Relaxant effects of selected sildenafil analogues in the rat aorta

Article abstract of DOI:10.3109/14756366.2015.1024674

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their relaxant effects in the rat aorta. Evaluation of prepared derivatives demonstrated that compound (8a) is probably a non-selective phosphodiesterase (PDE) inhibitor, as it induced aortic relaxation through endothelium-independent mechanism.

Full text of DOI:10.3109/14756366.2015.1024674

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1024674
!
RESEARCH ARTICLE
Relaxant effects of selected sildenafil analogues in the rat aorta
Mariusz Mojzych1, Monika Kubacka2, Szczepan Mogilski2, Barbara Filipek2, and Emilia Fornal3
1Department of Chemistry, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland, 2Department of Pharmacodynamics, Jagiellonian
3
University Medical College, Krakow, Poland, and Department of Chemistry, Laboratory of Separation and Spectroscopic Method Applications,
´
Center for Interdisciplinary Research, The John Paul II Catholic University of Lublin, Lublin, Poland
Abstract
Keywords
A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group
has been synthesized and characterized. The compounds were tested for their relaxant effects
in the rat aorta. Evaluation of prepared derivatives demonstrated that compound (8a) is
probably a non-selective phosphodiesterase (PDE) inhibitor, as it induced aortic relaxation
through endothelium-independent mechanism.
PDE5 inhibitors, pyrazolo[4,3-e][1,2,4]triazine,
relaxant effect, sildenafil analogues,
sulfonamides
History
Received 13 January 2015
Revised 25 February 2015
Accepted 26 February 2015
Published online 23 March 2015
Introduction
treatment of male erectile dysfunction but also for pulmonary
hypertension, we have evaluated the relaxant effects of the newly
synthesized compounds in the rat aorta.
Cyclic nucleotide phosphodiesterases (PDEs) are key enzymes
that control cellular concentrations of the second messengers
cyclic adenosine 30,50-monophosphate (cAMP) and cyclic guano-
sine 30,50-monophosphate (cGMP)1. To date, 11 families of PDEs
Experimental
have been classified according to the sequence homology and Chemistry
biochemical properties2,3. PDE molecules contain a variable
General
regulatory domain and a conserved catalytic domain. However,
each PDE family shows characteristic substrate specificity and Melting points were determined on a Mel-Temp apparatus and are
inhibitor selectivity. Phosphodiesterase 5 (PDE5) is widely uncorrected. 1H and 13C NMR spectra were recorded on a Varian
distributed in vascular and visceral smooth muscle cells, platelets, spectrometer (400 MHz for 1H and 100 MHz for 13C). The
kidney, lung and corpus cavernosum, and plays a key role in the chemical shift values are expressed in ppm (part per million) with
regulation of the cellular level of cGMP. The main therapeutic tetramethylsilane (TMS) as internal reference. The relative
indications for PDE5 inhibitors are the treatment of erectile integrals of peak areas agreed with those expected for the
dysfunction [sildenafil (Viagra), vardenafil (Levitra), and tadalafil assigned structures. Molecular weight of final compounds was
(Cialis), udenafil (Zydena)] and idiopathic pulmonary hyperten- assessed by electrospray ionization mass spectrometry (ESI/MS)
sion [sildenafil (Revatio)], although several other potentials have on a Agilent Technologies 6538 UHD Accurate Mass Q-TOF LC/
also been identified, such as memory improvement, anticancer MS (Lublin, Poland). Elemental compositions are within 0.4%
therapy and treatment of heart diseases4. PDE5 is a therapeutic of the calculated values. For preparation and spectroscopic data of
target of considerable research interest as can be seen by compounds 2a–5a and 8a–c see literature7,8
.
Synthesis of 1-(3-methylsulfanyl-1,2,4-triazin-5-yl)butan-1-one
oxime (2b). To a stirred suspension of powdered potassium
numerous publications regarding the design, synthesis and
optimization of new PDE5 inhibitors5,6
.
