3,5,7-Substituted Pyrazolo[4,3- d]pyrimidine Inhibitors of Cyclin-Dependent Kinases and Their Evaluation in Lymphoma Models

Article abstract of DOI:10.1021/acs.jmedchem.9b00189

Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines with various substitutions at position 5 with potent antiproliferative activity in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and the cocrystal with CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation of XIAP and MCL-1, and activation of caspases, which collectively confirmed ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a combination therapy with the BCL2-targeting venetoclax. These findings support further studies of combinatorial treatment based on CDK inhibitors.

Full text of DOI:10.1021/acs.jmedchem.9b00189

Subscriber access provided by IDAHO STATE UNIV
Article
3,5,7-Substituted pyrazolo[4,3-d]pyrimidine inhibitors of cyclin-
dependent kinases and their evaluation in lymphoma models
Radek Jorda, Libor Havlicek, Antonin Sturc, Diana Tuskova, Lenka Daumova, Mahmudul Alam,
Jana Skerlova, Michaela Nekardova, Miroslav Perina, Tomáš Pospíšil, Jitka Siroka, Lubor
Urbánek, Petr Pachl, Pavlina Rezacova, Miroslav Strnad, Pavel Klener, and Vladimir Krystof
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00189 • Publication Date (Web): 03 Apr 2019
Downloaded from http://pubs.acs.org on April 4, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
,5,7-Substituted Pyrazolo[4,3-d]pyrimidine Inhibitors of Cyclin-Dependent Kinases and
Their Evaluation in Lymphoma Models
a,#
b,#
b
c
c
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Radek Jorda , Libor Havlíček , Antonín Šturc , Diana Tušková , Lenka Daumová ,
c
d,e
d,f
a
Mahmudul Alam , Jana Škerlová , Michaela Nekardová , Miroslav Peřina , Tomáš
a
a
a
d
d,e
a
Pospíšil , Jitka Široká , Lubor Urbánek , Petr Pachl , Pavlína Řezáčová , Miroslav Strnad ,
c
a*
Pavel Klener , Vladimír Kryštof
a
Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany,
The Czech Academy of Sciences, Šlechtitelů 27, 783 71 Olomouc, Czech Republic
b
Isotope Laboratory, Institute of Experimental Botany, The Czech Academy of Sciences,
Vídeňská 1083, 14220 Prague, Czech Republic
c
Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague,
Czech Republic
d
Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
e
Institute of Molecular Genetics, The Czech Academy of Sciences, Vídeňská 1083, 14220
Prague, Czech Republic
f
Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 3, Prague 2,
121 16, Czech Republic
KEYWORDS
cyclin-dependent kinase, lymphoma, pyrazolo[4,3-d]pyrimidine, venetoclax
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By
convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-
pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines with various substitutions at position
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
with potent antiproliferative activity in non‐Hodgkin lymphoma cell lines. The most potent
derivative 4.35 also displayed activities across more than sixty cancer cell lines. The kinase
profiling confirmed high selectivity of 4.35 toward CDKs 2, 5 and 9, and the cocrystal with
CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated
with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1,
downregulation of XIAP and Mcl-1 and activation of caspases, which collectively confirmed
ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line
xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a
combination therapy with the BCL2-targeting venetoclax. These findings support further
studies of combinatorial treatment based on CDK inhibitors.
2
ACS Paragon Plus Environment

Page 3 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Non-Hodgkin lymphomas (NHLs) are the most frequent hematologic malignancies in the
Western hemisphere, comprising approximately 30% of all hematologic cancers. Based on the
behavior of the cancer, NHLs can be classified as indolent, aggressive and highly aggressive
lymphomas. The latter two NHL types include diffuse large B-cell lymphoma, Burkitt’s
lymphoma, mantle cell lymphoma and peripheral T-cell lymphomas. The deregulation of cell
cycle machinery together with the disruption of programmed cell death are hallmarks of
aggressive NHL. These hallmarks include mutated or overexpressed oncogenes (e.g., MYC,
BCL6, MYD88, cyclin D1, BCL2, CDK4/6) or the deletion of tumor suppressors (e.g., TP53,
TP63, RB1, CDKN2A, ATM). Double- and triple-hit lymphomas characterized by the
concurrent genetic aberrations of MYC, BCL-2 and/or BCL6 represent a subgroup of NHL
with an extremely dismal prognosis.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Some of the recurrently overexpressed or mutated genes encode drug targets, such as
BCL-2 protein, Bruton’s tyrosine kinase or phosphoinositide 3-kinase, which can be
modulated by the small molecule inhibitors venetoclax, ibrutinib or idelalisib, respectively.2-4
Another emerging pharmacological approach targets cyclin-dependent kinases (CDK), which
5
were first discovered for their role in regulating the cell cycle. Depending on their selectivity,
CDK inhibitors can interfere either with cell cycle progression by blocking RB protein
phosphorylation (governed by CDKs 2/4/6) or with global transcription by inactivating RNA
polymerase II (governed by CDKs 7/8/9/12). However, many inhibitors have broad specificity
and simultaneously obstruct both of these processes,6-8 providing the advantage of
overcoming possible compensatory functions among the CDK family.9
CDK inhibitors, including clinical candidate drugs such as flavopiridol, roscovitine,
dinaciclib and the recently approved drugs palbociclib and abemaciclib, have been
increasingly evaluated in various lymphomas.10-14 In NHL, similar to other cancers, the
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pharmacological inhibition of transcriptional CDKs leads to the downregulation of short-lived
mRNAs related to cell survival, such as those of MCL-1, XIAP, MYC and cyclin D1. The
downregulation of cyclin D1 and MCL-1 protein levels was observed in mantle cell
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
lymphomas and in aggressive MYC-driven B-cell lymphoma treated with roscovitine and
dinaciclib, respectively.1
1,12
Despite these compounds inhibit multiple CDKs, a widely
accepted mechanism of action involves CDK9, which was independently supported by
1
5
findings obtained with the highly CDK9-selective drug atuveciclib. Importantly, the loss of
MCL-1 as a consequence of CDK9 or CDK7 inhibition by these drugs has been found to
strongly enhance apoptosis induction in NHL by small-molecule antagonists of BCL-2.1
The pan-selective purine CDK inhibitor roscovitine, which was discovered in our
laboratory, was among the first CDK inhibitors that entered clinical trials.22,23 The further
6-21
exploration of roscovitine has been mainly oriented toward either modifications in its
substitutable positions2
4-26
or the redistribution of nitrogen atoms of the purine scaffold; the
latter approach culminated in nanomolar inhibitors based on pyrazolo[4,3-d]pyrimidines and
pyrazolo[1,5-a][1,3,5]triazine.2
7-30
The objective of this work was to synthesize novel potent pyrazolo[4,3-d]pyrimidine
CDK inhibitors, which proved to be more potent than related purines,28-30 and explore their
activity toward lymphoma models. The heterocyclic core was substituted with groups that
conferred high activity in purines, identified during our previous structure-activity studies24,26
;
i.e. isopropyl moiety at position 3, benzylamines at position 7 and alkyl- or cycloalkyl-
amines at position 5.29-30 The compounds were prepared using more convenient alkylation of
5-sulfanyl derivatives, providing substantially higher yields over the 5-amino-substituted
pyrazolo[4,3-d]pyrimidines described earlier.30,31 In line with our expectations, the newly
prepared derivatives displayed nanomolar potency against CDKs and cancer cell lines. Our
previous study demonstrated that roscovitine strongly sensitizes leukemia and lymphoma cells
4
ACS Paragon Plus Environment

