Synthesis of pyrazolo[4,3-e][1,2,4]triazine sulfonamides, novel Sildenafil analogs with tyrosinase inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2014.10.009

Tyrosinase is a multifunctional, glycosylated and copper-containing oxidase which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors, newly discovered from natural and synthetic sources. The inhibitory strength is comparable to that of the standard inhibitor kojic acid. Also their inhibitory mechanisms are discussed. The new obtained compounds were also tested as PDE5 inhibitors and did not show significant inhibitory effect.

Full text of DOI:10.1016/j.bmc.2014.10.009

Bioorganic & Medicinal Chemistry 22 (2014) 6616–6624
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of pyrazolo[4,3-e][1,2,4]triazine sulfonamides, novel
Sildenafil analogs with tyrosinase inhibitory activity
b
c
Mariusz Mojzych a, , Aleksandar Dolashki , Wolfgang Voelter
⇑
a Department of Chemistry, Siedlce University of Natural Sciences and Humanities, 3-go Maja 54, 08-110 Siedlce, Poland
b Institute of Organic Chemistry with Centre of Phytochemistry, G. Bonchev Str. 9, Sofia 1113, Bulgaria
c Interfacultary Institute for Biochemistry, Hoppe-Seyler-Str.4, 72076 Tubingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2014
Revised 29 September 2014
Accepted 9 October 2014
Available online 18 October 2014
Tyrosinase is a multifunctional, glycosylated and copper-containing oxidase which catalyzes the first two
steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged
fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzy-
matic browning in fruits are desirable. These phenomena have encouraged researchers to seek new
potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors,
newly discovered from natural and synthetic sources. The inhibitory strength is comparable to that of
the standard inhibitor kojic acid. Also their inhibitory mechanisms are discussed. The new obtained com-
pounds were also tested as PDE5 inhibitors and did not show significant inhibitory effect.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Denovo synthesis
Inhibitors
aza-Sildenafil
aza-isoViagra
1. Introduction
ether analogs which were found to show tyrosinase inhibitory
effect, apart from its well-known orally effective properties for
Azoloazines are biological interesting molecules, and their
chemistry is receiving considerable attention.1–3 Furthermore, the
multiple biological activities of pyrazole and its annelated deriva-
tives are of increasing interest as antimycotics,4 antidepressants,5
fungicidal6 or herbicidal agents.7 Compounds containing the pyraz-
olo[4,3-e][1,2,4]triazine moiety have attracted considerable atten-
tion due to their anticancer and antibacterial activity.8–10 The most
important members in this family of naturally occurring purine
analogs are pseudoiodinine,8 nostocine A9 and fluviols A–E10 pro-
duced by Pseudomona fluorescens var. pseudoiodinine and Nostoc
spongiaeforme, respectively. Despite the fact that pyrazolo[4,3-
e][1,2,4]triazines have been less studied in the group of condensed
pyrazolotriazines, some of them show moderate inhibition of pur-
ine nucleoside phosphorylase11 and cytotoxic activity in A549 lung
carcinoma cell line at submicromolar range,12 induction of cas-
pase-dependent cell death, and inhibition of cyclin-dependent
kinase 2 (CDK2).13 Moreover, this system, for example, pyrazol-
o[4,3-e][1,2,4]triazine is structurally related to pyrazolo[4,3-d]pyr-
imidin-7-one present in the structure of Sildenafil and its methyl
the treatment of male erectile dysfunction.14,15 It should be noted
that so far no analogs of Sildenafil containing the skeleton of 1H-
pyrazolo[4,3-e][1,2,4]triazine ring system have been described in
the literature. With regard to the above discussed facts, we envis-
age a high potential of the pyrazolo[4,3-e][1,2,4]triazine system
regarding the discovery of new biological activities.
In this context and in continuation of our program aimed at the
discovery and development of new bioactive molecules, we repre-
sent here the preparation of novel analogs of Sildenafil and
isoVigra16 (termed aza-analogs of Sildenafil 8a–j and aza-isoViagra
9) in which the carbonyl group, present in the structures of Silde-
nafil and isoViagra, is replaced with the nitrogen atom N7, present
on the triazine ring (Fig. 1), to test their PDE5 and tyrosinase inhib-
itory activity. Moreover, we have described synthesis, spectral
properties and biological activity of new bis-1H-pyrazolo[4,3-
e][1,2,4]triazine sulfonamides 10a–c related to lodenafil carbon-
ate17 (Fig. 2). Lodenafil carbonate, a new phosphodiesterase type
5 (PDE5) inhibitor is used in treatment of erectile dysfunction. It
is formulated as a dimer, composed of two lodenafil molecules
linked via a carbonate bridge and behaves as a prodrug which
releases lodenafil as active metabolite. Preclinical and clinical stud-
ies have shown that this dimer has higher oral bioavailability than
the parent drug (Lodenafil), low toxicity and is safe and efficient in
the treatment of erectile dysfunction.
⇑
Corresponding author. Tel.: +48 025 643 1105; fax: +48 025 6442045.
E-mail address: mmojzych@yahoo.com (M. Mojzych).
http://dx.doi.org/10.1016/j.bmc.2014.10.009
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
6617
H3C
N
O
N
O
N
the next step, using Guillaumet’s and co-workers palladium-cata-
lyzed cross-coupling reaction of 3-methylsulfanyl-1,2,4-triazine
with boronic acid derivatives,19 we have reacted 5-methylsulfa-
nyl-pyrazolo[4,3-e][1,2,4]triazines 2 and 3 with 2-ethoxyphenyl-
boronic acid in the presence of copper(I) 3-methylsalicylate to
obtain appropriate derivatives 4 and 5. Compound 4 was collected
in excellent yield (90%), but derivative 5 was produced in 30% yield
only. Chlorosulfonylation of compounds 4 and 5 in neat chlorosul-
fonic acid at 0 °C proceeded smoothly and selectively at 50-position
of the phenyl ring to give the desired products 6 and 7 in excellent
yield. However, it should be noted that chlorosulfonyl derivative 7
is not stable and after isolation was immediately reacted with
amine. The final analogs of Sildenafil 8a–j and aza-isoViagra 9 were
prepared by stirring chlorosulfonyl derivatives 6 and 7 with three
equivalents of appropriate amines in acetonitrile at room temper-
ature overnight. The structures of Sildenafil and isoViagra as well
as their new analogs are shown in Fig. 1.
The low yield of Suzuki cross coupling reaction of 3 with 2-eth-
oxyphenyl boronic acid prompted us to improve the efficiency of
the preparation of the useful intermediate 5 as illustrated in
Scheme 1 (pathway B). To avoid our problem, another possibility
to synthesize 5-20-ethoxyphenyl-2H-pyrazolo[4,3-e][1,2,4]-tri-
azine 5 is to use N-protecting groups. We decided to exploit previ-
ously elucidated ethyl vinyl ether and 3,4-dihydro-1H-pyran as
new protecting groups for NH-pyrazoles as published in the litera-
ture.20 Exposure of N-unsubstituted derivative 1 to both ethers
under heating in benzene at 40 °C with catalytic amounts of con-
centrated HCl gave the appropriate intermediates 11 and 12 in
good yield (80–90%). After chromatography and treatment with
2-ethoxyphenyl boronic acid under Guillaumet’s procedure,19
compounds 11 and 12 gave the isomeric derivatives 13 and 14 in
80–85% yield, and their deprotection in methanol with concen-
trated HCl at room temperature for 12 h gave 1-unsubstituted pyr-
azolo[4,3-e][1,2,4]triazine 15 in excellent yield. N-alkylation of 15
with methyl iodide in a mixture of EtOH/water (1:1, v/v) in the
presence of potassium carbonate furnished at room temperature
isomeric target molecules 4 and 5 in the ratio 1:1.2 with a total
yield of 90%.