To develop novel PDE5 inhibitors with improved therapeutic hydroxide (8.0 g) in dry dimethyl sulfoxide (20 mL) a solution of
efficacy based on the structure of sildenafil, we have prepared a the 1,2,4-triazine (1) (2.0 g, 15.74 mmol) and nitrobutane (3 mL,
series of sildenafil analogues in which the iminocarbonyl group 23.88 mmol) in dimethyl sulfoxide (1–2 mL) was added at once at
was replaced with the nitrogen atoms of the triazine ring. room temperature. The mixture was stirred at room temperature
Moreover, the methylpiperazine moiety was replaced with various for 2 h, then was poured into ice/water (200 mL) and acidified
amines (Figure 1). Because sildenafil is being used not only for with acetic acid (pH 6.9–7.0). The product (2b) was precipitated
and was collected by filtration and washed with water. The crude
product was purified by chromatography (silica gel; CH2Cl2/
EtOH 50:1) to afford (2) as a yellowish powder. Yield 86%; m.p.
Address for correspondence: Mariusz Mojzych, Department of
Chemistry, Siedlce University of Natural Sciences and Humanities,
3-go Maja 54, 08-110 Siedlce, Poland. E-mail: mmojzych@yahoo.com
1
126–127 ꢀC; H NMR (400 MHz, CDCl3) ꢀ: 0.97 (t, J ¼ 7.2 Hz,
3H), 1.57–1.66 (m, 2H), 2.67 (s, 3H), 2.86 (t, 2H, J ¼ 7.2 Hz),

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M. Mojzych et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
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the corresponding pyrazolotriazine (5b) in 61% yield. H NMR
(400 MHz, CDCl3) ꢀ: 1.00 (t, 3H, J ¼ 7.6 Hz), 1.83–1.90 (m, 2H),
2.70 (s, 3H), 2.98 (t, 2H, J ¼ 7.6 Hz), 4.23 (s, 3H). 13C NMR
(100 MHz, CDCl3) ꢀ: 13.9, 14.2, 21.6, 27.9, 34.6, 134.5, 144.3,
147.1, 166.8. HRMS (ESI, m/z) Calcd for C9H14N5S [M + H]
224.0969. Found [M + H] 224.0964. Anal. Calcd for C9H13N5S:
C, 48.41; H, 5.87; N, 31.36. Found: C, 48.33; H, 5.97; N, 31.20.
General method synthesis of derivatives (6ab). To a mixture of
(5a) or (5b) (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 eqiuv) in dry tetrahydrofuran (THF) (25 mL) under argon
Pd(PPh3)4 (0.36 g, 0.31 mmol, 0.1 equiv) were added. The reac-
tion mixture was stirred overnight at reflux. The reaction was
quenched with a Na2CO3 saturated 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
product (6) was obtained.
3-(2-Ethoxyphenyl)-1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]-
triazine (6a). Yellow powder. Yield 75%; m.p. 85–86 ꢀC. 1H NMR
(400 MHz, CDCl3): ꢀ 1.29–1.33 (t, 3H, J ¼ 14 Hz), 2.70 (s, 3H),
4.10–4.15 (q, 2H, J ¼ 14 Hz), 4.32 (s, 3H); 7.06–7.12 (m, 2H),
7.42–7.46 (m, 2H), 7.74–7.75 (d–d, 1H, J1 ¼ 2.4 Hz, J2 ¼ 8.8 Hz).
13C NMR (CDCl3): ꢀ 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 C14H15N5O [M+] 269.1276. Found
[M+] 269.1284. Anal. Calcd for C14H15N5O: C, 62.44; H, 5.61;
N, 26.01. Found: C, 62.30; H, 5.70; N, 25.93.
3-(2-Ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-e]
[1,2,4]-triazine (6b). Yellow oil. Yield 80%. 1H NMR (400 MHz,
CDCl3) ꢀ: 1.03 (t, 3H, J ¼ 7.6 Hz), 1.31 (t, 3H, J ¼ 7.2 Hz), 1.88–
1.98 (m, 2H), 3.08 (t, 2H, J ¼ 7.6 Hz), 4.12 (q, 2H, J ¼ 7.2 Hz),
4.32 (s, 3H), 7.06–7.12 (m, 2H), 7.42–7.46 (m, 1H), 7.78 (dd, 1H,
J1 ¼ 7.6 Hz, J2 ¼ 1.6 Hz). 13C NMR (100 MHz, CDCl3) ꢀ: 14.0,
14.8, 22.0, 28.2, 34.8, 64.7, 113.5, 120.9, 127.0, 131.2, 132.1,
134.5, 146.2, 147.1, 157.4, 160.2. HRMS (ESI, m/z) Calcd for
C16H20N5O [M + H] 298.1667. Found [M + H] 298.1662. Anal.
