Synthesis and kinase inhibitory activity of new sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazines

Article abstract of DOI:10.1016/j.ejmech.2014.03.054

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine has been synthesized and characterized. Their anticancer activity was tested in vitro against multiple human cancer cell lines and were found to have dose-dependent antiproliferative effects. Furthermore, some of the new compounds inhibited the Abl protein kinase with low micromolar IC₅₀ values and exhibited selective activity against the Bcr-Abl positive K562 and BV173 cell lines, providing starting points for the further development of this new kinase inhibitor scaffold.

Full text of DOI:10.1016/j.ejmech.2014.03.054

European Journal of Medicinal Chemistry 78 (2014) 217e224
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and kinase inhibitory activity of new sulfonamide
derivatives of pyrazolo[4,3-e][1,2,4]triazines
, ,
1
b,
Mariusz Mojzych a , Veronika Subertová 1, Anna Bielawska c, Krzysztof Bielawski c,
ꢀ
*
b
d
b
e
b,
*
ꢀ
ꢀ
Václav Bazgier , Karel Berka , Tomás Gucký , Emilia Fornal , Vladimír Krystof
a Department of Chemistry, Siedlce University of Natural Sciences and Humanities, ul. 3 Maja 54, Siedlce 08-110, Poland
b Centre of the Region Haná for Biotechnological and Agricultural Research, Laboratory of Growth Regulators, Faculty of Science, Palacký University,
ꢀ
ꢁ
Slechtitelu 11, 783 71 Olomouc, Czech Republic
c Department of Medicinal Chemistry and Drug Technology, Medical University of Bialystok, Bialystok, Poland
d Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University Olomouc,
17. listopadu 12, 77146 Olomouc, Czech Republic
e Department of Chemistry, Laboratory of Separation and Spectroscopic Method Applications, Center for Interdisciplinary Research,
The John Paul II Catholic University of Lublin, al. Krasnicka 102, 20-718 Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine has been synthesized and
characterized. Their anticancer activity was tested in vitro against multiple human cancer cell lines and
were found to have dose-dependent antiproliferative effects. Furthermore, some of the new compounds
inhibited the Abl protein kinase with low micromolar IC50 values and exhibited selective activity against
the Bcr-Abl positive K562 and BV173 cell lines, providing starting points for the further development of
this new kinase inhibitor scaffold.
Received 2 December 2013
Received in revised form
13 March 2014
Accepted 16 March 2014
Available online 18 March 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Bcr-Abl
Kinase
Inhibitor
Synthesis
Cancer
Leukaemia
Pyrazolo[4,3-e][1,2,4]triazine
1. Introduction
active tyrosine kinase that initiates the onset of chronic myeloge-
nous leukaemia [4].
Nitrogen-containing heterocycles have attracted huge interest
over the past decades because of their diverse pharmacological
activities, including protein kinase inhibition. Being involved in
nearly all aspects of life at the cellular level, protein kinases have
become the most important targets of drugs for various indications,
such as cancers and inflammations [1e3]. Medicinal chemists have
developed probes targeting a substantial fraction of the human
kinome, and dozens of kinase inhibitors have been evaluated in
clinical trials since the milestone approval of imatinib, the first anti-
kinase drug targeting the fusion protein Bcr-Abl, a constitutively
Despite the clear clinical success of imatinib, many tumours
become resistant to it, often due to point mutations in the kinase
domain of Bcr-Abl. Several second-generation drugs have been
developed that inhibit some imatinib-resistant Bcr-Abl mutants,
but there are still significant numbers of resistant mutants for
which no alternative drugs are available [5]. The same may well be
true for other pharmacologically relevant kinases. Consequently,
there is still a need for new compounds that will enrich the pool of
known inhibitors, especially if they possess novel scaffolds, binding
modes or mechanisms of action.
Pyrazolo[4,3-e][1,2,4]triazine is a scaffold that has not yet
received significant attention in drug discovery although some of
its derivatives are known to exhibit biological activity. Over the last
few decades, seven naturally occurring pyrazolo[4,3-e][1,2,4]tri-
azines have been isolated and characterized spectroscopically and
* Corresponding authors.
ꢀ
E-mail address: vladimir.krystof@upol.cz (V. Krystof).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2014.03.054
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

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M. Mojzych et al. / European Journal of Medicinal Chemistry 78 (2014) 217e224
structurally: pseudoiodinine, nostocine A, and fluviols AeE [6e9].
In three cases, the proposed structures were verified by total syn-
thesis [10]. Importantly, synthetic 1,3,5-substituted pyrazolo[4,3-e]
[1,2,4]triazines have been reported to exhibit significant activity
against human cancer cell lines and to weakly inhibit cyclin-
dependent kinase CDK2 [11].
[1,2,4]triazine core. The new compounds are shown to exhibit se-
lective toxicity towards Bcr-Abl positive cells.
2. Results and discussion
2.1. Chemistry
The reported biological activities of pyrazolo[4,3-e][1,2,4]tri-
azines might stem, at least in part, from their physicochemical
similarity to purine, another nitrogen-containing heterocycle that
is widely distributed in nature [12]. To date, chemical modification
of the purine core (via scaffold hopping) has led to the discovery of
various purine bioisosteres that have been used as scaffolds for the
synthesis and development of chemical probes and drugs [12,13],
including cyclin-dependent kinase (CDK) inhibitors [14]. The CDKs
are a family of Ser/Thr kinases which, in association with specific
cyclins, play critical roles as regulators of the different phases of the
cell cycle. These enzymes and their direct regulators are frequently
mutated, amplified, or deleted in malignant cells, suggesting that
pharmacological CDK inhibition may be an effective strategy for
treating cancer [14]. We have previously prepared and character-
ized many CDK inhibitors based on purine scaffolds [15e17] and
bioisosteric alternatives such as 8-azapurines and pyrazolo[4,3-d]
pyrimidines. Many of the compounds based on these bioisosteres
are comparable or superior to the corresponding purine derivatives
in terms of anticancer activity and kinase inhibition [18,19]. The
discovery of kinase inhibitors based on new scaffolds is a major goal
of our current research, and this prompted us to investigate the
structureeactivity relationships of pyrazolo[4,3-e][1,2,4]triazines.