Page 5 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
to TRAIL-induced apoptosis by altering the levels of BCL-2 proteins in the mitochondria32
and, together with other observations,11 this led us to explore anticancer activity of new
pyrazolo[4,3-d]pyrimidines against several lymphoma models. The compounds proved to
potently kill various lymphoma cell lines and also displayed significant in vivo activity both
in monotherapy and in a combination with venetoclax, which corresponds to the
abovementioned findings.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Some cyclin-dependent kinase inhibitors with activity in lymphoma.
Results and Discussion
Design and Synthesis
We recently described 5-alkylamino-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-
pyrazolo[4,3-d]pyrimidines that share some of the same substitutions as the purine CDK
inhibitor roscovitine but that showed substantially higher potency against CDKs and cancer
cell lines than did roscovitine.28,30 Due to the laborious synthesis of these derivatives, we
sought to improve the synthetic accessibility of these compounds while retaining or even
improving their biochemical potency. We focused on the substituted position 5 on the
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pyrazolo[4,3-d]pyrimidine scaffold and report here a new series of 3,5,7-trisubstituted
pyrazolo[4,3-d]pyrimidines in which the alkylamino group at the 5-position of the heterocycle
is replaced by a 5-alkylthio group. To verify the effect of this modification, we employed
molecular modeling. We applied the computational procedure delineated in our previous
work, in which we identified the pyrazolo[4,3-d]pyrimidine core as the most favorable central
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
heterocycle of the purine bioisosteres of CDK2 inhibitors. Importantly, we have shown that
the contributions of the central heterocyclic cores and the individual substituents, quantified
and evaluated in relation to conformations of the optimized protein-inhibitor complexes, are
not simply additive. Depending on the scaffold, the same substituents can be associated with
-1
different interaction “free” energies G’int (in kcal mol ). Our preliminary calculations
suggested that the replacement of the alkylamino group at the 5-position with the 5-alkylthio
group can increase the binding affinity to CDK2 (selected data are available in
Supplementary Information).
The new compounds have been prepared from 3-isopropyl-5-sulfanyl-1(2)H-
2
8
pyrazolo[4,3-d]pyrimidin-7-ol 1 (synthesized according to a described procedure ), which
was first converted to 5,7-dithiol 2 and subsequently substituted at the heterocycle position 7
with 4-(2-pyridyl)benzylamine. The final compounds 4 were prepared by the alkylation of the
reactive 5-sulfanyl functional group of 3 by various agents (Scheme 1); in the final step, we
successfully prepared 24 alkyl halogens (yielding compounds 4.2 - 4.4, 4.6 - 4.11, 4.17, 4.18,
4.21 - 4.23, 4.25, 4.26, 4.28, 4.30 - 4.33, 4.35), 8 epoxides (yielding compounds 4.1, 4.5, 4.12,
4
.16, 4.19, 4.29, 4.33, 4.34) and aziridines (yielding compounds 4.13, 4.24 and 4.35).
The reactivity of the sulfanyl functional group enables the use of different classes of
alkylating agents, eventually leading to the same compounds with similar yields (4.33 and
.35 were prepared not only by using alkyl halogens but also by using suitable epoxide or
4
aziridine, respectively). This new synthetic approach overcomes the less convenient
6
ACS Paragon Plus Environment

Page 7 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
nucleophilic aromatic substitution (S Ar, which requires difficult reaction conditions, makes
N
undesired by-reactions and subsequently by-products) described in our previous article
examining 5-alkylamino-1(2)H-pyrazolo[4,3-d]pyrimidine derivatives.30
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. General procedure for the synthesis of novel 5-alkylthio-3-isopropyl-7-[4-(2-
pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines.
Structure-Activity Relationships
The implementation of the most suitable substitution at the 5 position of the pyrazolo[4,3-
d]pyrimidine core was mostly inspired by previous SAR studies on trisubstituted
pyrazolo[4,3-d]pyrimidine CDK inhibitors,29,30 related purines26,34-37 and pyrazolo[1,5-
a]pyrimidines.38 The compounds we prepared included inhibitors with significant activity
against CDK2/E; all compounds exhibited an IC below 200 nM, and approximately 50% of
50
inhibitors blocked the activity of the simple 5-methylthio derivative 4.14 (IC = 48 nM). The
5
0
substituent in position 5 is mostly in contact with the flexible parts of the enzyme surface or
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with the solvent (water molecules) and therefore can be more variable, whereas the side
chains at positions 3 and 7 do not allow such variability. Despite low differences in the CDK2
inhibition measured with the studied compounds, we found some relationships that are
associated with the size/length of the substituent at position 5 or its polarity. Compound 3,
which contains an acidic sulfanyl functional group in position 5, was the weakest in the series,
with nearly three-fold lower potency than an analogous 3,7-disubstituted compound described
earlier.30 The methylation of the 5-sulfanyl functional group results in short lipophilic
substituents in 4.14, increasing the potency more than ten-fold.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The weakest CDK2 inhibition (IC50 between 100 - 200 nM) was observed for
compounds bearing rigid piperazine and morpholine moieties (4.2 and 4.1, respectively).
Additional groups with weak activity against CDK2 included compounds with less polar (4.4,
4
.7, 4.11, 4.14) or cycloalkyl (4.5, 4.12, 4.13) substituents at position 5 or those with tertiary
amino groups (4.8, 4.9) (IC = 48 - 138 nM). In contrast, the most potent inhibitors of the
50
series (IC = 2 - 39 nM) bear at least one polar group (mainly a hydroxy or primary amino
5
0
group) placed either on a short linear (4.35, 4.33, 4.22, 4.23, 4.28) or on a small branched
chain (4.34, 4.32, 4.30, 4.29, 4.27, 4.26). Further modification of 2-aminoethylthio in the
most active compound 4.35 led to the impairment of CDK2 inhibition; this effect was
observed when the terminal amino group was modified by methylation, acetylation and
conversion to urea or guanidine (4.28, 4.15, 4.9, 4.27 and 4.20, respectively) and is probably
due to steric hindrance due to possible hydrogen bonding, increased basicity of the amine
(
4.28 and 4.20), or a combination of both. Other attempts to modify the primary amine in 4.35
diminished its activity, such as its extension with aminomethyl (4.30) or its cyclization (4.13 a
.17). A similar scenario is also evident for the modified hydroxyethylthio derivatives, among
which only compound 4.34 reached the same potency as the analog 4.33; all other compounds
4
(4.32, 4.29, 4.19, 4.18, 4.16, 4.12, 4.11, 4.5, 4.3, 4.2, 4.1, 4.31) have worse CDK2 inhibition.
8
ACS Paragon Plus Environment