N
N
NH OC2H5
NH OC2H5
H3C N
SO2
N
SO2
N
N
CH3
N
CH3
Sildenafil
isoViagra
New analogues
H3C
N
N
N
H3C
N
N
OC2H5
N
N
N
N
H3C N
N
N
N
OC2H5
N
OC2H5
N
H3C
H3C
SO2
N
SO2
N
SO2
N
N
CH3
N
CH3
X
aza-Sildenafil
aza-analogues of Sildenafil
X = NH2, NH, N-CH3, O, OH, CH2
aza-isoViagra
Figure 1. Structures of Sildenafil, isoViagra and new analogs: aza-isoViagra, aza-
Sildenafil, and its aza-analogs.
O
N
O
N
H3C
N
CH3
N
NH
O
HN
O
O
N
O
N
O
N
N
O
S
S
N
N
O
Lodenafil carbonate
O
O
O
S N
N
S O
The dimeric sulfonamides 10a–c were also synthesized follow-
ing two synthetic pathways as shown in Scheme 2. In the first way,
two equivalents of chlorosulfonyl derivative 6 were reacted with
one equivalent of bifunctional amine (piperazine, homopiperazine
and ethylenediamine) (pathway A, Scheme 2), and in the second
way, an equal molar amount of derivative 6 and appropriate mono-
sulfonamide 8b and 8f–g were stirred in acetonitrile at room tem-
perature (pathway B, Scheme 2). Structures of lodenafil carbonate
and its new analogs 10a–c are shown in Fig. 2.
N
N
N
N
N
N
H3C
CH3
N
N
N
N
O
O
CH3
H3C
New analogues of lodenafil carbonate
Figure 2. Structure of lodenafil carbonate and its new analogs.
2. Results and discussion
2.1. Synthesis
2.2. Pharmacology
2.2.1. Effect of inhibitors on tyrosinase
For further characterization of the obtained inhibitors were
incubated with the enzyme L. sacchari tyrosinase, and their prop-
erties were examined (See Table 1).
The enzyme was purified from L. sacchari starting by anion-
exchange material (Servacell, DEAE 52), and the obtained superna-
tant was further purified by size exclusion chromatography, on a
The synthesis of aza-analogs of Sildenafil 8a–j and aza-isoViagra
9 are outlined in Scheme 1, and the preparation could be achieved
in two pathways (Scheme 1). The key starting derivative 1 was
obtained using our previously discovered procedure.18 Starting
our study on the intended synthesis of new sulfonamides with
the pyrazolo[4,3-e][1,2,4]triazine core, we decided to exploit
N1- and N2-methyl derivatives of the pyrazolo[4,3-e][1,2,4]triazine
ring system (2 and 3)18 to realize our target. Thus, unsubstituted
pyrazolotriazine 1 was smoothly converted into the corresponding
isomeric structures 2 and 3 upon the reaction with methyl iodide
in the presence of potassium carbonate in an EtOH/H2O mixture
(1:1, v/v) at rt. Derivative 2 can be prepared also by cyclization
of the methylhydrazone of 5-acyl-1,2,4-triazine under acidic
conditions according to our previously published procedure.18 In
FPLC system equipped with
a
Sephacryl S-100 column
(16 ꢀ 60 mm). The column was eluted with the same buffer at a
flow rate of 0.4 ml/min. The fraction with the highest activity
was additionally purified after chromatography on the same col-
umn and used for kinetic measurements. The inhibition was deter-
mined by measuring the enzymatic activity of this fraction and
5 mM L-DOPA in the presence of the inhibitor at different concen-
trations. L. sacchari tyrosinase was inhibited completely by 1 mM
pyrazolo[4,3-e][1,2,4]triazine, but to only 48% by 0.1 mM of the

6618
M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
H3C
N
N
N
N
N
N
N
N
H3C
N
N
SCH3
SCH3
H3C
a
pathway A
H3C
H
2
3
N
N
N
N
N
SCH3
H3C
1
pathway B
O
N
O
e
N
N
N
N
N
N
N
N
SCH3
N
SCH3
H3C
H3C
11
12
b
b
b
b
O
O
N
N
N
OEt
N
N
N
OEt
N
N
N
f
N
H3C
H3C
OEt
13
14
H
N
N
N
N
N
H3C
15
H3C
N
N
N
N
OEt
N
N
OEt
N
H3C
a
N
N
N
H3C
H3C
5
4
c
c
H3C
N
N
N
N
N
N
OEt
N
OEt
N
N
H3C
N
H3C
H3C
SO2Cl
SO2Cl
7
6
d
d
H3C
N
N
N
OEt
N
N
N
N
N
OEt
N
H3C
N
H3C
8a-j
H3C
SO2
R
SO2
N
NH
N
N
N
N
CH3
8f : R =
8g : R =
9
8a : R =
8b : R =
N
CH3
NH
N
NH2
H
N
O
N
H
N
O
8c : R =
8h : R =
8i : R =
8j : R =
N
N
N
8d : R =
8e : R =
N
OH
H
Scheme 1. Synthetic path to the aza-analogs of Sildenafil 8a–j. Reagents and conditions: (a) CH3I, EtOH/H2O/K2CO3; (b) ethoxyphenylboronic acid, Pd(PPh3)4, CuMeSal, THF,
under argon atmosphere, reflux, overnight; (c) ClSO3H, 0 °C to rt, 2 h; (d) appropriate amine, anhydrous MeCN, rt, overnight; (e) ether (ethyl vinyl ether or 2H-pyran, benzene,
conc. HCl, 40 °C, 8 h; (f) conc. HCl, MeOH, rt, 12 h.

M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
6619
H3C
N
N
N
N
OEt
N
H3C
SO2
N
H3C
H3C
N
N
N
a
N
b
OEt
N
N
OEt
N
N
pathway B
N
N
N
SO2
pathway A
H3C
H3C
H3C
6
SO2Cl
SO2
R
N
N
N
N
N
OEt
H3C
N
N
NH
8b : R =
10a-c
NH
8f : R =
8g : R =
10a-c
10a
10b
10c
N
NH2
N
H
N
N
HN
HN
N
N
N
Figure 3A. Kinetic measurements of tyrosinase activity with L-DOPA as substrate
and different concentrations of inhibitors in 50 mM phosphoric acid buffer solution
(pH 6.8, 1.8 mL), an aqueous solution of tyrosinase (1000 U/mL, 0.1 mL), and DMSO
(0.1 mL) at 25 °C for 10 min. The reaction was monitored at 475 nm for 10 min.
Scheme 2. Synthetic pathway to dimeric sulfonamides 10a–c. Reagents and
conditions: (a) appropriate amine, anhydrous MeCN, rt, overnight; (b) derivative
6, anhydrous MeCN, rt, overnight.
Table 1
Tyrosinase inhibitory activities of the tested compounds with concentration of
0.30 mg/ml
Compounds
IC50 (mg/ml)
Inhibition (%)
Activity (%)
aza-Sildenafil
0.12
0.11
0.08
0.15
0.12
0.09
0.10
0.07
0.15
0.12
0.10
0.14
nt
57.1
58.8
70.5
51.4
59.8
60.4
62.1
96.3
62.1
68.3
39.5
54.8
nt
42.9
41.2
29.5
48.6
40.2
39.6
33.5
3.7
37.9
31.7
60.5
45.2
nt
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
9
10a
10b
10c
nt
0.90
nt
79.7
nt
20.2
nt = not tested.