We have previously described the convenient synthesis and
structural analysis of a series of 3,5,7-trisubstituted pyrazolo[4,3-e]
[1,2,4]triazines [20e22]. Herein we describe the synthesis and
in vitro biological activity of sulfonamides with a pyrazolo[4,3-e]
The synthetic pathway leading to the title compounds is out-
lined in Scheme 1. Methylhydrazone 1, the precursor of pyrazolo
[4,3-e][1,2,4]triazine 2, was prepared using a standard procedures.
The cyclization of 1 to form 2 was then achieved under acidic
conditions using a literature method [20,21]. Compound 2 was
treated with potassium permanganate under phase transfer cata-
lytic conditions at room temperature for 1 h to give sulfone 3 in
nearly quantitative yield. Compound 3 was then reacted with
anhydrous aniline in a sealed tube at 150 ꢀC for 9 days. Chlor-
osulfonylation of the aniline-substituted pyrazolotriazine 4 in
chlorosulfonic acid at 0 ꢀC proceeded smoothly and selectively at
the 40-position of the phenyl ring to give the desired compound 5 in
excellent yield. The chlorosulfonyl derivative 5 was readily coupled
with amines to produce the target sulfonamides 6ael as shown in
Scheme 1.
The structures and purity of the newly synthesized compounds
were characterized using 1H and 13C NMR spectroscopy, HPLC-MS,
and elemental analysis. The spectral data confirmed that all of the
new sulfonamides had the expected structures and were of high
purity.
2.2. Antiproliferative activity in vitro
The antiproliferative activity of the new compounds against
several breast carcinoma (MCF-7, MDA-MB-231) and leukaemia
Scheme 1. Reagents and conditions: (a) p-TSA, T; (b) KMnO4, Bu4NBr, CH3COOH, benzene-H2O, rt, 2 h; (c) aniline, seal tube, 9 days, 150 ꢀC; (d) ClSO3H, 0 ꢀC to rt, 2 h; (e) NH3 or
amine, MeCN, rt.

M. Mojzych et al. / European Journal of Medicinal Chemistry 78 (2014) 217e224
219
(K562, BV173, HL60, CCRF-CEM) cell lines was measured using the
MTT assay after 24 h of incubation [23]. The results obtained are
summarized in Table 1. Concentration-dependent activity was
observed in all cases. The breast carcinoma lines were much less
sensitive to all of the compounds (with IC50 values ranging from
100 to 200 mM) than the leukaemia cell lines. However, in general,
the compounds had similar rankings with respect to activity in all
cell lines, with the most active being 6a, 6c, 6e and 6h. All of these
species have similar side chains (N-methylpiperazine, piperazine,
homopiperazine, 1,2-diaminoethane). Conversely, compounds 6d,
6f, 6i, and 6j exhibited very little activity.
The IC50 values for the tested leukaemia cell lines ranged from
20 to 70
for BV173. The IC50 range for the K562 line was even broader, with
the most potent compound 6e having a value of 21 M while 6j was
inactive even at 200 M. Surprisingly, the IC50 values for the most
mM for CCRF-CEM, 24 to 58 mM for HL60, and 27 to 72 mM
m
m
potent compounds against K562 e 6e and 6c e were 7 and 5 times
lower than their IC50 values against breast carcinoma cell lines,
respectively. This suggests that these compounds exhibit a degree
of selective toxicity towards the K562 cell line. A similar activity
pattern was observed in the BV173 cell line, with the most potent
compounds again being 6e and 6c.
The compounds’ antiproliferative activities are probably at least
partly due to their cytostatic effects: preliminary investigations
using breast carcinoma cell lines showed that they prevent the
incorporation of [3H]thymidine into living cells. As in the MTT
assay, all of the tested compounds exhibited concentration-
dependent activity with different potencies (Supplementary
Table S1). However, cytostatic effects alone cannot fully explain
their antiproliferative activity because in several cases, the number
of viable cells declined significantly after as little as 24 h of treat-
ment. We therefore investigated the influence of the most potent
compounds (6c, 6e and 6h) on the viability of K562 cells using the
propidium iodide exclusion assay. Treatment with compound 6c or
6e caused sharp increases in the number of cells with compromised
cell membranes (Fig. 1a). In parallel, we evaluated the test com-
pounds’ effects on cell cycling and found that 6c and 6e had no
effect on the distribution of cell cycle phases (data not shown) but
did cause pronounced increases in the sub-diploid population,
which is indicative of ongoing apoptotic cell death (Fig. 1b). Com-
pound 6h had only marginal effects on viability and did not in-
crease the sub-diploid cell population.
Fig. 1. Effect of test compounds on K562 cell viability after 24 h treatment. Propidium
iodide exclusion (a) and the sub-diploid cell population (b) were quantified by flow
cytometry in native or in ethanol-fixed cells, respectively.
Table 1
2.3. Kinase inhibitory activity
In vitro antiproliferative activity of new sulfonamide derivatives of pyrazolo[4,3-e]-
[1,2,4]triazine after 24 h incubation.
The enhanced potency of the new pyrazolo[4,3-e][1,2,4]tri-
azines against the K562 and BV173 cell lines (both of which are Bcr-
Abl positive) relative to other cell lines prompted us to explore their
inhibitory potency against a purified recombinant Abl kinase. The
seven tested compounds significantly inhibited Abl activity, with
IC50 values in the micromolar range (Table 2). The most potent
Compd.