Calcd for C16H19N5O: C, 64.63; H, 6.44; N, 23.55. Found: C,
64.55; H, 6.56; N, 23.42.
Figure 1. Structure of sildenafil and its new analogues. The data are the
means of four experiments S.E.M.
9.45 (s, 1H), 9.96 (bs, 1H). 13C NMR (100 MHz, CDCl3) ꢀ: 13.7,
14.2, 19.4, 24.9, 141.7, 153.0, 157.3, 173.7. HRMS (ESI, m/z)
Calcd for C8H13N4OS [M + H] 213.0810. Found [M + H]
213.0805. Anal. Calcd for C8H12N4OS: C, 45.27; H, 5.70; N,
26.39. Found: C, 45.30; H, 5.76; N, 26.30.
Synthesis of 1-(3-methylsulfanyl-1,2,4-triazin-5-yl)butan-1-one
(3b). The oxime (2b) (2.12 g, 10 mmol) was dissolved in dioxane
(40 mL) and treated with a solution of hydrosulfite Na2S2O4
(4.6 g, 26.4 mmol) in water (40 mL). The resulting mixture was
stirred at room temperature for 24 h. Dioxane was then removed
in vacuo and the residue was treated with 10% HCl (pH ꢁ3) and
bubbled by air for 10 min. Then the mixture was made neutral by
addition of solid NaHCO3 and extracted with CH2Cl2.
Evaporation of the organic solvent gave a crude product, which
was purified by chromatography (silica gel; CH2Cl2/hexane 1:2)
to afford ketone (3b) as a yellow oil. Yield 55%. 1H NMR
(400 MHz, CDCl3) ꢀ: 1.00 (t, 3H, J ¼ 7.6 Hz), 1.72–1.78 (m, 2H),
2.72 (s, 3H), 3.12 (t, 2H, J ¼ 7.6 Hz), 9.37 (s, 1H). 13C NMR
(100 MHz, CDCl3) ꢀ: 13.6, 14.0, 16.8, 39.4, 140.9, 147.3, 174.7,
200.9. HRMS (ESI, m/z) Calcd for C8H12N3OS [M + H]
198.0701. Found [M + H] 198.0696. Anal. Calcd for
C8H11N3OS: C, 48.71; H, 5.62; N, 21.30. Found: C, 48.80; H,
5.75; N, 21.10.
Synthesis of methylhydrazone of 5-butanoyl-3-methylsulfanyl-
1,2,4-triazine (4b). To a solution of (3b) (2.2 g, 11.16 mmol) and
methylhydrazine (0.77 g, 16.73 mmol) in 10 mL of ethanol
p-toluenosulfonic acid monohydrate (200 mg) was added and
the resulting reaction mixture was stirred for 15 min at room
temperature (rt). After that time the precipitated solid was filtered
off. The crude product was recrystallized from ethanol–water
mixture to obtain the pure product (4b) as a yellow solid. Yield
50%; m.p. 129–131 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ: 0.96
(t, 3H, J ¼ 7.6 Hz), 1.46–1.56 (m, 2H), 2.60–2.64 (m, 5H), 3.28
(s, 3H), 9.40 (s, 1H). 13C NMR (100 MHz, CDCl3) ꢀ: 13.7, 14.3,
18.0, 23.8, 38.3, 138.7, 141.6, 153.9, 172.1. HRMS (ESI, m/z)
Calcd for C9H16N5S [M + H] 226.1126. Found [M + H] 226.1121.
Anal. Calcd for C9H15N5S: C, 47.98; H, 6.71; N, 31.08. Found: C,
48.13; H, 6.90; N, 31.00.
Synthesis of 1-methyl-5-methylsulfanyl-3-propyl-1H-pyra-
zolo[4,3-e][1,2,4]triazine (5b). Method A: A solution of (4b)
(155 mg, 0.78 mmol) and 10% hydrochloric acid (0.1 mL) in
ethanol (5 mL) was refluxed for 1 h. After that time the solvent
was evaporated in vacuo and the crude product was purified by
column chromatography (silica gel, chloroform) to afford the final
product (5b) as a yellow oil (130 mg, 0.58 mmol, 58%).
General method synthesis of derivatives (7ab). Compound (6a)
or (6b) (1.88 mmol) was added portion wise 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 solution was
cautiously added to ice-water (15 mL), and the aqueous mixture
was extracted with dichloromethane. The combined extracts were
dried over anhydrous Na2SO4 and evaporated under vacuum to
give the required sulfonyl chloride (7a) or (7b), respectively.