Figure 3B. Kinetic measurements of tyrosinase activity with L-DOPA as substrate
and different concentrations of inhibitors in 50 mM phosphoric acid buffer solution
(pH 6.8, 1.8 mL), an aqueous solution of tyrosinase (1000 U/mL, 0.1 mL), and DMSO
(0.1 mL) at 25 °C for 10 min. The reaction was monitored at 475 nm for 10 min.
same inhibitor. The enzyme was inhibited strongly by glutathione,
b-mercaptoethanol, but EDTA and sodium chloride did not inhibit
the enzyme very efficiently. Thiol compounds, such as glutathione,
inhibit the enzyme and may also affect the subsequent non-enzy-
matic reactions by reducing the quinones to diphenols and cause
retarded browning. These findings are comparable with other tyr-
osinases from bacterial, fungal and plant origins.21,22 Low inhibi-
tion of L. sacchari tyrosinase by EDTA is similar to tyrosinases
from other organisms such as Trichoderma reesei.22,23
The inhibition effects of synthesized inhibitors on tyrosinase
activity were performed with L-DOPA as substrate and different
concentrations of inhibitors in 50 mM phosphoric acid buffer solu-
tion (pH 6.8, 1.8 mL). The activity of tyrosinase (1000 U/mL,
0.1 mL) was determined by spectrophotometric techniques after
an incubation time of 0.5 and 1.0 min (Fig. 3A and B).
morfoline ring attached to the skeleton, the enzyme activity
remains high: 48.6% and 37.9%, respectively. Very high enzymatic
activity (60.5%) was measured after treatment of tyrosinase with
the inhibitor 8j which contains a N-2-hydroxyethyl substituent
(Fig. 3B). However, our study showed that inhibitors 8b and 8f
with one NH group in the sulfonamide part are more active inhib-
itors and reduce the enzyme activity of tyrosinase to 29.5% and
33.5%, respectively. It is suggested, that NH or NH2 groups are
involved in the inhibition effect of tyrosinase activity. Our sugges-
tion was confirmed by the activity of inhibitor 10c (20.2%) with
two NH groups. The most active inhibitor in the tested group
was compound 8g that reduced the tyrosinase activity to 3.7%. In
consideration of our results, the –NHCH2CH2NH2 group in the
structure of the inhibitor 8g seems to be the most important resi-
due for the inhibition of tyrosinase activity.
Aza-Sildenafil and its isomeric analogs 8a and 9 are structurally
similar and represent comparable range of tyrosinase activity,
42.9%, 41.2% and 45.2%, respectively. Lower inhibition exhibited
derivatives 8e (39.6%) and 8d (40.2%) with a pyrrolidine and a
piperidine ring in the sulfonamide group, respectively. After the
treatment of tyrosinase with inhibitors 8c and 8h, that possess a
Tyrosinase contains one binuclear copper complex in the cata-
lytic centre and the catechol hydroxyl groups of the substrate are

6620
M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
suggested to be bound to the copper atoms during the enzymatic
oxidation process.24 Phenolic hydroxyl groups are generally capa-
ble to coordinate to copper atoms causing inhibition of the enzyme
in competition to the catechol substrate. The competitive inhibi-
tion the compounds 8g may arise with strong participation both
NH and NH2 group. However, other tested derivatives were only
weak tyrosinase inhibitors. Therefore, we suggest that primary
binding to one copper atom should be strengthened by additional
no hydrogen bond
N
hydrogen bond
O
H3C
H3C
H
N
N
N
O
N
O
N
N
N
N
R
SO2
N
SO2
N
interactions between the inhibitor (e.g., the NH group and
p elec-
trons of the ring) and another copper atom or the amino acid res-
idues of the enzyme’s active site to accomplish the inhibition.
Probably flexibility and structure –NHCH2CH2NH2 group allows
to create strong interactions between the inhibitor 8g and the
amino acid residues of the enzyme’s active site. Similar results
were obtained by molecular docking study for unsymmetrical
curcumin analogs.25
X
N
CH3
aza-analogues of Sildenafil
Sildenafil
Figure 4. Structure of Sildenafil and its new aza-analogs.
2.2.2. PDE5 assay
The newly synthesized aza-analogs of Sildenafil were sub-
jected also to the PDE5A (isolated from rabbit platelets) inhibition
assay for structure–activity relationship studies. The inhibition
3. Summary
A practical, high yielding, and scalable method for the prepara-
tion of new pyrazolo[4,3-e][1,2,4]triazine derivatives as new aza-
analogs of Sildenafil from inexpensive commercially available
starting materials is described. The new Sildenafil analogs were
designed by replacing the carbonyl group of the scaffold by a tri-
azine nitrogen atom. Biotesting experiments for Sildenafil and
lodenafil carbonate analogs showed good tyrosinase inhibitory
activity, but very low PDE5A inhibitory potency, and further mod-
ification of the structure are under way to increase the potency
against tyrosinase.
activities of the compounds were tested at 1 lM using a tritium
scintillation proximity assay (SPA) system (Amersham
Biosciences). PDE5A inhibitory potency of the aza-Sildenafil26
and compounds 8a–j are presented in Table 2 in comparison to
Sildenafil.
The tested new compounds did not show significant inhibitory
effects on PDE5 at 1 lM. The most active compounds are 8d, 8b,
and 8e (inhibition: 24.1–29.5%) and the dimers 10c and 10b (inhi-
bition: 26.7–28.1%). The aza-Sildenafil in which nitrogen atom N7
replaces the carbonyl group present in the pyrimidinone ring of Sil-
denafil shows very low inhibitory effect (inhibition 5.8%) in com-
parison with Sildenafil (inhibition 98.9%). The decrease in activity
of the tested new Sildenafil analogs probable is due to the lack of
two common features that define the scaffold of all known PDE5
inhibitors with the pyrazolo[4,3-d]pyrimidin-7-one core. Based
on the literature, the first common feature for most of the tested
PDE5 inhibitors is a planar ring structure of the molecule that is
held tightly in the active site by a pair of hydrophobic residues,
and the second feature is that the inhibitor always forms one or
two hydrogen bonds with the purine-selective glutamine position
at the enzyme.27 Taking the above into account, the low activity of
the new analogs of Sildenafil toward PDE5 might be due to the lack
of a planar structure of the sulfonamides resulting on the lack of an
intramolecular hydrogen bond between the ethoxy group at the
phenyl ring and nitrogen atom N6 in the triazine ring (Fig 4).
4. Experimental section
4.1. Materials and methods
Melting points were determined on a Mel-Temp apparatus and
are uncorrected. 1H and 13C NMR spectra were recorded on a Var-
ian spectrometer (400 MHz for 1H and 100 MHz for 13C, respec-
tively). The chemical shift values are expressed in ppm (part per
million) with TMS as internal reference. The relative integrals of
peak areas agreed with those expected for the assigned structures.
The molecular weights of final compounds were assessed by elec-
trospray ionization mass spectrometry (ESI/MS) on an Agilent
Technologies 6538 UHD Accurate Mass Q-TOF LC/MS instrument.
Elemental compositions are within 0.4% of the calculated values.