MTT assay, IC50
(
m
M)a
MCF-7 MDA-
K562
BV173
HL60
CCRF-CEM
MB-231
6a
6b
6c
6d
6e
6f
6g
6h
6i
102 ꢁ 2 99 ꢁ 2
66 ꢁ 5 40 ꢁ 8
90 ꢁ 8 58 ꢁ 5
27 ꢁ 4 22 ꢁ 6
49 ꢁ 2 36 ꢁ 2
39 ꢁ 1 69 ꢁ 8
55 ꢁ 2 20 ꢁ 2
>200 >200
150 ꢁ 2 130 ꢁ 2
>200 >200
140 ꢁ 3 155 ꢁ 2
>200 >200
compounds 6c and 6e bear piperazine (IC50 values of 5.8
mM) and
100 ꢁ 4 41 ꢁ 10 42 ꢁ 5 49 ꢁ 8
homopiperazine (IC50 values of 5.9 M) appendages, respectively.
m
21 ꢁ 5 22 ꢁ 4
38 ꢁ 1 36 ꢁ 12
Such rings, and especially the secondary amines within them, seem
to be important for Abl inhibition: analogues bearing a methyl-
substituted piperazine (6a) or deaza and oxo analogues (piperi-
dine and morpholine derivatives 6g and 6b, respectively) exhibited
dramatically lower activity (Table 2 and supplementary table S2).
The acyclic 2-aminoethylamine derivative 6h also exhibited
102 ꢁ 1 47 ꢁ 14 56 ꢁ 6 30 ꢁ 2
200 ꢁ 2 140 ꢁ 1
98 ꢁ 2 36 ꢁ 9
77 ꢁ 7 39 ꢁ 8
24 ꢁ 2 30 ꢁ 2
42 ꢁ 6 56 ꢁ 2
126 ꢁ 1 120 ꢁ 1
>200
>200
>200
>200
106 ꢁ 8 45 ꢁ 11 58 ꢁ 3 50 ꢁ 1
6j
6k
6l
>200
58 ꢁ 9
40 ꢁ 2 54 ꢁ 8
41 ꢁ 1 64 ꢁ 6
44 ꢁ 5 57 ꢁ 3
38 ꢁ 2 21 ꢁ 8
55 ꢁ 7 45 ꢁ 1
146 ꢁ 1 125 ꢁ 2
96 ꢁ 3 39 ꢁ 8
200 ꢁ 2 140 ꢁ 2 101 ꢁ 2 42 ꢁ 9
somewhat lower activity (IC50 ¼ 15
mM). All other derivatives were
Chlorambucil 97 ꢁ 2
93 ꢁ 2
84 ꢁ 6 34 ꢁ 8
13 ꢁ 2 20 ꢁ 6
Imatinib
n.a.
n.a.
either much weaker inhibitors (unsubstituted sulfonamide 6i and
hydroxyalkyl sulfonamides 6d and 6l) or entirely inactive at the
tested concentrations (6j and 6f).
a
The reported values represent the mean ꢁ S.D. for each compound based on four
independent experiments.

220
M. Mojzych et al. / European Journal of Medicinal Chemistry 78 (2014) 217e224
Table 2
compounds synthesized in this work did not inhibit either CDK2 or
CK2 within the tested concentration range, showing that they have
some degree of selectivity for Abl (Table 2). The only exception is
compound 6h, which exhibited weak activity towards CDK2
Inhibition of protein kinases by sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]
triazines.
Compd.
IC50
Abl
(mM)
(IC50 ¼ 34.9
mM). This may contribute to its marginal anti-
CDK2
CK2
proliferative effects (Table 1). These preliminary selectivity data are
however not sufficient to exclude the possibility that the new
compounds may bind to and inhibit other targets, including other
protein kinases.
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
>50
>25
5.8
39
5.9
>50
>25
15.0
33.0
>50
20
34.0
>100
0.3
>50
>25
>25
>50
>50
>50
>25
>50
>25
>25
>50
>50
>50
>25
>50
>50
>50
>50
>50
n.a.
To clarify the new compounds’ lack of activity towards CDK2
and guide future optimization, we investigated the interactions
of the pyrazolo[4,3-e][1,2,4]triazine derivatives with CDK2. Mo-
lecular docking experiments suggested that the mostly negative
results obtained in the biochemical assays are due to the rela-
tively unfavourable mode of binding adopted by the pyrazolo
[4,3-e][1,2,4]triazines in the CDK2 active site (Fig. 3). Well-known
purine CDK inhibitors such as olomoucine and roscovitine typi-
cally bear small hydrophobic chain at N9, an aromatic ring
attached to the secondary amino group at C6, and a polar alkyl
amine at C2. In addition, they have no substituents at N7
[13,14,16]. This substitution pattern, together with the donore
acceptor H-bond motif formed by the N7 group and the sec-
ondary amino group at C6 that interacts with the hinge region of
CDK2, gives the known CDK2 inhibitors a well-defined binding
orientation within the CDK2 active site. Because pyrazolo[4,3-e]
[1,2,4]triazines cannot be substituted at the corresponding posi-
tions, we anticipated, based on the well known structureeac-
tivity relationships for CDK inhibitors [15e17], that they might
instead bind to CDK2 in a ‘reversed’ orientation, similar to that
adopted by the guanine-based inhibitor NU6102 [25]. NU6102
binds to the hinge region via H-bonds involving the secondary
amino group at C2 and purine nitrogens N3 and N9 (Fig. 3).
However, it is likely that the methyl substitution of the pyrazole
ring would both abolish one of these H-bonds and also form an
unfavourable steric interaction that would discourage the bind-
ing of 6h in the active site (Fig. 3a).
34.9
>50
>50
>50
>50
0.1
>100
1.8
Roscovitine
Imatinib
Silmitasertib
n.a.