Page 9 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Recent experiments have shown that pharmacological CDK inhibition is an effective
and rational treatment option for aggressive MYC-driven lymphomas.11 We therefore
screened all prepared compounds against the non‐Hodgkin lymphoma cell lines HT, OCI-
LY2 and MINO.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The antiproliferative activity of the prepared compounds was consistent with enhanced
CDK2 inhibition (Table 1). The most potent compounds, 4.28, 4.29, 4.31, 4.32, 4.33, 4.34
and 4.35, had notably improved potency compared with the related purine CR8, with an
average GI value 5-fold higher than that of CR8.
5
0
Table 1. CDK2/E inhibitory and cellular activities of novel pyrazolo[4,3-d]pyrimidines.
N
HN
IC50 (µM)*
GI50 (µM)*
N
HN
N
R
N
Compound
R - substitution
CDK2/E
0.558
HT
OCI-LY2
3.240
MINO
2.175
3
2.760
HS
HO
S
4
.1
0.171
0.156
0.332
0.200
0.435
0.200
0.767
0.243
N
HN
HO
N
S
4
4
.2
.3
O
0.15
0.255
0.975
0.181
0.668
0.119
0.773
HO
S
S
4.4
0.138
S
HO
S
4
.5
0.134
0.132
0.636
0.690
0.680
0.585
0.530
0.641
HO
S
4.6
F
F
F
4
4
.7
.8
0.100
0.099
1.005
0.105
0.858
0.109
0.923
0.076
S
N
S
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
.9
N
0.077
0.054
0.061
0.093
S
4
4
.10
.11
0.077
0.059
0.980
0.340
1.001
0.285
1.307
0.243
HO
S
O
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
S
4
.12
0.052
0.052
0.567
0.139
0.373
0.136
0.450
0.157
H2N
S
4.13
4
4
.14
.15
0.048
0.041
0.783
0.071
1.207
0.054
1.050
0.042
S
H
N
S
O
HO
S
4
.16
0.039
0.032
0.273
0.155
0.280
0.145
0.427
0.135
N
4.17
N
S
S
H2N
4
4
.18
.19
0.031
0.030
0.055
0.507
0.055
0.610
0.064
0.477
OH
HO
S
H
N
H2N
4
4
.20
.21
S
0.030
0.027
0.39
0.812
0.163
0.39
NH
H2N
S
0.192
0.197
O
4
4
.22
.23
0.022
0.021
0.120
0.109
0.151
0.102
0.156
0.089
H2N
S
S
HO
H2N
4
4
.24
.25
S
0.020
0.019
0.085
0.080
0.100
0.053
0.064
0.069
HN
O
S
O
O
4
4
.26
.27
0.019
0.018
0.120
0.240
0.084
0.198
0.081
0.136
H2N
S
H
H2N
N
S
O
H
4.28
.29
N
0.013
0.012
0.056
0.062
0.092
0.152
0.040
0.081
S
HO
4
S
1
0
ACS Paragon Plus Environment

Page 11 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
H2N
S
4
4
.30
.31
0.012
0.011
0.480
0.063
0.780
0.099
0.770
0.051
NH2
H2N
HO
S
S
OH
OH
4
4
.32
.33
0.010
0.003
0.021
0.048
0.022
0.050
0.010
0.040
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
S
S
HO
4
4
.34
.35
0.003
0.002
0.190
0.022
0.141
0.028
0.160
0.041
H2N
S
CR8
0.062
0.002
>25
0.263
0.019
15.9
0.307
0.011
18.9
0.202
0.019
10.7
dinaciclib
ibrutinib
*
measured at least in triplicate
Crystal Structure of the Complex of CDK2/cyclin A2 with Pyrazolo[4,3-d]pyrimidine
.35
4
To obtain structural insight into the interaction of our inhibitors with the enzyme, we
determined the crystal structure of the active Thr160-phosphorylated CDK2 with a fragment
of cyclin A2 (residues 175-432) in complex with compound 4.35 at 2.15 Å resolution (PDB
code: 6GVA). For data collection and refinement statistics, see Supplementary Table 2. The
asymmetric unit contains one CDK2/cyclin A2 heterodimer. The residues 39-40 and C-
terminal residues 291-298 of CDK2 could not be modeled into the electron density map due
to disorder.
The inhibitor binds to the active site located in a narrow cleft between the N- and C-
terminal domains of CDK2 in the standard binding mode typical for pyrazolo[4,3-
d]pyrimidine-based inhibitors (see Figure 2). Specifically, the pyrazolo[4,3-d]pyrimidine core
is sandwiched between the hydrophobic side-chains of Ile10 and Leu134 and interacts via
three conserved hydrogen bonds with the hinge region residues (7-amino N-H···O:Leu83,
3
N2-H···O:Glu81, and a weak N1···HN:Leu83 bond). The R isopropyl group forms
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydrophobic interactions with the gatekeeper residue Phe80. The terminal amino group of the
5
R moiety forms a hydrogen bond with the carbonyl oxygen of Gln131 from the phosphate-
7
ribose pocket. The proximal phenyl ring of the R moiety is sandwiched between Ile10 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the backbone of His84, close to the specificity surface. The terminal pyridine ring sticks out
of the binding cleft, roughly in plane with the proximal phenyl ring. Moreover, N4 from the
pyrazolo[4,3-d]pyrimidine core of the inhibitor interacts through a chain of three water
molecules (W414, W435, and W436) with the side chains of Lys33 and Glu51 and with the
main chain of Asp145. Another chain of three water molecules (W409, W426, and W471)
5
provides additional hydrogen bonding of the amino group on the R moiety to the main chain
of Glu12 and to the side chains of Asp86 and Lys89 at the edge of the binding cleft.
Figure 2. Inhibitor 4.35 bound to the active site of CDK2 (PDB: 6GVA). CDK2 residues
interacting with the inhibitor directly (black dashed lines) or through water molecules (cyan
dashed lines and red spheres) are shown in green or gray stick representations, respectively. A
refined 2|F |-|F | electron density map for the inhibitor contoured at 1σ is shown.
o
c
1
2
ACS Paragon Plus Environment

Page 13 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Kinase Selectivity
As the most potent compound, 4.35 was then assayed on a panel of related CDKs. 4.35 had
low nanomolar activity not only against CDK2 but also against CDK5 and CDK9 (Table 2).
The compound displayed at least 10-fold increased potency toward most of the assayed CDKs
compared to the related purine CR8, with the highest increase (43-fold) toward CDK4/cyclin
D1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. CDK selectivity profile of pyrazolo[4,3-d]pyrimidine 4.35 and purine CR8 (assayed
as a control).
IC50 (µM)*
Kinase
4.35
CR8
Dinaciclib
CDK1/B1
CDK2/E1
CDK2/A2
CDK4/D1
CDK5/p25
0.090 ± 0.012
0.002 ± 0.001
0.006 ± 0.003
0.603 ± 0.318
0.015 ± 0.003
0.787 ± 0.100
0.062 ± 0.026
0.144 ± 0.002
26.09
0.072 ± 0.024
0.002 ± 0.001
0.003 ± 0.001
0.115 ± 0.018
0.067 ± 0.032
0.170 ± 0.008
0.015 ± 0.006
0.225 ± 0.013
1.769 ± 0.013
0.272 ± 0.038
CDK7/H/MAT 0.124 ± 0.007
CDK9/T1 0.025 ± 0.002
measured at least in triplicate
*
Next, preliminary selectivity profiling of 4.35 was performed on a panel of 50 additional
protein kinases, a representative sampling of the human kinome. The compound was assayed
at a single concentration of 1 µM. As shown in Supplementary Table 3, significant
inhibition was observed for CK1δ and CHK2; the residual activity of these kinases decreased
to 11 and 18%, respectively. We then determined the IC values for CK1δ and CHK2 (0.045
5
0
µM and 0.525 µM, respectively). Weak inhibition of PAK4, CAMK1 or RSK1 kinases was
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
also observed; these kinases are known to be sensitive to related CDK inhibitors with
pyrazolo[4,3-d]pyrimidine or purine scaffolds.29,39-41 These results confirmed the reasonable
selectivity of 4.35.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Antiproliferative Effects
We next assessed the anticancer activity of 4.35 against additional lymphoma cell lines as
well as other human cancer cell lines (Table 3). 4.35 displayed a broad range of activity
across all assayed cell lines, with the average concentration to reduce cell proliferation by
5
0% (GI value) of 21.6 nM. The most sensitive cell lines were the diffuse large B–cell
50
lymphomas OCI-LY3, RIVA and HBL-1. The clinical candidate dinaciclib, assayed in
parallel, displayed similar potency.
Table 3: Effect of pyrazolo[4,3-d]pyrimidine 4.35 on various human cancer cell lines.
Cancer type
cell line
IC50 (µM)*
4.35
Dinaciclib
0.031
n.d.
lymphoma (DLBCL-ABC)
RIVA
OCI-LY3
U2932
0.011
0.007
0.030
0.010
0.012
0.023
0.014
0.017
0.015
0.019
0.043
0.014
0.011
0.013
0.017
0.011
0.002
n.d.
HBL-1
TMD8
lymphoma (DLBCL-GCB)
OCI-LY2
OCI-LY7
SU-DHL10
SU-DHL4
HT
0.010
0.019
0.013
0.014
lymphoma (MCL)
HBL-2
JEKO-1
1
4
ACS Paragon Plus Environment