Derivatives 1–3 were prepared according to the literature
procedure.18a
Table 2
4.2. General method for the synthesis of derivatives 4 and 5
PDE5A Inhibitory potency of aza-Sildenafil analogs
Compound
Concentration (
lM)
Inhibition (%)
To a mixture of 2 or 3 (0.612 g, 3.14 mmol, 1.0 equiv), CuMeSal
(1.68 g, 7.85 mmol, 2.5 equiv), 2-ethoxyphenylboronic acid (1.3 g,
7.85 mmol, 2.5 equiv) in dry THF (25 mL), Pd(PPh3)4 (0.36 g,
0.31 mmol, 0.1 equiv) was added under argon. The reaction mix-
ture was stirred overnight under reflux. The reaction was
quenched with a saturated Na2CO3 solution and extracted with
dichloromethane. The combined organic phases were dried over
MgSO4 and concentrated in vacuo. After purification by column
chromatography on silica gel (hexane: CH2Cl2, 5:1), the desired
products 4 or 5 were obtained.
aza-Sildenafil
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
9
10a
10b
10c
Sildenafil
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
5.8
6.0
26.5
0
29.5
24.1
0.6
0
13.3
16.4
Not tested
0
11.4
26.7
28.1
98.9
4.2.1. 5-(2-Ethoxyphenyl)-1,3-dimethyl-1H-pyra-zolo[4,3-
e][1,2,4]triazine (4)
Yield 90%, yellow powder, mp 85–86 °C. 1H NMR (400 MHz,
CDCl3) d: 1.30 (t, 3H, J = 14.0 Hz), 2.70 (s, 3H), 4.13 (q, 2H,

M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
6621
J = 14.0 Hz), 4.32 (s, 3H); 7.06–7.12 (m, 2H), 7.42–7.46 (m, 1H),
7.75 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz). 13C NMR (CDCl3) d: 11.05,
14.69, 34.68, 64.61, 113.47, 120.79, 126.88, 131.09, 131.92,
134.48, 141.97, 146.96, 157.31, 160.10. HRMS (ESI, m/z) calcd. for
J = 2.4 Hz). 13C NMR (CDCl3) d: 11.04, 14.43, 34.73, 45.54, 45.76,
53.96, 65.00, 112.84, 127.11, 127.29, 131.01, 132.03, 134.49,
142.22, 146.91, 158.46, 160.79; HRMS (ESI, m/z) calcd. for C19H26-
N7O3S [M+H] 432.1812. Found [M+H] 432.1813. Anal. calcd. for
C
C
14H15N5O [M+] 269.1276. Found [M+] 269.1284. Anal. calcd. for
14H15N5O: C, 62.44; H, 5.61; N, 26.01. Found: C, 62.30; H, 5.70;
C19H25N7O3S: C, 52.88; H, 5.84; N, 22.72. Found: C, 52.77; H,
5.90; N,22.60.
N, 25.93.
4.4.2. 5-[2-Ethoxy-5-(piperazin-1-ylsulfonyl)-phenyl]-1,3-
dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazine (8b)
4.2.2. 5-(2-Ethoxyphenyl)-1,3-dimethyl-2H-pyra-zolo[4,3-
e][1,2,4]triazine (5)
Yield 92%, yellow powder, mp 172–176 °C. 1H NMR (CDCl3) d:
1.38 (t, 3H, J = 14.0 Hz), 2.71 (s, 3H), 2.96–3.09 (m, 8H), 4.20 (q,
2H, J = 14.0 Hz), 4.33 (s, 3H), 7.16 (d, 1H, J = 8.8 Hz), 7.85 (dd, 1H,
J1 = 8.8 Hz, J2 = 2.4 Hz), 8.15 (d, 1H, J = 2,4 Hz). 13C NMR (CDCl3) d:
11.02, 14.41, 34.73, 44.92, 46.35, 64.98, 112.85, 127.16, 127.28,
131.02, 131.96, 134.48, 142.19, 146.90, 158.43, 160.78; HRMS
(ESI, m/z) calcd. for C18H24N7O3S [M+H] 418.1656. Found [M+H]
418.1656. Anal. calcd for C18H23N7O3S: C, 51.78; H, 5.55; N,
23.49. Found: C, 51.63; H, 5.66; N, 23.40.
Yield 30%, yellow oil. 1H NMR (400 MHz, CDCl3) d: 1.30 (t, 3H,
J = 7.2 Hz), 2.75 (s, 3H), 4.11 (q, 2H, J = 7.2 Hz), 4.29 (s, 3H), 7.05–
7.11 (m, 2H), 7.41–7.45 (m, 1H), 7.78 (dd, 1H, J1 = 7.6 Hz,
J2 = 1.6 Hz). 13C NMR (CDCl3) d: 9.12, 14.69, 39.29, 64.61, 113.44,
120.74, 127.21, 128.83, 131.04, 131.84, 133.52, 154.47, 157.42,
159.32. HRMS (ESI, m/z) calcd. for C14H15N5O [M+] 269.1276. Found
[M+] 269.1284. Anal. calcd for C14H15N5O: C, 62.44; H, 5.61; N,
26.01. Found: C, 62.22; H, 5.74; N, 25.89.
4.3. General method for the synthesis of derivatives 6 and 7
4.4.3. 5-[2-Ethoxy-5-(morpholin-1-ylsulfonyl)-phenyl]-1,3-
dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazine (8c)
Compounds 4 or 5 (594 mg, 2 mmol) was added portionwise to
stirred and cooled chlorosulfonic acid (2 mL) in an ice bath under
argon atmosphere. The reaction mixture was then warmed to room
temperature gradually for 2 h after the addition. The reaction solu-
tion was cautiously added to ice water (15 mL), and the aqueous
mixture extracted with dichloromethane. The combined extracts
were dried over anhydrous Na2SO4 and evaporated under vacuum
to give the required sulfonyl chloride 6 or 7. The crude products
were purified by column chromatography (silicagel, CH2Cl2).
Yield 87%, yellow powder, mp 170–179 °C. 1H NMR (CDCl3) d:
1.37 (t, 3H, J = 14.0 Hz), 2.71 (s, 3H), 3.05 (t, 4H, J = 9,2 Hz), 3.74
(t, 4H, J = 9.2 Hz), 4.22 (q, 2H, J = 14.0 Hz), 4.34 (s, 3H), 7.18 (d,
1H, J = 8.8 Hz), 7.86 (dd, 2H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.17 (d, 1H,
J = 2.4 Hz). 13C NMR (CDCl3) d: 11.04, 14.43, 34.76, 46.04, 65.00,
66.06, 112.84, 126.74, 127.30, 131.15, 132.06, 134.51, 142.23,
146.91, 158.38, 160.89; HRMS (ESI, m/z) calcd. for C18H23N6O4S
[M+H] 419.1496. Found [M+H] 419.1497. Anal. calcd. for C18H22N6-
O4S: C, 51.66; H, 5.30; N, 20.08. Found: C, 51.50; H, 5.47; N.19.94.
4.3.1. 4-Ethoxy-3-(1,3-dimethyl-1H-pyrazolo[4,3-
e][1,2,4]triazin-5-yl)benzene-1-sulfonyl chloride (6)
4.4.4. 5-[2-Ethoxy-5-(piperidin-1-ylsulfonyl)-phenyl]-1,3-
dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazine (8d)
Yield 85%, yellow crystals, mp 119–120 °C. 1H NMR (400 MHz,
CDCl3) d: 1.39 (t, 3H, J = 14.0 Hz), 2.72 (s, 3H), 3.64 (s, 3H), 4.26
(q, 2H, J = 14.0 Hz), 7.21 (d, 1H, J = 9.2 Hz), 7.46 (d, 1H, J = 2.4 Hz),
8.14 (dd, 1H, J1 = 8.8 Hz, J2 = 2.8). 13C NMR (CDCl3) d: 11.21,
14.48, 34.99, 65.57, 113.15, 127.92, 130.75, 131.71, 134.64,
136.12, 142.45, 147.13, 157.87, 162.69. HRMS (ESI, m/z) calcd. for
Yield 92%, yellow powder, mp 160–162 °C. 1H NMR (CDCl3) d:
1.36 (t, 3H, J = 14.0 Hz), 1.42 (t, 2H, J = 11.6 Hz), 1.61–1.67 (m,
4H), 2.71 (s, 3H), 3.02 (t, 4H, J = 10.8 Hz), 4.20 (q, 2H, J = 14.0 Hz),
4.34 (s, 3H), 7.14 (d, 1H, J = 8.8 Hz), 7.86 (dd, 2H, J1 = 8.8 Hz,
J2 = 2.4 Hz), 8.15 (d, 1H, J = 2,4 Hz). 13C NMR (CDCl3) d: 11.04,
14.45, 23.47, 25.12, 34.76, 46.96, 64.90, 127.08, 127.94, 130.29,
131.00, 131.85, 134.51, 142.20, 146.93, 158.58, 160.48; HRMS
(ESI, m/z) calcd. for C19H25N6O3S [M+H] 417.1703. Found [M+H]