0.1
n.a.
To better understand the molecular basis for the inhibitory ac-
tivity of the pyrazolo[4,3-e][1,2,4]triazine derivatives, we used
molecular modelling to rationalize their binding to Abl. We hy-
pothesized that compounds 6c and 6e might bind to the ATP-
binding site of the Abl kinase in a manner similar to that of pyr-
ido[2,3-d]pyrimidine PD180970, interacting with the protein pri-
marily via non-polar interactions and H-bonds with the backbone
NH group of the M318 residue [24]. Our studies confirmed that both
6c and 6e dock into the Abl kinase in this way. The piperazine ring
of 6c fits in to a pocket of the active site that is lined by the gate-
keeper residue T315. This is the pocket occupied by the dichlor-
ophenyl group of PD180970 and the methylbenzene moiety of
imatinib. In addition, we observed a possible H-bond between I313
and the secondary amino groups on the sulfonamide appendages of
6c and 6e (Fig. 2). This H-bond has not previously been observed in
any Abl co-crystal structure but its presence is consistent with the
SAR data presented above: the analogous 2-aminoethylamine de-
rivative 6h is less active despite having a primary amine at about
the right distance from I313 to form a similar H-bond, probably due
to the greater flexibility of its side chain. The same is true for the
even less active hydroxy derivative 6d. The inactivity of compounds
that lack a secondary amine or other H-bond acceptor capable of
forming an interaction of this sort (e.g. the methyl-substituted
compound 6a, piperidine 6g, and morpholine 6b) further sup-
ports this hypothesis.
2.5. Cellular inhibition of tyrosine kinases
Compounds 6c and 6e emerged as most potent Abl inhibitors
from this series. Because of their strong antiproliferative activity in
Bcr-Abl positive cell lines (K562 and BV173), we decided to char-
acterize their cellular effects in more detail. Derivative 6h was also
included in these experiments due to its lower activity and broader
selectivity. We treated K652 cells with increasing doses of 6c, 6e
and 6h for 1 h and immunoblotted the cell lysates with phospho-
specific antibodies towards phosphorylated forms of STAT5 and
CrkL, known substrates of Bcr-Abl kinase that both contribute to the
sustained proliferation of leukaemic cells (Fig. 4a). All three
2.4. Kinase inhibition selectivity
We have recently demonstrated that certain pyrazolo[4,3-e]
[1,2,4]triazines inhibit CDK2/cyclin E [22]. However, most of the
Fig. 2. The binding poses of compound 6e (a) and PD180970 (b) in c-Abl (PDBID: 2hzi). The compounds’ interactions with I313 and M318 are indicated by dotted lines.

M. Mojzych et al. / European Journal of Medicinal Chemistry 78 (2014) 217e224
221
Fig. 3. The binding poses of compound 6h (a) and NU6102 (b) in CDK2 (PDBID: 2c60). Interactions with D86, K89 and with the backbone of L83 and E81 are indicated by dotted
lines.
derivatives caused doseeresponse decreases in the phosphoryla-
tion of STAT5, but only 6c reduced the phosphorylation of CrkL.
We also evaluated the influence of the most potent compounds
on the global tyrosine phosphorylation status of the proteins in
K562 cells. This was done by immunoblotting their lysates with an
anti-phosphotyrosine antibody (Fig. 4b). In untreated cells, several
proteins phosphorylated on tyrosine residues were detected.
Treatment with derivatives 6c and 6e reduced the phosphorylation
of some of these proteins in a dose-dependent manner, including
some that formed intense bands with molecular weights between
150 and 250 kDa and probably corresponded to Abl and Bcr-Abl
[26]. In addition, at least three additional phosphoproteins with
molecular weights between 40 and 60 kDa (which yielded weaker
bands) exhibited dose-dependent reductions in signal intensity.
Importantly, treatment with the less active derivative 6h did not
reduce the phosphorylation of any of these proteins whereas
imatinib (which was used as a positive control in both experiments)
rapidly inhibited the phosphorylation of several proteins.
3. Conclusions
We have described an efficient method for the preparation of
sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazines (6).
Approximately half of the prepared compounds exhibited modest
activity towards cancer cell lines. In addition, our study identified
two compounds, 6c and 6e, that exhibit micromolar inhibition of
the Abl protein kinase and preferential antiproliferative activity in
leukaemia cell lines that overexpress the oncogenic kinase Bcr-Abl.
These compounds represent new scaffolds for protein kinase in-
hibitors, which are still needed in oncological drug discovery,
especially because of the emerging resistance to existing drugs.
Further studies aimed at optimizing the structures of the hits in
order to increase their potency against the Bcr-Abl kinase and
cancer cell lines in vitro are currently underway in our laboratories.
4. Experimental section
4.1. Chemistry
Melting points were determined on a Mel-Temp apparatus and
are uncorrected. 1H and 13C NMR spectra were recorded on a Varian
spectrometer (400 and 100 MHz, respectively). Chemical shift
values are expressed in ppm (part per million) relative to tetra-
methylsilane as an internal standard. The relative integrals of peak
areas agreed with those expected for the assigned structures. The
molecular weights of the final compounds were determined by
electrospray ionization mass spectrometry (ESI/MS) performed
using an Agilent Technologies 6538 UHD Accurate Mass Q-TOF LC/
MS. All elemental compositions were within ꢁ0.4% of the calcu-
lated values.