Page 15 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
MAVER-1
MINO
UPF1H
UPF7U
Z138
0.013
0.016
0.025
0.012
0.013
0.044
0.038
0.036
0.033
0.019
0.015
0.016
0.017
0.022
0.036
0.017
0.011
0.013
0.019
n.d.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
lymphoma (BL)
Ramos
Raji
0.015
0.025
0.011
0.007
0.007
0.009
0.006
leukemia (CML)
leukemia (ALL)
leukemia (AML)
multiple myeloma
breast
K562
CEM
THP-1
RPMI8226
MCF-7
HeLa
cervix
0.013
0.007
0.016
colon
HCT-116
G361
melanoma
*
measured at least in triplicate; DLBCL–ABC, Diffuse large B–cell lymphoma, subtype – activated B–cell;
DLBCL–GCB, Diffuse large B–cell lymphoma, subtype – germinal center B–cell; MCL – mantle cell
lymphoma; BL – Burkitt lymphoma; CML, chronic myelogenous leukemia; ALL, acute lymphoblastic leukemia;
AML, acute monocytic leukemia
To further assess the anticancer selectivity of 4.35, we also screened the NCI60 cell line
panel. Notably, 4.35 robustly inhibited proliferation across the entire cancer cell line panel
with a mean GI of 23 nM (Supplementary Table 4). In general, 4.35 was effective against
5
0
leukemia, melanoma and breast cancer cells but showed decreased activity in ovarian and
renal cells.
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Mechanism of Action in Cells
Compound 4.35 emerged as the most potent CDK inhibitor in this series, and because of its
superior proapoptotic activity in several lymphoma cell lines (Supplementary Figures S6
and S7), we sought to characterize its cellular effects. We therefore performed
immunoblotting analyses of MINO cells exposed to 4.35 both at different doses and for
different lengths of time. The treated cells contained clear markers of ongoing apoptotic cell
death, including the appearance of the PARP-1 fragment (89 kDa), diminution of full-length
PARP-1, activation of caspases 3 and 7 and reduction in the levels of the anti-apoptotic
proteins Mcl-1 and XIAP (Figure 3A, B). On the other hand, the anti-apoptotic proteins BCL-
2 and BCL-XL did not show any change, probably due to their high stability. Clear markers
of apoptosis were also detected in UPF1H and MAVER-1 cells (Supplementary Figure S8).
These results are consistent with the known mechanisms of the pro-apoptotic action of other
CDK inhibitors.1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,12,24,30
The kinetics of CDK inhibition by 4.35 was studied in MINO cells over the period of
2
4 h (Figure 3C). The dose of 12.5 nM was selected because it induced a strong response
within 24 h in previous experiments (Figure 3A, B), and it was anticipated that CDK
inhibition would be evident at earlier time points. Importantly, a gradual decrease in the
phosphorylation of the C-terminus of RNA polymerase II (at serines 2 and 5) and
retinoblastoma protein (at serines 608 and 780) in a time-dependent manner was observed,
suggesting that at least CDK2, CDK7 and CDK9 were effectively inhibited.
1
6
ACS Paragon Plus Environment

Page 17 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Immunoblotting analysis of expression of CDK substrates and apoptosis-related
proteins in MINO lymphoma cells treated with 4.35 in a dose (A) and time-dependent (B, C)
manner.
In Vivo Activity: Monotherapy
The in vivo efficacy of 4.35 was then assessed in murine models of aggressive lymphomas
based on subcutaneous xenotransplantation of three established lymphoma cell lines (MINO,
MAVER-1 and HBL-2) and one patient-derived lymphoma xenograft (PDX - VFN-M1) in
immunodeficient mice. Due to relatively rapid pharmacokinetics and limited oral availability
(for details see Supplementary Information), compound 4.35 was administered daily by i.v.
injections. Treatment with 4.35 was associated with a significant reduction in the growth of
all three xenografted lymphoma cell lines compared to controls (Figure 4). Importantly, anti-
lymphoma activity was also clearly achieved in the PDX mouse model VFN-M1, which
represents a surrogate model for primary lymphoma cells from patients (Figure 4).
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Determination of tumor volume of vehicle- or 4.35-treated (10 mg/kg) mice after
i.v. dosing in MINO (n = 10), MAVER-1 (n = 12), Hbl-2 (n = 8) and VFN-M1 patient-
derived xenograft (PDX, n = 10).
Ex vivo analysis of selected pharmacodynamic targets of 4.35 in subcutaneously grown
lymphoma tumors 24 h after a single i.v. administration of 4.35 confirmed the induction of
apoptosis and the downregulation of MCL-1 and XIAP proteins but had no impact on the
expression of BCL-2 (Figure 5, Supplementary Figure S10). Notably, the changes were also
evident in the PDX model VFN-M1, a reliable surrogate for the primary cells of patients
(Figure 5C). In addition, reduced RB and RNA polymerase II phosphorylation at several
phosphorylation sites was also observed (Supplementary Figure S11).
1
8
ACS Paragon Plus Environment

Page 19 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Inhibitory effect of pyrazolo[4,3-d]pyrimidine 4.35 on the growth of lymphoma
xenografts derived from the MINO (A) and MAVER-1 (B) cell lines and from patient-derived
VFN-M1 cells (C). Tumor-bearing mice were intravenously injected with 4.35. After 24 h, the
mice were euthanized, and the tumors were removed and investigated for the expression of
apoptosis markers. Each lane represents a different animal.
In Vivo Activity: A Combination with Venetoclax
We and others have previously demonstrated that the concurrent inhibition of BCL-2 and
MCL-1 results in marked cytotoxic synergy in lymphoma models.42,43 A loss of the
antiapoptotic protein MCL-1 observed in the cell line and xenograft lymphoma models treated
with 4.35 (Figures 3 and 5) suggests that the depletion may confer the lymphoma cell
dependency on other BCL2 family members. Therefore, we investigated the response of a
MAVER-1 xenograft to the combination 4.35 and venetoclax (ABT199), an inhibitor of BCL-
2
family proteins. The individual treatment of both compounds, 4.35 and venetoclax,
significantly inhibited tumor growth. Importantly, the inhibitory effect was more pronounced
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
when the mice were treated with a combination of these compounds, and the tumors did not
start to expand over the observed time period (Figure 6). The results are in line with recent
observations made with the CDK inhibitor dinaciclib, which clearly sensitized MYC-BCL-2
double-hit lymphomas to the BCL-2 inhibitor venetoclax.17,19
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. A combination of 4.35 and venetoclax in MAVER-1 xenografts. (A) Mean
calculated tumor volumes (± SD) of MAVER-1 s.c. tumors from the initiation of therapy (day
1
). MAVER-1 cells were xenografted to NSG mice and treated with vehicle (control, n = 6),
4.35 (9 mg/kg, n = 8), venetoclax (20 mg/kg, n = 8) or 4.35 + venetoclax combination (n =
0). (B) Evaluation of the mean calculated tumor weight (± SD) of MAVER-1 s.c. tumors
1
from the abovementioned therapies (= day 13).
Conclusion
Relatively slow progression of pan-CDK inhibitors in clinical trials, which is generally
attributed to their complicated mechanisms of action, narrow therapeutic windows and the
lack of robust patient selection criteria, collectively emphasize the necessity to develop and
explore new compounds with altered kinase profile. Herein, we describe the synthesis and
biological activity of new pyrazolo[4,3-d]pyrimidines that potentially inhibited cyclin-
2
0
ACS Paragon Plus Environment