417.1703. Anal. calcd. for C19H24N6O3S: C, 54.79; H, 5.81; N,
20.18. Found: C, 54.70; H, 5.90; N.20.04.
C14H14N5O3ClS [M+] 367.0505. Found [M] 367.0514. Anal. Calcd.
for C14H14N5O3ClS: C, 45.72; H, 3.84; N, 19.04. Found: C, 45.60;
H, 3.90; N, 18.90.
4.3.2. 4-Ethoxy-3-(1,3-dimethyl-2H-pyrazolo[4,3-
e][1,2,4]triazin-5-yl)benzene-1-sulfonyl chloride (7)
Yield 80%, yellow-brown oil. 1H NMR (400 MHz, CDCl3) d: 1.38
(t, 3H, J = 7.2 Hz), 2.79 (s, 3H), 4.24 (q, 2H, J = 7.2 Hz), 4.35 (s,
3H), 7.20 (d, 1H, J = 9.2 Hz), 8.12 (dd, 1H, J1 = 9.2 Hz, J2 = 2.4 Hz),
8.48 (d, 1H, J = 2.8 Hz).
4.4.5. 5-[2-Ethoxy-5-(4-pyrrolidin-1-ylsulfonyl)-phenyl]-1,3-
dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazine (8e)
Yield 89%, yellow powder, mp 190–193 °C. 1H NMR (CDCl3) d:
1.35 (t, 3H, J = 13.6 Hz), 1.76–1.79 (m, 4H), 2.71 (s, 3H), 3.26 (t,
4H, J = 13.6 Hz), 4.20 (q, 2H, J = 14.0 Hz), 4.34 (s, 3H), 7.15 (d, 1H,
J = 8.4 Hz), 7.93 (dd, 2H, J1 = 8.4 Hz, J2 = 2.4 Hz), 8.17 (d, 1H,
J = 2,4 Hz). 13C NMR (CDCl3) d: 11.04, 14.45, 25.17, 34.76, 47.97,
64.90, 112.71, 127.11, 128.67, 130.83, 131.75, 134.51, 142.20,
146.92, 158.58, 160.49; HRMS (ESI, m/z) calcd. for C18H23N6O3S
[M+H] 403.1547. Found [M+H] 403.1548. Anal. calcd. for C18H22N6-
O3S: C, 53.72; H, 5.51; N, 20.88. Found: C, 53.60; H, 5.66; N, 20.70.
4.4. Synthesis of sulfonamides 8a–j and 9
A mixture of sulfonyl chloride 6 or 7 (100 mg, 0.29 mmol) and
appropriate amine (1 mmol) in anhydrous acetonitrile (5 mL) was
stirred overnight at room temperature and then concentrated in
vacuo to afford the crude sulfonamide. The residue was purified
on silica gel using a mixture of CH2Cl2/EtOH (25:1) as eluent to give
the required compounds as a yellow solids.
4.4.6. 5-(5-(1,4-Diazepan-1-ylsulfonyl)-2-ethoxy-phenyl]-1,3-
dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazine (8f)
Yield 88%, yellow powder, mp 146–157 °C. 1H NMR (CDCl3) d:
1.35 (t, 3H, J = 13.6 Hz), 1.83 (t, 2H, J = 11.6 Hz), 2.71 (s, 3H),
2.92–2.99 (m, 4H), 3.10–3.13 (m, 2H), 3.35–3.41 (m, 2H), 4.19 (q,
2H, J = 13.6 Hz), 4.33 (s, 3H), 7.13 (d, 1H, J = 8.8 Hz), 7.85 (dd, 2H,
J1 = 8.8 Hz, J2 = 2.4 Hz), 8.16 (d, 1H, J = 2.4 Hz). 13C NMR (CDCl3) d:
10.97, 14.38, 30.34, 34.69, 47.14, 47.46, 49.72, 50.56, 64.88,
112.84, 127.16, 130.17, 130.92, 131.08, 134.41, 142.10, 146.85,
4.4.1. 5-[2-Ethoxy-5-(4-methylpiperazin-1-ylsulfon-yl)phenyl]-
1,3-dimethyl-1H-pyrazolo[4,3-e]-[1,2,4]triazine (8a)
Yield 91%, yellow powder, mp 148–153 °C. 1H NMR (CDCl3) d:
1.36 (t, 3H, J = 14.0 Hz), 2.17 (s, 3H), 2.29 (s, 3H), 2.52 (bs, 4H),
3.13 (bs, 4H), 4.20 (q, 2H, J = 14.0 Hz), 4.34 (s, 3H), 7.16 (d, 1H,
J = 8.0 Hz), 7.85 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.18 (d, 1H,

6622
M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
158.46, 160.33. HRMS (ESI, m/z) calcd. for C19H26N7O3S [M+H]
432.1812. Found [M+H] 432.1811. Anal. calcd. for C19H25N7O3S:
C, 52.88; H, 5.84; N, 22.72. Found: C, 52.78; H, 5.90; N, 22.60.
Anal. calcd. for C19H25N7O3S: C, 52.88; H, 5.84; N, 22.72. Found:
C, 52.70; H, 5.95; N, 22.57.
4.5. Synthesis of derivatives 10a–c
4.4.7. N-(2-Aminoethyl)-3-(1,3-dimethyl-1H-pyrazolo[4,3-
e][1,2,4]triazin-5-yl)-4-ethoxybezene-sulfonamide (8g)
Method A: A mixture of the sulfonyl chloride 6 (1 mmol)
and amine (piperazine, homopiperazine or ethylenediamine)
(0.5 mmol) in anhydrous acetonitrile (5 mL) was stirred at room
temperature overnight. Next, the reaction mixture was concen-
trated in vacuo to afford the crude bisulfonamides 10a–c. The res-
idues were purified on silica gel using a mixture of CH2Cl2/EtOH
(100:1) as eluent to give the required compounds as a yellow
solids.
Yield 65%, yellow powder, mp 149–155 °C. 1H NMR (CDCl3) d:
1.33 (t, 3H, J = 14.0 Hz), 2.69 (s, 3H), 2.95 (t, 2H, J = 10.8 Hz), 3.10
(t, 2H, J = 10.8 Hz), 4.18 (q, 2H, J = 14.0 Hz), 4.31 (s, 3H), 7.16 (d,
1H, J = 8.8 Hz), 8.00 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.25 (d, 1H,
J = 2.4 Hz). 13C NMR (CDCl3) d: 11.05, 14.44, 34.77, 40.65, 44.18,
64.96, 113.00, 127.11, 130.53, 131.33, 131.39, 134.51, 142.20,
146.88, 158.51, 160.49. HRMS (ESI, m/z) calcd. for C16H22N7O3S
[M+H] 392.1499. Found [M+H] 392.1501. Anal. calcd. for C16H21N7
O3S: C, 49.09; H, 5.41; N, 25.05. Found: C, 48.98; H, 5.55; N, 24.91.