4.2. Synthesis of precursors
Fig. 4. Phosphoprotein analysis of K562 cells treated with test compounds. The cells
were incubated for 1 h with the indicated doses of 6c, 6e and 6h (in mM), lysed and
immunoblotted with anti-phospho-STAT5, anti-STAT5, anti-phospho-CrkL and anti-
tubulin antibodies (a). In a second experiment, the immunoblotting was performed
with an anti-phosphotyrosine antibody (b). Imatinib (0.1, 1 or 10 mM) was used as a
4.2.1. 1,3-Dimethyl-5-phenylamino-1H-pyrazolo[4,3-e][1,2,4]
triazine (4)
A mixture of 3 (1.68 g, 7.37 mmol) and freshly distilled aniline
(10 ml, 110 mmol) was introduced into a sealed tube and heated at
positive control in both experiments.

222
M. Mojzych et al. / European Journal of Medicinal Chemistry 78 (2014) 217e224
150 ꢀC for 9 days. The excess aniline was then distilled out from the
reaction mixture, and the resulting residue was purified by column
chromatography on silica gel, eluting with a mixture of ethyl ace-
tate/hexane (1:1). The combined fractions were evaporated,
157.32. HRMS (ESI, m/z) calc. for C16H21N8O2S [MþH] 389.1503.
Found 389.1502. Anal. Calcd for C16H20N8O2S: C, 49.47; H, 5.19; N,
28.85. Found: C, 49.17; H, 5.23; N, 28.67.
providing the crude product
4
as an orange solid (0.74 g,
: 2.49
4.3.4. 4-(1,3-Dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-
3.07 mmol). Yield 42%, mp 215e217 ꢀC. 1H NMR (DMSO-d6)
d
ylamino)-N-(2-hydroxyethyl)-benzenesulfonamide (6d)
(s, 3H), 4.09 (s, 3H), 6.98 (t, 1H, J ¼ 7.6 Hz), 7.32 (t, 2H, J ¼ 7.4 Hz),
Yield 65%, mp 242e244 ꢀC. 1H NMR (DMSO-d6)
d: 2.53 (s, 3H),
7.81 (d, 2H, J ¼ 9.0 Hz), 10.26 (s, 1H, NH). 13C NMR (DMSO-d6)
d:
2.78 (t, 2H, J ¼ 6.0 Hz), 3.36 (q, 2H, J ¼ 6.0 Hz), 4.16 (s, 3H), 4.64 (t,
10.72, 34.67, 118.54, 121.93, 128.95, 133.05, 138.33, 140.42, 146.17,
158.36. HRMS (ESI, m/z) calcd. for C12H13N6 [MþH] 241.1196. Found
241.1195. Anal. Calcd. for C12H12N6: C, 59.99; H, 5.03; N, 34.98.
Found: C, 59.76; H, 5.06; N, 34.84.
1H, J ¼ 6.0 Hz, OH), 7.37 (bs, 1H, NH), 7.75 (d, 2H, J ¼ 8.4 Hz), 8.02 (d,
2H, J ¼ 8.4 Hz), 10.82 (bs, 1H, NH). 13C NMR (DMSO-d6)
d: 10.53,
34.52, 45.10, 59.93, 117.45, 127.72, 132.26, 132.73, 138.38, 143.88,
146.08, 157.39. HRMS (ESI, m/z) calcd. for C14H18N7O3S [MþH]
364.1186. Found 364.1186. Anal. Calcd. for C14H17N7O3S: C, 46.27; H,
4.72; N, 26.98. Found: C, 46.04; H, 4.74; N, 26.84.
4.2.2. 4-(1,3-Dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-ylamino)
benzene-1-sulfonyl chloride (5)
Compound 4 (1.03 g, 4.28 mmol) was added portionwise to
stirred and ice-cooled chlorosulfonic acid (9.03 g, 77.48 mmol)
under an argon atmosphere, after which the reaction mixture was
gradually warmed to room temperature over 2 h. The reaction so-
lution was cautiously poured over crushed ice (40 g) and the
aqueous mixture was extracted with dichloromethane. The com-
bined extracts were dried over anhydrous Na2SO4 and evaporated
under reduced pressure to give the required sulfonyl chloride 5 as a
yellow solid (1.37 g, 4.03 mmol). Yield 94%, mp 233 ꢀC. 1H NMR
4.3.5. N-[4-(1,4-Diazepan-1-ylsulfonyl)phenyl]-1,3-dimethyl-1H-
pyrazolo[4,3-e][1,2,4]triazin-5-amine (6e)
Yield 70%, mp 168e174 ꢀC. 1H NMR (DMSO-d6)
d: 1.76e1.80 (m,
2H), 2.52 (s, 3H), 2.72 (t, 2H, J ¼ 12.0 Hz), 2.77 (t, 2H, J ¼ 10.0 Hz),
3.20 (t, 2H, J ¼ 10.0 Hz), 3.25 (t, 2H, J ¼ 12.0 Hz), 4.15 (s, 3H), 4.18 (bs,
1H, NH), 7.73 (d, 2H, J ¼ 8.8 Hz), 8.05 (d, 2H, J ¼ 8.8 Hz), 10.92 (bs,
1H, NH). 13C NMR (DMSO-d6)
d: 10.50, 27.87, 34.51, 45.74, 46.82,
47.89, 48.10, 117.52, 128.02, 129.89, 132.67, 138.36, 144.36, 146.07,
157.29. HRMS (ESI, m/z) calc. for C17H23N8O2S [MþH] 403.1659.
Found 403.1660. Anal. Calcd. for C17H22N8O2S: C, 50.73; H, 5.51; N,
27.84. Found: C, 50.52; H, 5.53; N, 27.72.
(DMSO-d6)
d
: 2.49 (s, 3H), 4.10 (s, 3H), 7.57 (d, 2H, J ¼ 8.4 Hz), 7.79
(d, 2H, J ¼ 8.8 Hz), 10.43 (s, 1H, NH). 13C NMR (DMSO-d6)
d: 10.56,
34.52, 117.17, 126.25, 132.86, 138.17, 140.69, 140.78, 146.04, 157.95.