Page 21 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
dependent kinase 2 as demonstrated by biochemical assays and crystallographic analysis. Our
findings confirmed the in vitro and in vivo sensitivity of aggressive non-Hodgkin lymphomas
to CDK inhibitors and provide rationale for their future clinical evaluation. In addition, the
combination of CDK inhibitors selected based on biological mechanism (targeting
predominantly MCL-1) and the BCL-2 targeting agent venetoclax was proven to exert
synergistic effects exhibit synthetic lethality in vivo and should be considered for
combinatorial treatment approaches in patients.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental Section
NMR spectra were recorded on a JEOL ECA-500 spectrometer operating at the frequencies of 500.16
1
13
1
13
MHz ( H) and 125.76 MHz ( C). H NMR and C NMR chemical shifts were referenced to the solvent
1
signals; for H, (residual CHCl ) = 7.25 ppm, (residual DMSO - d ) = 2.50 ppm, (residual CD OD) =
3
5
3
1
3
3
.31 ppm; C: (CDCl ) = 77.23 ppm, (DMSO-d ) = 39.52 ppm, and (CD3OD) = 49.15 ppm. Chemical
3
6
shifts are provided in the  scale [ppm] and the coupling constants are provided in Hz.
Melting points were determined on a Kofler block and are uncorrected. Reagents were of
analytical grade from standard commercial sources or were synthesized according to the referenced
procedure. Thin layer chromatography (TLC) was performed using aluminum sheets with silica gel
F254 from Merck. Spots were visualized under UV light (254 nm). ESI or APCI mass spectra were
determined using a Waters Micromass ZMD mass spectrometer (sample solution in MeOH, direct
inlet, coin voltage was in the range of 10–30 V, trace amounts of HCOOH or NH OH was used to
4
influence ionization). Column chromatography was performed using Merck silica gel Kieselgel 60
(230–400 mesh). The purity of all synthesized compounds was determined by HPLC–PDA (200–500
nm) and gave satisfactory results (> 95 %). Specific optical rotation was measured on a polarimeter
polAAr 3001 (wave length: 589.0 nm, tube length: 50 mm, at 23 °C). All compounds gave satisfactory
elemental analyses (± 0.4%).
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
-Isopropyl-5-sulfanyl-1(2)H-pyrazolo[4,3-d]pyrimidine-7-ol (1). This compound was prepared
according to literature.28
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3-Isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine-5,7-dithiol
(2).
3-Isopropyl-5-sulfanyl-1(2)H-
pyrazolo[4,3-d]pyrimidin-7-ol (1) (74 g, 0,35 mol) was added to a solution of phosphorus pentasulfide
93 g, 0.42 mol) in pyridine (550 mL) at 75 °C. The reaction mixture under nitrogen was refluxed for 3
(
h. Then, the mixture was concentrated by the distillation of pyridine at atmospheric pressure to a
syrupy consistency. Water (demineralized, approx. 400 mL) was added dropwise at 65 – 75 °C. The
resulting foamy mixture was heated at 95 °C for 1.5 h, and then the mixture was cooled to r.t. and
the solid product was filtered off and washed with water and EtOH (200 mL). The product was dried
under vacuum at 75 °C for a total of 61.7 g and a 76% yield. The analytical sample was crystallized
from THF (m.p. 283–293 °C; UV (MeOH, nm): 274 max, 320 max, 372 sh). MS APCI + m/z 227.1
+
- 1
(M+H) , APCI and ES – m/z 225.1 (M-H) . H (500 MHz; DMSO-d ): 1.20 (d, J = 7.03 Hz, 6H, -CH(CH ) );
6
3 2
3
1
.30 (bs, 1H); 13.33 (bs, 1H); 13.45 (bs, 1H); 13.84 (bs, 1H). 13C (125 MHz; DMSO-d ): 21.9; 24.8;
6
21.5; 133.0; 143.4; 170.1; 175.1.
3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine-5-thiol
pyridyl)benzylamine (2) (26.2 g; 0.14 mol) was dissolved in 2-methoxyethanole (150 mL) at 50 °C, and
-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidin-5,7-dithiol (2) (23 g, 0.10 mol) was added under nitrogen
(3).
4-(2-
3
gas. The reaction mixture was then refluxed for 8 h. The product crystallized after slow cooling at r.t.
and then at 5–10 °C. The product was filtered off, washed with 2-methoxyethanole and then with
THF and dried under vacuum at 75 °C. The product (30 g) was obtained as nearly white fine-grain
crystals with a 78% yield. Titration in AcOH with 0.1 N HClO gave 100% of the product. Analytical
4
samples were crystallized from DMSO/H O and dried under vacuum at 75 °C (m.p. 263–272 °C; UV
2
+
- 1
(MeOH, nm): 255 max, 289 max, 336 max). MS ESI + 377.1 (M+H) , ESI – 375.1 (M-H) . H (500 MHz;
DMSO-d ): 1.24 (d, J = 7.03 Hz, 6H); 3.32 (bs, 1H); 3.45 (bs, 1H); 4.80 (bs, 2H); 7.31–7.33 (m, 1H); 7.46
6
2
2
ACS Paragon Plus Environment