Method B: An equimolar mixture of 6 and sulfonamide 8c or 8f–
g was allowed to react according to the procedure as described
above for 10a–c.
4.4.8. 3-(1,3-Dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazin-5-yl)-4-
ethoxy-N-(2-morpholinethyl)-benzenesulfonamide (8h)
4.5.1. 1,4-Bis(3-(1,3-dimethyl-1H-pyrazolo[4,3-e]-[1,2,4]triazin-
5-yl)-4-ethoxyphenylsulfonyl)-piperazine (10a)
Yield 91%, yellow powder, mp 151–153 °C. 1H NMR (CDCl3) d:
1.36 (t, 3H, J = 14.0 Hz), 2.31 (t, 4H, J = 8.8 Hz), 2.56 (t, 2H,
J = 11.2 Hz), 2.72 (s, 3H), 3.04 (t, 2H, J = 11.2 Hz), 3.68 (t, 4H,
J = 8.8 Hz), 4.20 (q, 2H, J = 14.0 Hz), 4.34 (s, 3H), 7.15 (d, 1H,
J = 8.8 Hz), 7.99 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.27 (d, 1H,
J = 2.4 Hz). 13C NMR (CDCl3) d: 11.05, 14.44, 34.79, 38.77, 53.01,
56.48, 65.01, 66.34, 112.82, 127.37, 130.51, 131.17, 131.33,
134.42, 142.19, 146.85, 158.45, 160.58. HRMS (ESI, m/z) calcd. for
Yield 92%, mp 172–176 °C. 1H NMR (CDCl3) d: 1.36 (t, 6H,
J = 7.2 Hz), 2.70 (s, 6H), 3.16 (s, 8H), 4.19 (q, 4H, J = 7.2 Hz), 4.32
(s, 6H), 7.16 (d, 2H, J = 8.8 Hz), 7.81 (dd, 2H, J1 = 8.8 Hz,
J2 = 2.4 Hz), 8.14 (d, 2H, J = 2,4 Hz). 13C NMR (CDCl3) d: 11. 04,
14.40, 34.73, 45.47, 65.03, 112.99, 126.81, 127.42, 130.91, 131.88,
134.46, 142.21, 146.90, 158.43, 161.02. HRMS (ESI, m/z) calcd. for
C32H36N12O6S2 [M+] 748.2317. Found [M+] 748.2316. Anal. calcd.
C
20H28N7O4S [M+H] 462.1918. Found [M+H] 462.1920. Anal. calcd.
for C32H36N12O6S2: C, 51.33; H, 4.85; N, 22.45. Found: C, 51.20;
H, 5.00; N, 22.25.
for C20H27N7O4S: C, 52.05; H, 5.90; N, 21.24. Found: C, 51.97; H,
6.15; N, 21.18.
4.5.2. 1,4-Bis(3-(1,3-dimethyl-1H-pyrazolo[4,3-e]-[1,2,4]triazin-
5-yl)-4-ethoxyphenylsulfonyl)-1,4-diazepane (10b)
4.4.9. 3-(1,3-Dimethylo-1H-pyrazolo[4,3-e][1,2,4]-triazin-5-yl)-
4-ethoksy-N,N-diethylbenzene-sulfonamide (8i)
Yield 88%, mp 146–157 °C. 1H NMR (CDCl3) d: 1.34 (t, 6H,
J = 6.8 Hz), 1.99 (m, 2H), 2.69 (s, 6H), 3.40 (t, 4H, J = 6.0 Hz), 3.44
(s, 4H), 4.18 (q, 4H, J = 6.8 Hz), 4.31 (s, 6H), 7.13 (d, 2H,
J = 8.8 Hz), 7.84 (dd, 2H, J1 = 2.0 Hz, J2 = 8.8 Hz), 8.16 (d, 2H,
J = 2.4 Hz). 13C NMR (CDCl3) d: 11.00, 14.39, 29.03, 34.70, 47.34,
51.07, 64.94, 112.99, 127.30, 130.07, 130.75, 131.07, 134.41,
142.14, 146.87, 158.39, 160.53. HRMS (ESI, m/z) calcd. for C33H39-
N12O6S2 [M+H] 763.2551. Found [M+H] 763.2548. Anal. calcd. for
Yield 92%, yellow powder, mp 167–170 °C. 1H NMR (CDCl3) d:
1.15 (t, 6H, J = 14.4 Hz), 1.35 (t, 3H, J = 14.0 Hz), 2.72 (s, 3H), 3.25
(q, 4H, J = 14.4 Hz), 4.20 (q, 2H, J = 14.0 Hz), 4.34 (s, 3H), 7.13 (d,
2H, J = 8.8 Hz), 7.91 (dd, 2H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.22 (d, 2H,
J = 2.4 Hz). 13C NMR (CDCl3) d: 11.05, 14.23, 14.47, 34.78, 42.14,
64.90, 112.76, 127.08, 130.34, 131.33, 132.14, 134.56, 142.23,
146.91, 158.62, 160.23. HRMS (ESI, m/z) calcd. for C18H25N6O3S
[M+H] 405.1703. Found [M+H] 405.1704. Anal. calcd. for C18H24N6-
O3S: C, 53.45; H, 5.98; N, 20.78. Found: C, 53.33; H, 6.07; N, 20.63.
C33H38N12O6S2: C, 51.96; H, 5.02; N, 22.03. Found: C, 51.78; H,
5.16; N, 21.91.
4.5.3. N,N-(Ethane-1,2-diyl)bis(3-(1,3-dimethyl-1H-
pyrazolo[4,3-e][1,2,4]triazin-5-yl)-4-ethoxy-
benzenesulfonamide (10c)
4.4.10. 3-(1,3-Dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazin-5-yl)-
4-ethoxy-N-(2-hydroxyethyl)benzene-sulfonamide (8j)
Yield 93%, yellow powder, mp 149–152 °C. 1H NMR (CDCl3) d:
1.35 (t, 3H, J = 14.0 Hz), 2.71 (s, 3H), 3.14 (t, 2H, J = 8.8 Hz), 3.68
(q, 2H, J = 9.6 Hz), 4.20 (q, 2H, J = 14.0 Hz), 4.33 (s, 3H), 5.37 (bs,
1H, OH), 7.14 (d, 1H, J = 8.8 Hz), 7.96 (dd, 2H, J1 = 8.8 Hz,
J2 = 2.4 Hz), 8.25 (d, 1H, J = 2,4 Hz). 13C NMR (CDCl3) d: 11.05,
14.43, 34.84, 45.34, 61.10, 65.01, 112.89, 126.95, 130.60, 131.35,
131.48, 134.69, 142.26, 146.75, 158.33, 160.53. HRMS (ESI, m/z)
calcd. for C16H21N6O4S [M+H] 393.1340. Found [M+H] 393.1341.
Anal. calcd. for C16H20N6O4S: C, 48.97; H, 5.14; N, 21.42. Found:
C, 48.90; H, 5.20; N, 21.21.
Yield 65%, mp 149–155 °C. 1H NMR (CDCl3) d: 1.32 (t, 6H,
J = 6.8 Hz), 2.68 (s, 6H), 3.10 (s, 4H), 4.17 (q, 4H, J = 6.8 Hz), 4.30
(s, 6H), 7.12 (d, 2H, J = 8.8 Hz), 7.90 (dd, 2H, J1 = 8.8 Hz,
J2 = 2.4 Hz), 8.21 (d, 2H, J = 2.4 Hz). 13C NMR (CDCl3) d: 11.04,
14.42, 34.79, 42.91, 65.00, 113.04, 126.93, 130.59, 131.26, 131.32,
134.62, 142.24, 146.73, 158.30, 160.58. HRMS (ESI, m/z) calcd. for
C30H34N12O6S2 [M+] 722.2160. Found [M+H] 722.2156. Anal. calcd.
for C30H34N12O6S2: C, 49.85; H, 4.74; N, 23.25. Found: C, 49.93; H,
4.85; N, 23.13.