4.3.6. 1,3-Dimethyl-N-[4-(pyrrolidin-1-ylsulfonyl)phenyl]-1H-
4.3. General procedure for preparation of the sulfonamides (6ael)
pyrazolo[4,3-e][1,2,4]triazin-5-amine (6f)
Yield 88%, mp 284e286 ꢀC. 1H NMR (DMSO-d6)
d: 1.63e1.66 (m,
A mixture of chlorosulfonyl derivative (5) (100 mg, 0.29 mmol)
and the appropriate amine (1.00 mmol) in anhydrous acetonitrile
(10 ml) was stirred overnight at room temperature, after which the
reaction mixture was concentrated under reduced pressure to
afford crude sulfonamides, 6ael as yellow solids. The residues were
purified by crystallization from EtOH or by column chromatography
on silica using a mixture of CH2Cl2:EtOH (25/1, v/v) as the eluent.
4H), 2.52 (s, 3H), 3.12e3.15 (m, 4H), 4.15 (s, 3H), 7.76 (d, 2H,
J ¼ 8.8 Hz), 8.07 (d, 2H, J ¼ 8.8 Hz), 10.88 (s, 1H, NH). 13C NMR
(DMSO-d6)
d: 10.67, 24.67, 34.65, 47.79, 117.40, 127.61, 128.54,
132.70, 138.39, 144.47, 146.10, 157.33. HRMS (ESI, m/z) calcd. for
C
16H20N7O2S [MþH] 374.1394. Found 374.1396. Anal. Calcd. for
C16H19N7O2S: C, 51.46; H, 5.13; N, 26.26. Found: C, 51.30; H, 5.15; N,
26.19.
4.3.1. 1,3-Dimethyl-N-[4-(4-methylpiperazin-1-ylsulfonyl)phenyl]-
4.3.7. 1,3-Dimethyl-N-[4-(piperidin-1-ylsulfonyl)phenyl]-1H-
1H-pyrazolo[4,3-e][1,2,4]triazin-5-amine (6a)
pyrazolo[4,3-e][1,2,4]triazin-5-amine (6g)
Yield 71%, mp 246e251 ꢀC. 1H NMR (CDCl3)
d: 2.27 (s, 3H), 2.50e
Yield 85%, mp 241e248 ꢀC. 1H NMR (DMSO-d6)
d: 1.29e1.38 (m,
2.52 (m, 4H), 2.63 (s, 3H), 3.07e3.08 (m, 4H), 4.25 (s, 3H), 7.77 (d,
2H), 1.60e1.66 (m, 4H), 2.53 (s, 3H), 2.95e2.98 (m, 4H), 4.13 (s, 3H),
2H, J ¼ 8.8 Hz), 7.93 (d, 2H, J ¼ 8.8 Hz), 8.12 (s, 1H, NH). 13C NMR
7.66 (d, 2H, J ¼ 8.4 Hz), 8.06 (d, 2H, J ¼ 8.4 Hz),10.89 (bs,1H, NH). 13
C
(CDCl3)
d
: 10.87, 34.80, 45.68, 45.95, 54.07, 117.62, 127.98, 129.36,
NMR (DMSO-d6) d: 10.70, 22.12, 24.70, 43.54, 46.66, 117.43, 126.80,
133.97, 139.76, 143.53, 146.64, 157.14. HRMS (ESI, m/z) calcd. for
128.61, 132.70, 138.41, 144.53, 146.11, 157.32. HRMS (ESI, m/z) calcd.
for C17H22N7O2S [MþH] 388.1550. Found 388.1510. Anal. Calcd for
C
C
17H23N8O2S [MþH] 403.1659. Found 403.1661. Anal. Calcd. for
17H22N8O2S: C, 50.73; H, 5.51; N, 27.84. Found: C, 50.50; H, 5.53; N,
C17H21N7O2S: C, 52.70; H, 5.46; N, 25.31. Found: C, 52.45; H, 5.49; N,
27.71.
25.18.
4.3.2. 1,3-Dimethyl-N-(4-(morpholinosulfonyl)phenyl)-1H-
4.3.8. N-(2-Aminoethyl)-4-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]
pyrazolo[4,3-e][1,2,4]triazin-5-amine (6b)
triazin-5-ylamino)benzenesulfonamide (6h)
Yield 72%, mp 280e285 ꢀC. 1H NMR (CDCl3)
d: 2.64 (s, 3H), 3.03
Yield 78%, mp 238e242 ꢀC. 1H NMR (DMSO-d6)
d: 2.51 (s, 3H),
(t, 4H, J ¼ 4.8 Hz), 3.76 (t, 4H, J ¼ 4.8 Hz), 4.26 (s, 3H), 7.77 (d, 2H,
2.65 (t, 2H, J ¼ 6.6 Hz), 2.82 (t, 2H, J ¼ 6.4 Hz), 4.08 (bs, 2H, NH2),
J ¼ 6.8 Hz), 7.97 (d, 2H, J ¼ 6.8 Hz), 8.19 (s, 1H, NH). 13C NMR (CDCl3)
4.14 (s, 3H), 7.75 (d, 2H, J ¼ 8.8 Hz), 8.03 (d, 2H, J ¼ 8.8 Hz),10.86 (bs,
d: 10.87, 34.81, 46.05, 66.13, 117.65, 127.66, 129.43, 133.39, 139.75,
1H, NH). 13C NMR (DMSO-d6)
d: 10.53, 34.53, 43.30, 117.48, 127.79,
143.75, 146.65, 157.12. HRMS (ESI, m/z) calcd. for C16H20N7O3S
[MþH] 390.1343. Found 390.1342. Anal. Calcd for C16H19N7O3S: C,
49.35; H, 4.92; N, 25.18. Found: C, 49.09; H, 4.95; N, 25.04.