Page 23 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
bd, J = 6.42 Hz, 2H); 7.83–7.86 (m, 1H); 7.91–7.93 (m, 1H); 8.04 (bd, J = 7.34 Hz, 2H); 8.64 (bd, J =
3
1
.97 Hz, 1H). 13C (125 MHz; DMSO-d ): 21.8; 42.5; 54.8; 120.0, 122.4, 126.4, 127.8, 137.0, 149.4,
6
55.7.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-[3-(1-Piperazinyl)-2-hydroxy-1-propyl]thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-
pyrazolo[4,3-d]pyrimidine (4.1). The precursor of the synthesis, 3-(4-Boc-piperazin-1-yl)-1,2-
epoxypropane, was prepared by equimolar reaction of epichlorohydrin with N-Boc-piperazine in
acetonitrile in the presence of K CO (2 equivalents); the reaction mixture was stirred at 10 °C for 2
2
3
days. The crude product (used in subsequent synthesis) was isolated by filtration and then dried
under vacuum at a temperature below 45 °C.
The mixture of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine-5-
thiol 3 (380 mg, 1 mmol), tetramethylammonium hydroxide (0.2 mL 25% solution in MeOH),
trimethylamine (0.2 mL, 2.2 mmol) and 3-(4-Boc-piperazin-1-yl)-1,2-epoxypropane (0.45 g, 1.8 mmol)
in MeOH (5 mL) was stirred at room temperature for 2 days. The crude Boc product was isolated by
precipitation after adding water. The precipitate was dissolved in 3 mL TFA and was left 2 h at room
temperature. The solution was evaporated under vacuum, and the product was purified stepwise
(5%, 8% and 10%) with MeOH in CHCl with a trace amount of aqueous NH OH by column
3
4
chromatography. Chromatography provided (after evaporation under vacuum) an amorphous
+
- 1
colorless glass foam (0.25 g, 48% yield). ESI + 519.1 (M+H) , ESI – 517.1 (M-H) . H (500 MHz; DMSO-
d ): 1.36 (d, J = 7.03 Hz, 6H, -CH-CH ); 1.37 (d, J = 7.03 Hz, 6H, -CH-CH ); 2.33–2.42 (m, 6H, N-CH -CH-
6
3
3
2
OH + 2 x -CH -); 2.77–2.79 (m, 4H, 2 x -CH -); 3.01 (dd, J = 13.45 Hz, J = 7.03 Hz, 1H, -S-CH H -); 3.27
2
2
α β
(sept., J = 7.03 Hz, 1H, -CH(CH ) ); 3.35 (dd, J = 13.60 Hz, J = 4.58 Hz, 1H, -S-CH H -); 3.90 (m, 1H, -CH -
3
2
α
β
2
CH-OH); 4.78 (bs, 2H, -NH-CH -); 7.33 (qd, J = 7.41 Hz, J = 4.89 Hz, J = 1.22 Hz, 1H, H ); 7.49 (d, J =
2
Ar
8
.25 Hz, 2H, H ); 7.86 (td, J = 7.95 Hz, J = 1.83 Hz, 1H, H ); 7.93 (dt, J = 8.25 Hz, J = 1.1 Hz, 1H, H );
Ar
Ar
Ar
8.06 (d, J = 8.25 Hz, 2H, H ); 8.34 (bs, 1H, -NH-CH -); 8.65 (qd, J = 4.86 Hz, J = 1.83 Hz, J = 0.92 Hz, 1H,
Ar
2
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
H ); 12.80 (bs, 1H, -NH-). C (125 MHz; DMSO-d ): 21.6; 21.7; 25.7; 36.0; 42.9; 44.7; 53.1; 63.9; 67.2;
Ar
6
1
20.0; 122.4; 126.5; 127.9; 137.1; 137.5; 138.3; 139.8; 149.4; 150.1; 155.7; 157.9; 161.2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5[(3-(4-Morpholinyl)-2-hydroxy-1-propyl]thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-
pyrazolo[4,3-d]pyrimidine (4.2). 1-Chloro-3-morpholinopropan-2-ol (0.40 g, 2.2 mmol; prepared
4
4
according to the literature ) was added to a mixture of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-
(2)H-pyrazolo[4,3-d]pyrimidine-5-thiol 3 (0.38 g, 1 mmol) and K CO (0.17 g) in MeOH (10 mL), and
1
2
3
the mixture was stirred at 45 °C for 24 h. The crude product was precipitated by adding water and
then purified stepwise (3%, 4% and 5%) with MeOH in CHCl by column chromatography.
3
Chromatography provided (after evaporation under vacuum) an amorphous colorless glass foam
+
+
-
(
0.31 g, 60% yield). MS ESI + 520.1 (M+H) 15%, 542.1 (M+Na) 100%, ESI – 518.1 (M-H) 30%, 554.2
-
1
(
M+Cl) 100%. H (500 MHz; CDCl ): 1.19–1.21 (m, 6H, -CH(CH ) ); 2.31–2.35 (m, 3H, -CH-CH H -N- + -
3
3 2
α β
N-(CH H ) -); 2.42–2.46 (m, 2H, -N-(CH H ) -); 2.53 (dd, J = 12.53 Hz, J = 8.56 Hz, 1H, -CH-CH H -N);
α
β 2
α
β 2
α β
3
.11–3.19 (m, 2H, -CH(CH ) + S-CH H -CH-); 3.27 (d, J = 12.53 Hz, 1H, S-CH H -CH-); 3.50 (bs, 4H, O-
3
2
α
β
α β
(
CH ) -); 4.09–4.10 (m, 1H, -CH -CH-CH -); 4.65 (bs, 2H, -NH-CH -); 7.17 (dd, J = 7.18 Hz, J = 5.20 Hz,
2
2
2
2
2
1
H, H ); 7.23 (d, J = 7.95 Hz, 2H, H ); 7.42 (bs, 1H, -NH-); 7.59 (d, J = 7.95 Hz, 1H, H ); 7.69 (td, J =
Ar
Ar
Ar
1
3
7.79 Hz, J = 1.83 Hz, 1H, H ); 7.73 (d, J = 7.95 Hz, 2H, H ); 8.58 (d, J = 4.58 Hz, 1H, H ). C (125 MHz;
Ar
Ar
Ar
CDCl ): 21.5; 21.6; 26.1; 36.3; 44.2; 53.8; 63.9; 66.5; 68.5; 120.7; 122.2; 127.0; 128.1; 137.0; 138.3;
3
1
38.6; 149.4; 156.8; 162.9.
5-(1-Hydroxy-2-butyl)thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine (4.3). The precursor of the synthesis, 2-bromo-1-butanol, was obtained by introducing
diborane gas into a THF solution of 2-bromobutanoic acid at -10 °C for 5 h, then at 0 °C for 5 h, and
finally at 25 °C for 2 h. The excess diborane was consumed by adding 50% aq. HCOOH, and the
mixture was then concentrated by evaporation (15 torr, 25 °C). The remaining solution was
neutralized by aq. NaHCO and the product was extracted by EtOAc. The combined organic phase
3
,
2
4
ACS Paragon Plus Environment