4.6. Synthesis of derivatives 11 and 12
4.4.11. 5-[2-Ethoxy-5-(4-methylpiperazin-1-ylsulfo-nyl)-
phenyl]-2,3-dimethyl-2H-pyrazolo[4,3-e]-[1,2,4]triazine (9)
Yield 75%, yellow powder, mp 80–83 °C. 1H NMR (400 MHz,
CDCl3) d: 1.33 (t, 3H, J = 6.8 Hz), 2.47 (t, 4H, J = 4.8 Hz), 2.76 (s,
3H), 3.10 (bs, 4H), 3.67 (s, 3H), 4.16 (q, 2H, J = 6.8 Hz), 4.30 (s,
3H), 7.13 (d, 1H, J = 8.8 Hz), 7.81 (dd, 1H, J1 = 8.8 Hz, J2 = 2.0 Hz),
8.18 (d, 1H, J = 2.0 Hz). 13C NMR (100 MHz, CDCl3) d: 9.13, 14.39,
39.42, 45.48, 45.73, 53.90, 64.97, 112.79, 127.03, 127.59, 128.86,
130.93, 131.90, 134.01, 154.40, 157.64, 160.89. HRMS (ESI, m/z)
calcd. for C19H25N7O3S [M+] 431.17396. Found [M+] 431.17359.
To a solution of 1 (1.81 g, 10 mmol) in benzene (140 mL), ethyl
vinyl ether (2 ml, 20 mmol) or 3,4-dihydro-2H-pyran and one
drop of HCl (38%) was added. The reaction mixture was stirred
at 40 °C for 8 hours, and then a saturated solution of NaHCO3
was added. The benzene layer was separated, dried over
anhydrous MgSO4 and concentrated in vacuo. The product was
purified by column chromatography using silica gel and chloro-
form as eluent.

M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
6623
4.6.1. 1-(1-Ethoxyethyl)-3-methyl-5-methylsulfanyl-1H-
4.9. Reaction of 15 with methyl iodide
pyrazolo[4,3-e][1,2,4]triazine (11)
Yield 90%, mp 51 °C. 1H NMR (CDCl3) d: 1.14 (t, 3H, J = 7.0 Hz),
1.92 (d, 3H, J = 6.0 Hz), 2.65 (s, 3H), 2.74 (s, 3H), 3.19–3.34 (m,
1H), 3.49–3.64 (m, 1H), 6.30 (q, 1H, J = 6.0 Hz); HRMS (ESI) calcd.
for C10H15N5OSNa [M+Na] 276.0890. Found [M+Na] 276.0910.
A mixture of potassium carbonate (138 mg, 1 mmol) and deriv-
ative 15 (123 mg, 0.5 mmol) in ethanol/water mixture (EtOH/H2O,
4:1, v/v) was stirred for 10–15 min at room temperature. Then a
solution of methyl iodide (0.1 mL, 1 mmol) was added and stirred
overnight at room temperature. The excess of base was then
destroyed by the addition of a saturated solution of ammonium
chloride and the solvent was removed in vacuo. The crude product
was submitted to column chromatography on silica gel using
dichloromethane as eluent to give 4 as the first product. Further
elution with CH2Cl2/EtOH (25:1) gives pure 5. Total yield 90% with
a ratio of the isomers 4 and 5 1:1.2.
4.6.2. 1-(Tetrahydro-2H-pyran-2-yl)-3-methyl-5-
methylsulfanyl-1H-pyrazolo[4,3-e][1,2,4]triazine (12)
Yield 80%, yellow oil. 1H NMR (CDCl3) d: 1.65–1.68 (m, 2H),
1.81–1.87 (m, 2H), 2.08–2.12 (m, 1H), 2.16–2.20 (m, 1H), 2.63 (s,
3H), 2.73 (s, 3H), 2.73–2.81 (m, 1H), 4.13–4.19 (m, 1H), 6.27 (dd,
1H, J1 = 10.4 Hz, J2 = 2.4 Hz). 13C NMR (CDCl3) d: 11.03, 14.27,
22.94, 24.83, 29.31, 68.49, 84.11, 135.97, 142.09, 147.14, 167.93.
HRMS (ESI, m/z) calcd. for C11H15N5OS [M+] 265.0997. Found [M+]
265.0998.
4.10. Purification of enzyme
The enzyme was purified from L. sacchari starting with 1000 ml
of the culture filtrate after saturation by 60% (NH4)2SO4 under con-
tinuous stirring at 4 °C. Then, 10 g anion-exchange material (Serva-
cell, DEAE 52) was suspended in 500 ml 25 mM sodium acetate
buffer, pH 5.5, and equilibrated overnight at 4 °C. The upper layer
was decanted, and the sediment was mixed with 10 ml of the sam-
ple. The mixture was then adjusted to pH 7.0. The sediment was
centrifuged at 2500g for 15 min at 4 °C and the obtained superna-
tant used for further purification by size exclusion chromatogra-
phy. A FPLC system was used, equipped with a Sephacryl S-100
column (16 ꢀ 60 mm), equilibrated with 25 mM PIPES buffer, pH
7.0, containing 150 mM NaCl. The column was eluted with the
same buffer at a flow rate of 0.4 ml/min. The fraction with the
highest activity was additionally purified after chromatography
on the same column.
4.7. Synthesis of derivatives 13 and 14
Compounds 13 and 14 were synthesized following the proce-
dure for the preparation of derivatives 4 and 5 as described above.
4.7.1. 1-(1-Ethoxyethyl)-5-(2-ethoxyphenyl)-3-methyl-1H-
pyrazolo[4,3-e][1,2,4]triazine (13)
Yield 85%, yellow powder, mp 149–155 °C.1H NMR (CDCl3) d:
1.17 (t, 3H, J = 7.2 Hz), 1.34 (t, 3H, J = 7.2 Hz), 1.96 (d, 3H,
J = 6.0 Hz), 2.73 (s, 3H), 3.33–3.39 (m, 1H), 3.58–3.64 (m, 1H),
4.15 (q, 2H, J = 7.2 Hz), 6.43 (q, 1H, J = 6.0 Hz), 7.07–7.13 (m, 2H),
7.43–7.48 (m, 1H), 7.80 (dd, 1H, J1 = 7.0 Hz, J2 = 2.0 Hz). 13C NMR
(CDCl3) d: 11.27, 14.70, 14.78, 20.57, 64.61, 64.70, 83.25, 113.59,
120.85, 126.73, 131.21, 132.02, 135.13, 143.25, 147.36, 157.34,
160.78. HRMS (ESI, m/z) calcd. for C17H21N5O2 [M+] 327.1690.
Found [M+] 327.1691. Anal. calcd. for C17H21N5O2: C, 62.37; H,
6.47; N, 21.39. Found: C, 62.25; H, 6.52; N, 21.30.