4-Ethoxy-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]-triazin-5-
yl)benzene-1-sulfonyl chloride (7a). Yellow crystals. Yield 85%;
m.p. 119–120 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ: 1.37–1.40
(t, 3H, J ¼ 14 Hz), 2.72 (s, 3H), 3.64 (s, 3H), 4.23–4.28 (q, 2H,
J ¼ 14 Hz), 7.20–7.22 (d, 1H, J ¼ 9.2 Hz), 3.45–8.46 (d, 1H,
J ¼ 2.4 Hz), 8.12–8.15 (dd, 1H, J1 ¼ 2.8 Hz, J2 ¼ 8.8 Hz); 13C
NMR (CDCl3) ꢀ: 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 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-Ethoxy-3-(1-methyl-3-propyl-1H-pyrazolo[4,3-e][1,2,4]-tria-
zin-5-yl)benzene-1-sulfonyl chloride (7b). Yellow solid. Yield
95%; m.p. 65–66 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ: 1.01
(t, 3H, J ¼ 7.2 Hz), 1.36 (t, 3H, J ¼ 7.2 Hz), 1.87–1.94 (m, 2H),
3.07 (t, 2H, J ¼ 7.2 Hz), 4.23 (q, 2H, J ¼ 7.2 Hz), 7.21 (d, 1H,
J ¼ 8.8 Hz), 8.14 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz), 8.47 (d, 1H,
J ¼ 2.4 Hz). 13C NMR (100 MHz, CDCl3) ꢀ: 13.8, 14.2, 21.7,
Method B: A mixture of (4b) (1 mmol) and p-toluenosufonic
acid (2 mmol) was mixed and heated at 140 ꢀC for 1 min under
solvent free reaction condition. After cooling, the solid residue
was treated with chloroform and then the major product was
isolated by chromatography on silica gel with chloroform to give

DOI: 10.3109/14756366.2015.1024674
Relaxant effects of selected sildenafil analogues
3
27.9, 34.7, 65.3, 112.9, 127.5, 130.4, 131.4, 134.2, 135.7, 146.1, Pharmacology
146.8, 157.4, 162.4. HRMS (ESI, m/z) Calcd for C16H19ClN5O3S
[M + H] 396.0897. Found [M + H] 396.0892.
Materials and methods
Synthesis of sulfonamides (8d–g). A mixture of chlorosulfonyl Animals. The experiments were carried out using male Wistar
chloride (7a) (100 mg, 0.29 mmol) and amine (100 mg, 1 mmol) in rats (Krf:(WI) WU), 200–250 g. The animals were housed in
anhydrous acetonitrile (5 mL) was stirred overnight at room constant temperature facilities exposed to 12:12 h light/dark
temperature, and then the reaction mixture was concentrated cycles and were maintained on a standard pellet diet with tap
in vacuo to afford the crude sulfonamide, as a yellow solid. The water given ad libitum. All procedures were conducted according
residue was purified on silica gel using a mixture of CH2Cl2:EtOH to the Animal Care and Use Committee Guidelines and approved
(25:1) as eluent to give the titled compounds as a yellow solid.
(S)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-yl)-4-
ethoxy-N-(1-hydroxy-propan-2-yl)benzenesulfonamide (8d). Yield
by the Local Ethics Committee of the Jagiellonian University in
´
Krakow.