Page 25 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was dried (Na SO ) and evaporated (15 torr, 25 °C). The crude product ( = 1.38 g/mL) was directly
2
4
used in the subsequent alkylation reaction.
The mixture of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine-5-
thiol 3 (0.225 g, 0.66 mmol), DMF (5 mL), K CO (0.2 g) and 2-bromo-1-butanol (120 L, 1.0 mmol)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
was stirred at 50 °C for 8 h. Subsequently, the reaction mixture was evaporated at a temperature
below 50 °C, and the residue was partitioned between EtOAc and H O. The combined organic phase
2
was dried (MgSO ) and evaporated under vacuum. The product was finally purified stepwise (2%, 3%
4
and 4%) with MeOH in CHCl by column chromatography and was crystallized from absolute Et O
3
2
+
-
1
(
0.07 g, 22% yield, m.p. 126–129 °C). MS ESI + 449.1 (M+H) , ESI – 447.1 (M-H) . NMR: H (500 MHz,
DMSO-d ) δ 0.96 (t, J = 7.4 Hz, 3H, -CH ); 1.35 – 1.39 (m, 6H, -CH(CH ) ); 1.55 – 1.63 (m, 1H, -CH H -
6
3
3 2
α β
CH ); 1.81 – 1.88 (m, 1H, -CH H -CH ); 3.24 (m, J = 6.9 Hz, 1H, -CH(CH ) ); 3.50 – 3.54 (m, 1H, -S-CH-);
3
α
β
3
3 2
3.70 – 3.75 (m, 2H, -CH -OH); 4.77 (t, J = 4.9 Hz, 2H, NH-CH -); 4.87 (bs, 1H, -OH), 7.32 – 7.35 (m, 1H,
2
2
H ); 7.45 – 7.55 (m, 2H, H ); 7.85 – 7.95 (m, 2H, H ); 8.00 – 8.09 (m, 3H, H + -NH); 8.65 – 8.66 (m,
Ar
Ar
Ar
Ar
1
3
1
4
H, H ); 12.16 (s, 1H, -NH). C (125 MHz, DMSO-d ) δ 11.3, 21.5, 21.6, 21.7, 23.6, 24.9, 26.3, 40.4,
Ar
6
3.1, 48.5, 63.1, 120.1, 120.4, 122.3, 122.5, 126.3, 126.6, 127.7, 128.0, 130.4, 137.1, 137.6, 138.6,
139.6, 139.9, 140.6, 148.6, 148.7, 149.4, 153.7, 155.6, 155.8, 161.1.
5
-(2-Methylthio-1-ethyl)thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine (4.4). To a solution of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidin-5-thiol 3 (0.38 g, 1.0 mmol) in DMF (6 mL) K CO (0.2 g) and 2-chloroethyl methyl sulfide
2
3
(101 L, 1 mmol) were added, and the mixture was stirred at room temperature for 16 h. The
reaction mixture was evaporated at a temperature below 50 °C, and the residue was partitioned
between CHCl and H O. The combined organic phase was dried with sodium sulfate and evaporated
3
2
under vacuum. The product was purified stepwise (2%, 3% and 4%) with MeOH in CHCl by column
3
chromatography, and the product was crystallized from abs. Et O (0.35 g, 74% yield, m.p. 140 – 141
2
+
- 1
°
C). APCI + 474.3 (M+H) , ESI – 472.2 (M-H) . H (500 MHz; CDCl ): 1.33 (d, J = 7.03 Hz, 6H, -CH(CH ) );
3
3 2
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
.12 (s, 3H, -S-CH ); 2.80–2.83 (m, 2H, H C-S-CH -); 3.26–3.30 (m, 3H, -S-CH - + -CH(CH ) ); 4.62 (bs,
3
3
2
2
3 2
H, -NH-CH -); 6.66 (bs, 1H, -NH-CH -); 7.15–7.22 (m, 3H, H ); 7.61 (d, J = 8.25 Hz, 1H, H ); 7.70–7.74
2
2
Ar
Ar
1
3
(m, 3H, H ); 8.59 (d, J = 4.89 Hz, 1H, H ); 12.13 (bs, 1H, -NH-). C (125 MHz; CDCl ): 15.2; 21.6; 26.2;
Ar
Ar
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
30.6; 34.0; 44.0; 121.0; 122.4; 127.0; 127.9; 137.2; 138.1; 138.7; 149.3; 157.0; 161.7.
5
-(2-Hydroxycyclohexyl)thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine (4.5). The 1,2-epoxycyclohexane (0.12 mL, 1.15 mmol) in MeOH (2 mL) was added to a
stirred mixture of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine-5-thiol 3
(0.38 g, 1 mmol) and tetramethylammonium hydroxide (catalytic amount, 80 mg) in MeOH (6 mL),
and the mixture was stirred at 40 °C for 7 h. The reaction mixture was evaporated at a temperature
below 50 °C, and the residue was partitioned between 2-methyltetrahydrofuran and H O. The
2
combined organic phase was dried with magnesium sulfate and evaporated under vacuum. The
product was purified stepwise (2%, 3% and 4%) with MeOH in CHCl by column chromatography.
3
Chromatography provided (after evaporation under vacuum) an amorphous colorless glass foam
+
- 1
(0.22 g, 46% yield). MS ESI + 475.1 (M+H) , ESI – 473.1 (M-H) . H (500 MHz; CDCl ): 1.18 (d, J = 7.03
3
Hz, 3H, -CH ); 1.20–1.22 (m, 2H, -CH H -CH -CH-S + -CH H -CH -CH-OH); 1.24 (d, J = 7.03 Hz, 3H, -
3
α
β
2
α
β
2
CH ); 1.33–1.40 (m, 2H, -CH H -CH-S- + -CH H -CH-OH); 1.68–1.70 (m, 2H, -CH H -CH -CH-S- + -CH H -
3
α
β
α
β
α
β
2
α β
CH -CH-OH); 2.08–2.17 (m, 2H, -CH H -CH-S- + -CH H -CH-OH); 3.19 (sept., J = 7.03 Hz, 1H, -
2
α
β
α β
CH(CH ) ); 3.45 (ddd, J = 13.21 Hz, J = 10.09 Hz, J = 3.97 Hz, 1H, -S-CH-); 3.54 (td, J = 10.70 Hz, J = 4.26
3
2
Hz, 1H, -CH-OH); 4.65 (bs, 2H, -NH-CH -); 7.17–7.21 (m, 3H, H ); 7.32 (bs, 1H, -NH-CH -); 7.58 (d, J =
2
Ar
2
1
3
7.95 Hz, 1H, H ); 7.68–7.72 (m, 3H, H ); 8.58 (d, J = 4.89 Hz, 1H, H ). C (125 MHz; CDCl ): 21.3;
Ar
Ar
Ar
3
2
1
1.7; 24.0; 26.0; 26.2; 30.2; 31.6; 36.5; 44.0; 51.2; 77.4; 120.8; 121.1; 127.0; 127.9; 137.0; 138.2;
38.6; 149.3; 157.0; 163.6.
5-(3,3,3-Trifluoro-2-hydroxy-1-propyl)thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-
pyrazolo[4,3-d]pyrimidine (4.6). 2-Bromo-1,1,1-trifluoro-2-propanol (0.11 mL, 1.05 mmol) was
2
6
ACS Paragon Plus Environment