The purity of the protein was checked after each step of purifi-
cation by sodium dodecyl sulfate polyacrylamide gel electrophore-
sis (SDS–PAGE) (10% polyacrylamide) according to Laemmli.28 The
protein concentration was measured after each step of purification
by the method of Bradford.29
4.7.2. 5-(2-Ethoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-
methyl-1H-pyrazolo[4,3-e][1,2,4]-triazine (14)
Yield 80%, yellow oil. 1H NMR (CDCl3) d: 1.34 (t, 3H, J = 6.8 Hz),
1.65–1.68 (m, 2H), 1.81–1.87 (m, 2H), 2.08–2.12 (m, 1H), 2.16–2.20
(m, 1H) 2.73 (s, 3H), 2.73–2.81 (m, 1H), 4.12 (q, 2H, J = 6.8 Hz),
4.13–4.19 (m, 1H), 6.27 (dd, 1H, J1 = 10.4 Hz, J2 = 2.4 Hz), 7.07–7.14
4.11. Tyrosinase inhibition assay
Tyrosinase activity assays were performed with L-DOPA as sub-
strate, as previously described,30 with slight modifications. Different
inhibitor concentrations (8a–j, 9 and 10c) in 50 mM phosphoric acid
buffer solution (pH 6.8, 1.8 mL) and an aqueous solution of tyrosi-
nase (1000 U/mL, 0.1 mL) and DMSO (0.1 mL) were pre-incubated
for 10 min at 25 °C. Then, a 1.5 mM L-DOPA solution (1 mL) was
added and the reaction monitored at 475 nm for 10 min. The
detected absorbance increases accompanying the oxidation of the
substrate (L-DOPA). The final concentration of dopaquinone was
(m, 2H), 7.45–7.48 (m, 1H), 7.79 (dd, 1H, J1 = 7.2 Hz, J2 = 2.0 Hz). 13
C
NMR (CDCl3) d: 11.26, 14.72, 22.98, 24.87, 29.44, 64.72, 68.61,
83.96, 113.45, 120.89, 128.44, 128.56, 131.55, 131.99, 132.06,
132.16, 143.82, 157.35. HRMS (ESI, m/z) calcd. for C18H21N5O2 [M+]
339.1695. Found [M+] 339.1696. Anal. calcd. for C18H21N5O2: C,
63.70; H, 6.24; N, 20.64. Found: C, 63.80; H, 6.32; N, 20.45.
4.8. Deprotection of 13 and 14
measured using the molar absorption coefficient of 3700 Mꢁ1 cmꢁ1
.
The percentage of inhibition of tyrosinase activity was calculated as
inhibition (%) = (A ꢁ B)/A ꢀ 100, where A represents the difference
in the absorbance of control sample between 0.5 and 1.0 min of an
incubation time, and B the difference in absorbance of the test sam-
ple between an incubation time of 0.5 and 1.0 min. The activity of
tyrosinase was determined by spectrophotometric techniques (Shi-
madzu UV-2100). DMSO inhibitory activity, was determined to be
2.5% under the same experimental conditions
A mixture of 13 or 14 (206 mg, 1 mmol) and concentrated HCl
(0.5 mL) in methanol (10 mL) was stirred at room temperature
for 12 h. Then the solvent was removed and the residue separated
by column chromatography (silica gel, eluent/chloroform/ethanol,
20:1) to afford 124 mg (92%) of 15 as a yellow solid.
4.8.1. 3-Methyl-5-(2-ethoxyphenyl)-1H-pyrazolo-[4,3-
e][1,2,4]triazine (15)
Yield 75%, yellow oil. 1H NMR (CDCl3) d: 1.31 (t, 3H, J = 7.2 Hz),
2.75 (s, 3H), 4.16 (q, 2H, J = 7.2 Hz), 7.08–7.14 (m, 2H), 7.43–7.48
(m, 1H), 7.80 (dd, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz), 11.70 (s, 1H, NH).
13C NMR (CDCl3) d: 11.21, 14.66, 64.62, 113.36, 120.84, 126.23,
131.42, 131.96, 135.14, 144.02, 147.97, 157.26, 160.28. HRMS
4.12. PDE5 assay
PDE5A (BPS Bioscience Inc) activity was analyzed using a
tritium scintillation proximity assay (SPA) system, and the assay
was performed according to the manufacturer’s instructions
(Amersham Biosciences). Briefly, assays were performed in the
presence of 50 mM Tris–HCl (pH 7.5) containing 8.3 mM MgCl2
(ESI, m/z) calcd. for
C13H13N5O [M+] 255.1115. Found [M+]
255.1116. Anal. calcd. for C13H13N5O: C, 61.17; H, 5.13; N, 27.43.
Found: C, 61.00; H, 5.29; N, 27.25.
and 1.7 mM EGTA. Each assay was performed in a 100 ll reaction

6624
M. Mojzych et al. / Bioorg. Med. Chem. 22 (2014) 6616–6624
2. Desenko, S. M.; Komykhov, S. A.; Orlov, V. D.; Meier, H. J. Heterocycl. Chem.
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3. Kurasawa, Y.; Mitomi, T.; Okamoto, Y.; Kim, H. S. J. Heterocycl. Chem. 1998, 35,
1333.
volume containing the above buffer (Tris–HCl, pH 7.5), enzyme and
around 0.05
Ci [3H]cGMP. The reaction was carried out at 30 °C
for 30 minutes and stopped by the addition of 50 l PDE SPA beads
l
l
4. Youngdle, G. A. U.S. Patent, 4 288 440, 1980; Chem. Abstr. 1982, 96, 6596c.
5. Todd, A. H. Br. Patent, 1203 149, 1970; Chem. Abstr. 1970, 73, 120 508b
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(1 mg) suspended in 18 mM zinc sulfate. The reaction mixture was
left to settle at room temperature for 20 min before counting in a
MicroBeta TriLux instrument (Perkin–Elmer Life Sciences, USA).
For compound inhibition study on PDE5A, a stock solution of the
compounds was prepared in 100% DMSO, diluted in water to the
appropriate concentrations, and added to the assay buffer to give
a final concentration of <1% DMSO. The amount of enzyme used
in each reaction was such that the hydrolysis of substrates did
not exceed 15%, so that the amount of product increased linearly
with time. Duplicates were run in each assay.
PDE inhibitory activity was calculated from equation below:
À
Á
ꢀ
ꢁ
CPMsample ꢁ CPMblank
%PDE inhibition ¼ 1 ꢁ
ꢀ 100
ðCPMcontrol ꢁ CPMblank
Þ
Whereby, CPMsample represents the radioactive value of sample
obtained from the assay (with enzyme), CPMcontrol the radioactive
value of solvent (water) which was used for diluting the sample
obtained from the assay (with enzyme), and CPMblank the radioac-
tive value of the mixture of only buffer and substrate (without
enzyme).
Acknowledgements
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Westerholm-Parvinen, A.; Kalkkinen Buchert, N. J.; Kruus, K. FEBS J. 2006,
273, 4322.
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S.; Record, E.; O’Beirne, D.; Buchert, J.; Kruus, K. J. Biotechnol. 2007, 130, 471.
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Molecules 2013, 18, 3948.
26. Mojzych, M.; Karczmarzyk, Z.; Kalicki, P.; Ceruso, M.; Supuran, C. T. Bioorg. Med.
Chem. under preparation.
This research was funded by the National Science Centre,
Poland (Grant NN405 092340); Bulgarian Ministry of Education,
projects DHRC/01/6 and ‘Young researchers’ DMU 03/26; Grant
NoBG051PO001-3.3.06-0025, financed by the European Social
Fund and Operational Programme Human Resources Development
(2007–2013) and co-financed by Bulgarian Ministry of Education,
Youth and Science. Mariusz Mojzych is thankful to prof. JingShan
Shen from Shanghai Institute of Materia Medica, Chinese Academy
of Science for PDE5 assay.
27. Card, G. L.; England, B. P.; Suzuki, Y.; Fong, D.; Powell, B.; Lee, B.; Luu, C.;
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