83, 125.33, 118.20, 116.94, 98.71, 30.48 (CH2), 30.14
(CH2), 28.50 (CH2), 21.76 (CH2), 13.86 (CH3) ppm.
4.8.4. N-(4-fluoro-2-methylphenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40)
White solid; yield: 90%; mp 261-263 C; FTIR (ATR, cm-1)max: 3100, 2953, 1577, 1490, 1206,
1
1181; H-NMR (400 MHz, DMSO-d6) δ: 13.29 (s, 1H, NH), 9.69 (s, 1H, ArH), 7.33-7.36 (m,
1H, ArH), 7.20 (t, J = 4.76 Hz, 1H, ArH), 7.01-7.11 (m, 1H, ArH), 2.93 (s, 2H, CH2), 2.18 (s,
3H, CH3), 1.54 (s, 2H, CH2), 1.22 (s, 4H, (CH2)2), 0.84 (t, J = 6.96 Hz, 3H, CH3) ppm; 13C NMR
(100 MHz, DMSO-d6) δ: 167.72, 155.80, 132.83, 132.39, 129.41, 117.01, 116.79, 113.15,
112.90, 30.59 (CH2), 29.84(CH2), 29.13 (CH3), 21.76 (CH2), 17.87 (CH2), 13.87 (CH3) ppm.
4.8.5. N-(3-bromo-4-methylphenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (41)
Brown solid; yield: 86%; mp 237-239 C; FTIR (ATR, cm-1)max: 3099, 2923, 1623, 1556, 1474,
1395, 1301, 1178; 1H-NMR (400 MHz, DMSO-d6) δ: 10.63 (s, 1H, NH), 8.30 (s, 1H, NH), 8.23
28

(d, J = 1.84 Hz, 1H, ArH), 7.65 (dd, J = 8.28, 2.08 Hz, 1H, ArH), 7.34 (d, J = 8.32 Hz, 1H,
ArH), 3.12 (t, J = 7.28 Hz, 2H, CH2), 1.62-1.70 (m, 2H, CH2), 1.22-1.40 (m, 4H, (CH2)2), 0.84 (t,
J = 7.22 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.54, 153.23, 138.05, 132.30,
131.98, 130.82, 124.29, 123.61, 120.19, 98.52, 30.50 (CH2), 30.13 (CH2), 28.72 (CH2),
21.75(CH2), 21.73(CH3), 13.86 (CH3) ppm; HRMS (ESI) for C17H19N5FS, [M+H]+ calcd:
344.1348, found: 344.1345.
4.8.6. N-(2-chloro-4-fluorophenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (42)
White solid; yield: 80%; mp 247-249 C; FTIR (ATR, cm-1)max: 3054, 2925, 2716, 1625, 1573,
1
1488, 1382, 1257, 1181; H-NMR (400 MHz, DMSO-d6) δ: 10.44 (s, 1H, NH), 8.04 (s, 1H,
ArH), 7.59-7.62 (m, 2H, ArH), 7.29-7.34 (m, 1H, ArH), 2.89 (t, J = 7.42 Hz, 2H, CH2), 1.45-
13
1.52 (m, 2H, CH2), 1.16-1.22 (m, 4H, (CH2)2), 0.82 (t, J = 6.88 Hz, 3H, CH3) ppm; C NMR
(100 MHz, DMSO-d6) δ: 167.20, 161.54, 159.08, 155.10, 131.86, 131.01, 117.13, 116.87,
115.01, 114.78, 98.01, 30.59 (CH2), 30.09 (CH2), 29.19 (CH2), 21.76 (CH2), 13.86 (CH3) ppm.
4.8.7. N-(3,4-dimethoxyphenyl)-6-(pentylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (43)
Brown solid; yield: 78%; mp 247-249 C; FTIR (ATR, cm-1)max: 3055, 2951, 2725, 1625, 1507,
1260, 1233, 1026; 1H-NMR (400 MHz, DMSO-d6) δ: 10.52 (s, 1H, NH), 8.43 (s, 1H, NH), 7.40
(s, 1H, ArH), 7.28 (s, 1H, ArH), 6.97 (d, J = 8.72 Hz, 1H, ArH), 3.76 (d, J = 2.12 Hz, 6H,
(OCH3)2), 3.10 (t, J = 7.38 Hz, 2H, CH2), 1.59-1.64 (m, 2H, CH2), 1.20-1.34 (m, 4H, (CH2)2),
0.83 (t, J = 7.08 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 166.72, 153.97, 148.59,
131.38, 114.58, 111.74, 107.58, 98.57, 55.73 (OCH3), 55.59 (OCH3), 30.50 (CH2), 30.28 (CH2),
28.81 (CH2), 21.72 (CH2), 13.83 CH3) ppm; HRMS (ESI) for C18H22N5O2S, [M+H]+ calcd:
372.1496, found: 372.1494.
5.
Biological activity
5.1. CDK2 and Abl kinase inhibition assays
CDK2/cyclin E and Abl kinases were produced in Sf9 insect cells via baculoviral infection
and purified on a NiNTA column. The kinase reactions were assayed with suitable substrates (1
mg/mL histone H1 for CDK2 and 500 µM peptide GGEAIYAAPFKK for Abl) in the presence
of 15 or 10 µM ATP for CDK2 and Abl, respectively, 0.05 µCi [γ-33P]ATP, and the test
29