131.76, 132.78, 138.39, 144.03, 146.14, 157.38. HRMS (ESI, m/z) calcd.
for C14H19N8O2S [MþH] 363.1346. Found 363.1346. Anal. Calcd. for
C
14H18N8O2S: C, 46.40; H, 5.01; N, 30.92. Found: C, 46.20; H, 5.03; N,
30.81.
4.3.3. 1,3-Dimethyl-N-[4-(piperazin-1-ylsulfonyl)phenyl]-1H-
pyrazolo[4,3-e][1,2,4]triazin-5-amine (6c)
4.3.9. 4-(1,3-Dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-ylamino)
Yield 86%, mp 300e307 ꢀC. 1H NMR (DMSO-d6)
d
: 2.52 (s, 3H),
benzenesulfonamide (6i)
2.79e2.81 (m, 8H), 4.15 (s, 3H), 7.68 (d, 2H, J ¼ 8.8 Hz), 8.09 (d, 2H,
Yield 82%, mp 317e320 ꢀC. 1H NMR (DMSO-d6)
d: 2.49 (s, 3H),
J ¼ 8.8 Hz), 10.92 (s, 1H, NH). 13C NMR (DMSO-d6)
d: 10.54, 34.56,
4.12 (s, 3H), 7.18 (s, 2H, NH2), 7.76 (d, 2H, J ¼ 8.4 Hz), 7.96 (d, 2H,
44.27, 46.14, 117.45, 126.04, 128.94, 132.78, 138.47, 144.76, 146.18,
J ¼ 8.4 Hz), 10.74 (s, 1H, NH). 13C NMR (DMSO-d6)
d: 10.66, 34.65,

M. Mojzych et al. / European Journal of Medicinal Chemistry 78 (2014) 217e224
223
117.55, 126.87, 132.95, 136.38, 138.55, 143.52, 146.24, 157.60. HRMS
(ESI, m/z) calcd. for C12H14N7O2S [MþH] 320.0924. Found 320.0923.
Anal. Calcd. for C12H13N7O2S: C, 45.13; H, 4.10; N, 30.70. Found: C,
44.83; H, 4.16; N, 30.53.
number of viable cells to 50% of the control level) were determined
from the doseeresponse curves.
4.6. Flow cytometry
4.3.10. 4-(1,3-Dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-
ylamino)-N-(1-hydroxy-3-methylbutan-2-yl)benzenesulfonamide
(6j)
K562 cells (5 ꢂ 105 cells/ml) were incubated with test com-
pounds for 24 h. After incubation, cells were centrifuged, washed in
PBS and split into two subsamples for viability and cell cycle
analysis as described previously [17,19]. For viability analyses, the
cells were directly stained with propidium iodide and the per-
centage of viable cells (i.e. those excluding propidium iodide) was
determined by flow cytometry using a 488 nm laser (Cell Lab
Quanta SC, Beckman Coulter). For cell cycle analysis, the washed
cells were fixed with 70% ethanol, washed, stained with propidium
iodide and analyzed by flow cytometry using a 488 nm laser (Cell
Lab Quanta SC, Beckman Coulter). The sub-diploid fraction was
designated as apoptotic.
Yield 75%, mp 306e307 ꢀC. 1H NMR (DMSO-d6)
d: 0.71 (d, 3H,
J ¼ 6.8 Hz), 0.75 (d, 3H, J ¼ 6.8 Hz), 1.80e1.86 (m, 1H), 2.53 (s, 3H),
2.87e2.91 (m, 1H), 3.10e3.17 (m, 2H), 4.15 (s, 3H), 4.49 (t, 1H,
J ¼ 5.4 Hz, OH), 7.14 (d, 1H, J ¼ 8.4 Hz, NH), 7.75 (d, 2H, J ¼ 9.2 Hz),
8.01 (d, 2H, J ¼ 9.2 Hz), 10.84 (bs, 1H, HN). HRMS (ESI, m/z) calcd. for
C
C
17H24N7O3S [MþH] 406.1656. Found 406.1655. Anal. Calcd for
17H23N7O3S: C, 50.36; H, 5.72; N, 24.18. Found: C, 50.09; H, 5.74; N,
24.07.
4.3.11. (1,3-Dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-ylamino)-
N-(2-morpholinoethyl)-benzenesulfonamide (6k)
4.7. Kinase inhibition assay
Yield 76%, mp 218e220 ꢀC. 1H NMR (DMSO-d6)
d: 2.26e2.31 (m,
6H), 2.49 (s, 3H), 2.84 (q, 2H, J ¼ 6.4 Hz), 3.49 (t, 4H, J ¼ 4.4 Hz), 4.13
Kinase assays were performed according to established pro-
tocols [17,19]. Briefly, CDK2/cyclin E and Abl kinases were produced
(s, 3H), 7.29 (t, 2H, J ¼ 5.2 Hz), 7.74e7.76 (d, 2H, J ¼ 8.8 Hz), 8.00 (d,
2H, J ¼ 8.8 Hz),10.79 (s, 1H, NH). 13C NMR (DMSO-d6)
d
: 10.51, 15.25,
in Sf9 insect cells and purified on a NiNTA column (Qiagen). CK2
was purchased from ProQinase. CDK2 was assayed with 1 mg/ml
histone H1 in the presence of 15 M ATP, 0.05 Ci [
33P]ATP and of
the test compound in a final volume of 10 L, all in a reaction buffer
(60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3
orthovanadate, 1.2 mM DTT, 2.5 g/50 l PEG20.000). CK2
a1
34.52, 53.05, 57.09, 65.97, 117.44, 127.73, 131.76, 132.78, 138.36,
144.03, 146.14, 157.39. HRMS (ESI, m/z) calcd. for C18H25N8O3S
[MþH] 433.1765. Found 433.1763. Anal. Calcd. for C18H24N8O3S: C,
49.99; H, 5.59; N, 25.91. Found: C, 49.75; H, 5.62; N, 25.74.
m
m
g-
m
m
a
M Na-
1 was
m
m
4.3.12. N-(2,3-Dihydroxypropyl)-4-(1,3-dimethyl-1H-pyrazolo[4,3-
assayed in the same buffer but with casein as a substrate (0.2 mg/
ml). Abl was assayed with 500 of a synthetic peptide
(GGEAIYAAPFKK) 10 M of [
33P]ATP and the appropriate quantity
of the test compound in a final volume of 10 L, all in a reaction
buffer (25 mM Tris, pH 7.5, 5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT,
0.01% Brij35). The reactions were stopped by adding 5 L of 3% aq.