92%; m.p. 131–136 ꢀC. 1H NMR (CDCl3) ꢀ: 1.09 (d, 3H, Drugs and chemicals. The materials used included 3-isobutyl-1-
J ¼ 6.4 Hz), 1.35 (t, 3H, J ¼ 6.8 Hz), 2.63 (bs, 1H, OH, exchanged methylxanthine (IBMX), acetylcholine hydrochloride, glibencla-
with D20), 2.70 (s, 3H), 3.39–3.46 (m, 2H), 3.56 (d, 1H, mide, isoprenaline hydrochloride, N!-nitro-L-arginine methyl
J ¼ 7.6 Hz), 4.19 (q, 2H, J ¼ 6.4 Hz), 4.31 (s, 3H), 5.11 (d, 1H, ester hydrochloride (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]qui-
J ¼ 6.0 Hz, NH, exchanged with D2O), 7.14 (d, 1H, J ¼ 9.2 Hz), noxalin-1-one (ODQ), phentolamine hydrochloride, sildenafil
7.98 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 2.4 Hz), 8.28 (d, 1H, citrate
(Sigma-Aldrich,
Germany),
thiopental
sodium
J ¼ 2.4 Hz).13C NMR (CDCl3) ꢀ: 11.05, 14.43, 17.93, 34.82, (Biochemie GmbH, Austria). Other chemicals used were obtained
51.55, 64.94, 65.99, 112.79, 126.97, 130.48, 131.39, 132.15, from POCH (Polish Chemical Reagents, Poland). The drugs were
134.56, 142.21, 146.78, 158.42, 160.46. HRMS (ESI, m/z) Calcd dissolved in dimethyl sulfoxide for the in vitro studies or
for C17H22N6O4S [M+] 406.1423. Found [M+] 406.1427. Anal. suspended in 0.5% methylcelulose for the in vivo studies
Calcd for C17H22N6O4S: C, 50.23; H, 5.46; N, 20.68. Found: C, immediately before use.
50.00; H, 5.49; N, 20.50.
(R)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-yl)-4-
ethoxy-N-(1-hydroxy-propan-2-yl)benzenesulfonamide (8e).
Influence on isolated rat thoracic aorta contracted by KCl. Rats
were anesthetized with thiopental sodium (60 mg/kg i.p.) and the
thoracic aorta was dissected and placed in a Krebs–Henseleit
solution (NaCl 119 mM, KCl 4.7 mM, CaCl2 1.9 mM, MgSO4
1.2 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, glucose 11 mM,
EDTA 0.05 mM) and cleaned of surrounding fat tissues and cut
into approximately 4 mm long rings. Special care was taken to
avoid any damage to the endothelium. In some aortic rings, the
endothelial layer was mechanically removed by gently rubbing the
luminal surface of the aortic ring back and forth several times
with plastic tubing. The aorta rings were incubated in 30 mL
chambers filled with a Krebs–Henseleit solution at 37 ꢀC and pH
7.4 with constant oxygenation (O2/CO2, 19:1). Two stainless steel
pins were inserted through the lumen of each arterial segment:
one pin was attached to the bottom of the chamber and the other to
an isometric FDT10-A force displacement transducer (BIOPAC
Systems, Inc., COMMAT Ltd., Turkey). The aortic rings were
stretched and maintained at optimal tension of 2 g and allowed to
equilibrate for 2 h. The aortic rings were contracted to
submaximal tension with KCl (60 mM). The depolarising solution
KCl (60 mM) was obtained by equimolar substitution of NaCl for
KCl. Once the plateau was attained, concentration–relaxation
curves were obtained by the addition of cumulative doses of tested
compounds to the precontracted preparations.
Yield 89%; m.p. 119–122 ꢀC. 1H NMR (CDCl3) ꢀ: 1.09 (d,
3H, J ¼ 6.4 Hz), 1.34 (t, 3H, J ¼ 6.8 Hz), 2.69 (s, 3H), 2.84
(bs, 1H, OH, exchanged with D20), 3.38–3.45 (m, 2H), 3.55
(d, 1H, J ¼ 7.6 Hz), 4.18 (q, 2H, J ¼ 6.4 Hz), 4.32 (s, 3H), 5.24 (d,
1H, J ¼ 6.4 Hz, NH, exchanged with D2O), 7.13 (d, 1H,
J ¼ 8.8 Hz), 7.98 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz), 8.27 (d, 1H,
J ¼ 2.4 Hz). 13C NMR (CDCl3) ꢀ: 11.04, 14.42, 17.89, 34.80,
51.55, 64.92, 65.96, 112.78, 126.91, 130.47, 131.35, 132.18,
134.57, 142.19, 146.74, 158.40, 160.42. HRMS (ESI, m/z) Calcd
for C17H22N6O4S [M+] 406.1423. Found [M+] 406.1426. Anal.
Calcd for C17H22N6O4S: C, 50.23; H, 5.46; N, 20.68. Found: C,
49.89; H, 5.49; N, 20.55.