Page 27 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
added dropwise to a mixture of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine-5-thiol 3 (0.38 g, 1.0 mmol) in DMF (6 mL) and K CO (0.12 g), and the reaction mixture
2
3
was stirred at room temperature for 4 h. The reaction mixture was evaporated at a temperature
below 50 °C, and the residue was partitioned between CHCl and H O. The product was purified
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
stepwise (2%, 3% and 4%) with MeOH in CHCl by column chromatography. The product was
3
+
-
crystallized from DCM (0.15 g, 31% yield, m.p. 174–177 °C). MS ESI + 489.1 (M+H) , ESI – 487.1 (M-H)
1
.
NMR: Two tautomers: 2:1 (integration showed in relative values) H (500 MHz; DMSO-d ): 1.35 (d, J
6
= 7.03 Hz, 6H, -CH(CH ) form I); 1.38 (d, J = 7.03 Hz, 6H, -CH(CH ) form II); 2.83–2.92 (m, 1H, -S-
3
2
3 2
CH H - form I and II); 3.21–3.27 (m, H, -CH(CH ) , form I and II); 3.62 (d, J = 13.75 Hz, 1H, -S-CH H ,
α
β
3 2
α β
form II); 3.68 (dd, J = 13.75 Hz, J = 2.14 Hz, 1H, -S-CH H , form I); 4.23 (bs, 1H, S-CH -CH-form I and II);
α
β
2
4
.72 (d, J = 5.81 Hz, 2H, NH-CH -, form II); 4.78 (d, J = 5.50 Hz, 2H, NH-CH -, form I); 6.59 (d, J = 6.72
2
2
Hz, 1H, CH-OH, forn I and II); 7.32–7.35 (m, 1H, H , form I and II); 7.45 (d, J = 7.95 Hz, 2H, H , form
Ar
Ar
II); 7.50 (d, J = 8.25 Hz, 2H, H , form I); 7.82–7.87 (m, 1H, H , form I and II); 7.90–7.95 (m, 1H, H ,
Ar
Ar
Ar
form I and II); 8.01 (d, J = 7.95 Hz, 2H, H , form II); 8.08–8.10 (m, 3H, H + -NH-CH -, form I); 8.64–
Ar
Ar
2
8
1
.66 (m, 1H, H , form I and II); 8.79 (bt, J = 5.81 Hz, 1H,-NH-CH -, form II); 12.22 (bs, 1H, -NH-, form I);
Ar
2
1
3
3.80 (bs, 1H, -NH-, form II). C (125 MHz; DMSO-d ): mixture of two forms 21.3; 21.4; 21.5; 25.0;
6
26.3; 26.4; 31.2; 42.4; 43.1; 67.9 (q, -CF , form I); 68.0 (q, -CF , form II); 120.0; 120.1 120.5; 122.3;
3
3
1
1
22.5; 124.2; 126.3; 126.5; 126.6; 127.6; 128.0; 130.5; 134.8; 137.10; 137.15; 137.2 137.7; 138.9;
39.3; 139.7; 140.3; 148.8; 149.43; 149.48; 154.0; 155.6; 155.8; 160.0.
5-(Prop-2-en-1-yl)thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine
4.7). A 1N solution of NaOH (1.4 mL) and allyl bromide (0.13 mL, 1.1 mmol) was added to a
suspension of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine-5-thiol 3
0.38 g, 1.00 mmol) in H O (20 mL), and the mixture was stirred at room temperature for 6 h. The
(
(
2
product was filtered off and recrystallized from CHCl /Et O (0.28 g, 67% yield, m.p. 148–150 °C). MS
3
2
+
- 1
ESI + 417.2 (M+H) , ESI – 415.3 (M-H) . H (500 MHz; DMSO-d ): 1.38 (d, J = 6.72 Hz, 6H, -CH(CH ) );
6
3 2
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
5
5
.28 (sept., J = 7.03 Hz, 1H, -CH(CH ) ); 3.74 (d, J = 7.03 Hz, 2H, -S-CH -CH); 4.77 (bs, 2H, NH-CH -);
3
2
2
2
.02 (dd, J = 9.78 Hz, J = 0.92 Hz, 1H, CH H =CH-); 5.24 (dd, J = 16.96 Hz, J = 1.53 Hz, 1H, CH H =CH-);
α
β
α β
.95 (ddt, J = 17.1 Hz, J = 10.1 Hz, J = 7.03 Hz, 1H, CH =CH-CH -); 7.32 (ddd, J = 7.39 Hz, J = 4.89 Hz, J =
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.22 Hz, 1H, H ); 7.49 (d, J = 8.56 Hz, 2H, H ); 7.84 (td, J = 7.64 Hz, J = 1.83 Hz, 1H, H ); 7.92 (d, J =
Ar
Ar
Ar
7
.95 Hz, 1H, H ); 8.06 (d, J = 8.25 Hz, 2H, H ); 8.27 (bs, 1H, -NH-CH -); 8.65 (ddd, J = 4.86 Hz, J = 1.83
Ar
Ar
2
1
3
Hz, J = 0.92 Hz, 1H, H ); 12.58 (bs, 1H, -NH-). C (125 MHz; DMSO-d ): 21.6; 25.8; 33.0; 42.9; 116.7;
Ar
6
1
20.0; 122.4; 126.5; 127.8; 134.9; 137.1; 137.5; 139.8; 149.4; 155.7; 160.1.
5[3-(Dimehylamino)-1-propyl]thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine (4.8). K CO (0.6 g) and 3-chloro-N,N-dimethylpropylamine hydrochloride (182 mg, 1.15
2
3
mmol) were added to a solution of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine-5-thiol 3 (0.38 g, 1 mmol) in DMSO (10 mL), and the mixture was stirred at 60 °C for 24
h. The reaction mixture was evaporated at a temperature below 50 °C, and the residue was
partitioned between CHCl and H O. The product was purified stepwise (5%, 8% and 10%) with
3
2
MeOH in CHCl by column chromatography. Chromatography provided (after evaporation under
3
2
+
vacuum) an amorphous colorless glass foam (0.33 g, 72% yield). MS ESI + 231.6 (M+2H) 50%, 462.1
+
- 1
(M+H) 100%, ESI – 460.1 (M-H) . H (500 MHz; CDCl ): 1.34 (d, J = 6.72 Hz, 6H, -CH(CH ) ); 1.92 (pent.,
3
3 2
J = 7.03 Hz, 2H, -CH -CH -CH -); 2.22 (s, 6H, -N(CH ) ); 2.44 (t, J = 7.34 Hz, 2H, -CH -); 3.09 (t, J = 7.03
2
2
2
3 2
2
Hz, 2H, -CH -); 3.29 (sept., J = 6.72 Hz, 1H, -CH(CH ) ); 4.68 (bs, 2H, NH-CH -); 6.87 (bs, 1H, -NH-);
2
3 2
2
7
.18–7.20 (m, 1H, H ); 7.27 (d, J = 7.64 Hz, 2H, H ); 7.60 (d, J = 7.95 Hz, 1H, H ); 7.70 (t, J = 7.93 Hz,
Ar
Ar
Ar
1
3
1H, H ); 7.76 (d, J = 7.64 Hz, 2H, H ); 8.60 (d, J = 3.97 Hz, 1H, H ). C (125 MHz; CDCl ): 21.7; 26.2;
Ar
Ar
Ar
3
2
1
7.4; 28.9; 29.6; 43.9; 45.1; 58.5; 120.8; 122.2; 127.0; 127.9; 137.0; 138.0; 139.0; 149.3; 150.6; 156.9;
62.0.
5-[2-(Dimethylamino)-1-ethyl)]thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine (4.9). K CO (0.6 g) and 2-chloro-N,N-dimethylethylamine hydrochloride (166 mg, 1.15
2
3
2
8
ACS Paragon Plus Environment

Page 29 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mmol) were added to a solution of 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidin-5-thiol 3 (0.38 g, 1 mmol) in DMSO (10 mL), and the mixture was stirred at 60 °C for 24 h.
The reaction mixture was evaporated at a temperature below 50 °C, and the residue was partitioned
between CHCl and H O. The product was purified stepwise (5%, 8% and 10%) with MeOH in CHCl by
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
3
column chromatography. Chromatography provided (after evaporation under vacuum) an
2
+
+
amorphous colorless glass foam (0.30 g, 67% yield). MS ESI + 224.5 (M+2H) 70%, 448.1 (M+H)
-
1
1
00%, ESI – 446.1 (M-H) . H (500 MHz; CDCl ): 1.31 (d, J= 7.03 Hz, 6H, -CH-(CH ) ); 2.24 (s, 6H, 2x –
3
3 2
CH ); 2.66 (t, J = 7.64 Hz, 2H, -CH -); 3.20 (t, J = 7.34, 2H, -CH -); 3.27 (sept.; J = 7.03 Hz, 1H, -CH-
3
2
2
(CH ) ); 4.62 (bs, 2H, -NH-CH -); 6.97 (bs, 1H, -NH-CH-); 7.15–7.16 (m, 1H, ArH); 7.17 (d, J = 7.95 Hz,
3
2
2
2
2
1
H, ArH); 7.55 (d, J = 8.25 Hz, 1H, ArH); 7.66 (dd, J = 7.64 Hz, J= 1.83 Hz, 1H, ArH); 7.69 (d, J = 7.95 Hz,
1
3
H, ArH); 8.55 (d, J = 3.97 Hz, 1H, ArH). C (125 MHz; CDCl ): 21.6; 26.2; 28.0; 43.9; 44.9; 59.0; 120.8;
3
22.2; 126.9; 127.9; 137.0; 138.0; 138.9; 149.3; 150.6; 156.9; 161.6.
5
-(1-Hydroxy-3-methyl-2-buthyl)thio-3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-
d]pyrimidine (4.10). The precursor 2-bromo-3-methyl-1-butanol was prepared from 3-methylbutenal
4
5
by bromination with a bromine-dioxane complex (1:1) according to the literature. The obtained 2-
bromo-3-methylbutanal was then reduced to the desired 2-bromo-3-methyl-1-butanol in the usual
manner using NaBH and was used for alkylation without purification.
4
K CO (0.6 g) and 2-bromo-3-methyl-1-butanol (0.25 g, 1.5 mmol) were added to a solution of
2
3
3
-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine-5-thiol 3 (0.38 g, 1 mmol)
in DMSO (10 mL), and the mixture was stirred at room temperature for 48 h. The reaction mixture
was kept at a temperature below 50 °C. The residue was partitioned between CHCl and H O, and the
3
2
product was purified stepwise (2%, 3% and 4%) with MeOH in CHCl by column chromatography.
3
Chromatography provided (after evaporation under vacuum) an amorphous colorless glass foam
+
- 1
(0.31 g, 67% yield). MS ESI + 463.1 (M+H) , ESI – 461.1 (M-H) . H (500 MHz; CDCl ): 0.92 (d, J = 6.72
3
Hz, 3H, -CH ); 0.97 (d, J = 6.72 Hz, 3H, -CH ); 1.23 (d, J = 6.72 Hz, 3H, -CH ); 1.24 (d, J = 7.03 Hz, 3H, -
3
3
3
2
9
ACS Paragon Plus Environment