H3PO4. Aliquots were spotted onto P-81 phosphocellulose (What-
man), washed 3ꢂ with 0.5% aq. H3PO4 and finally air-dried. Kinase
inhibition was quantified using a FLA-7000 digital image analyzer
(Fujifilm). The concentration of the test compounds required to
decrease the kinase activity by 50% was determined from dosee
e][1,2,4]triazin-5-ylamino)-benzenesulfonamide (6l)
mM
Yield 87%, mp 235e236 ꢀC. 1H NMR (DMSO-d6)
d: 2.50 (s, 3H),
m
g-
2.60 (dd, 1H, J1 ¼ 7.2 Hz, J2 ¼ 1.32 Hz), 2.85 (dd, 1H, J1 ¼ 5.2 Hz,
J2 ¼ 1.28 Hz), 3.24e3.28 (m, 2H), 3.42e3.50 (m, 1H). 4.13 (s, 3H),
4.45 (t, 1H, J ¼ 3.6 Hz, OH), 4.70 (d, 1H, J ¼ 4.4 Hz, OH), 7.23 (bs, 1H,
NH), 7.74 (d, 2H, J ¼ 8.4 Hz), 8.00 (d, 2H, J ¼ 8.4 Hz), 10.80 (bs, 1H,
m
m
NH). 13C NMR (DMSO-d6)
d: 10.52, 34.51, 46.05, 63.52, 70.30, 117.40,
127.74, 132.15, 132.75, 138.37, 143.83, 146.11, 157.40. HRMS (ESI, m/z)
calcd. for C15H19N7O4S [MþH] 394.1292. Found 394.1292. Anal.
Calcd. for C15H19N7O4S: C, 45.79; H, 4.87; N, 24.92. Found: C, 45.52;
H, 5.03; N, 24.79.
response curves and identified as the IC50
.
4.4. Cell cultures
4.8. Immunoblotting
Human cancer cell lines MCF-7, MDA-MB-231, K562, CCRF-CEM,
HL60 and BV173 (purchased from the American Type Culture
Collection and the German Collection of Microorganisms and Cell
Cultures) were maintained in DMEM supplemented with 10% foetal
Immunoblotting analyses were performed as in our previous
works [16e19]. Specific antibodies were purchased from Sigmae
Aldrich (anti-a-tubulin and peroxidase-labelled secondary anti-
bodies) and Cell Signaling Technology (STAT5, STAT5 phosphory-
lated at Y694, CrkL phosphorylated at Y207), and Millipore (anti-
phosphotyrosine 4G10). Briefly, cellular lysates were prepared by
harvesting cells in Laemmli sample buffer. Proteins were separated
on SDS-polyacrylamide gels and electroblotted onto nitrocellulose
membranes. After blocking, the membranes were incubated with
specific primary antibodies overnight, washed and then incubated
with peroxidase-conjugated secondary antibodies. Finally, peroxi-
dase activity was detected with ECLþ reagents (AP Biotech) using a
CCD camera LAS-4000 (Fujifilm).
bovine serum (FBS), 50 U/ml penicillin, 50 mg/ml streptomycin at
37 ꢀC. Cells were cultured in Costar flasks and subconfluent cells
were detached with trypsin (in case of breast cell lines), counted
and plated at 5 ꢂ105 cells per well in 6-well plates (Nunc) in 2 ml of
growth medium. Cells reached 80% confluency on day 3 and in most
cases such cells were used for the assays.
4.5. MTT assay
The assay was performed according to the literature method
[23]. Cells exposed for 24 h to various concentrations of the studied
compounds in microtitre plates were stained with MTT solution at
37 ꢀC. The solution was then removed and 1 ml of 0.1 M HCl in
absolute isopropanol was added to the attached cells. The absor-
bance of the converted dye in living cells was measured at a
wavelength of 570 nm. Cell viability was calculated as a percentage
of the viable control cells. All experiments were performed in
triplicate. IC50 values (the drug concentrations that reduced the
4.9. Molecular modelling
3D structures of the studied compounds were prepared using
Marvin 5.10.3, 2012, ChemAxon (http://www.chemaxon.com). The
crystal structures of CDK2 with NU6102 (PDBID: 2c6o) and Abl with
PD180970 (PDBID: 2hzi) were used as the protein docking tem-
plates. Polar hydrogens were added to all ligands and proteins with
the AutoDock Tools program [27]. Molecular docking was

224
M. Mojzych et al. / European Journal of Medicinal Chemistry 78 (2014) 217e224
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Acknowledgements
This research was funded by the National Science Centre, Poland
(grant NN405 092340), the Czech Science Foundation (grant P305/
12/0783), the Centre of the Region Haná for Biotechnological
Agricultural Research (grant no. ED0007/01/01), Palacky University
(Student Project PrF_2014_023), the Operational Program Research
and Development for Innovations e European Regional Develop-
ment Fund (CZ.1.05/2.1.00/03.0058). The authors are grateful to
Prof. Robert Kawe˛cki and Dr. Konrad Zdanowski (Siedlce University
of Natural Sciences and Humanities) for providing NMR spectra and
Prof. W.T. Miller (Stony Brook University, NY) for providing the Abl
construct.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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