(S)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-yl)-4-
ethoxy-N-(1-hydroxy-3-methylbutan-2-yl)benzenesulfonamide
(8f). Yield 91%; m.p. 115–122 ꢀC. 1H NMR (CDCl3) ꢀ: 0.83 (dd,
6H, J1 ¼ 6.8 Hz, J2 ¼ 2.8 Hz), 1.34 (t, 3H, J ¼ 6.8 Hz), 1.82 (sec,
1H, J ¼ 6.8 Hz), 2.69 (s, 3H), 3.07–3.11 (m, 1H), 3.52–3.55 (m,
2H), 4.17 (q, 2H, J ¼ 6.8 Hz), 4.32 (s, 3H), 5.23 (d, 1H,
J ¼ 8.4 Hz, NH, exchanged with D2O), 7.11 (d, 1H, J ¼ 8.8 Hz),
7.96 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.8 Hz), 8.27 (d, 1H, J ¼ 2.8 Hz).
13C NMR (CDCl3) ꢀ: 11.03, 14.40, 18.56, 19.12, 29.62, 34.80,
60.98, 62.50, 64.91, 112.65, 126.79, 130.45, 131.42, 132.49,
134.57, 142.18, 146.69, 158.40, 160.33. HRMS (ESI, m/z) Calcd
for C19H26N6O4S [M+] 434.1736. Found [M+] 434.1741. Anal. Influence on isolated rat thoracic aorta contracted by
Calcd for C19H26N6O4S: C, 52.51; H, 6.03; N, 19.35. Found: C, phenylephrine. Rats were anesthetized with thiopental sodium
52.18; H, 6.06; N, 19.22.
(R)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-yl)-4-
ethoxy-N-(1-hydroxy-3-methylbutan-2-yl)benzenesulfonamide
(60 mg/kg i.p.) and the aorta was isolated, cut, mounted and
incubated as described in a method above. Special care was taken
to avoid any damage to the endothelium. In some aortic rings, the
(8g). Yield 72%; m.p. 115–120 ꢀC. 1H NMR (CDCl3) ꢀ: 0.84 (dd, endothelial layer was mechanically removed by gently rubbing the
6H, J1 ¼ 6.8 Hz, J2 ¼ 3.6 Hz), 1.35 (t, 3H, J ¼ 6.8 Hz), 1.82 (sec, luminal surface of the aortic ring back and forth several times
1H, J ¼ 6.8 Hz), 2.70 (s, 3H), 3.08–3.11 (m, 1H), 3.50–3.58 (m, with plastic tubing. The aorta rings were stretched and maintained
2H), 4.18 (q, 2H, J ¼ 6.8 Hz), 4.32 (s, 3H), 5.16 (d, 1H, at optimal tension of 2 g and allowed to equilibrate for 2 h.
J ¼ 8.8 Hz, NH, exchanged with D2O), 7.11 (d, 1H, J ¼ 8.8 Hz), Endothelial integrity or functional removal was verified by the
7.96 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz), 8.28 (d, 1H, J ¼ 2.4 Hz). presence or absence of the relaxant response to acetylcholine
13C NMR (CDCl3) ꢀ: 11.04, 14.41, 18.56, 19.13, 29.65, 34.81, (1 mM) on the phenylephrine (1 mM) contracted vessels.
60.98, 62.53, 64.92, 112.66, 126.81, 130.46, 131.44, 132.47,
In the first series of experiments, the effects of compound (8a)
134.57, 142.20, 146.72, 158.40, 160.36. HRMS (ESI, m/z) Calcd on vascular tension were defined. Aortic rings were contracted
for C19H26N6O4S [M+] 434.1736. Found [M+] 434.1741. Anal. with phenylephrine (10 mM) to obtain maximal response. Once
Calcd for C19H26N6O4S: C, 52.51; H, 6.03; N, 19.35. Found: C, the maximal response to phenylephrine had been obtained, the
52.18; H, 6.10; N, 19.15.
aortic rings were exposed to cumulative doses of compound (8a)

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M. Mojzych et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
and the responses were recorded. Additionally, to observe whether expressed as a percentage of inhibition of the maximal tension
the vasorelaxant effects of tested compounds are affected by obtained with the contractile agent (Emax ¼ 100%). Data are the
IBMX, a nonselective PDE inhibitor, the action of compound (8a) means S.E.M. of four separate experiments. The concentrations
in the presence of IBMX (1 mM) was investigated.
In another experiment, the effect of compound (8a) on the calculated.
vasorelaxant responses to isoprenaline, b-adrenoceptor-
needed to produce 50% of the maximum relaxation (IC50) were
a
mediated cAMP-dependent vasodilator was investigated by Results
incubating the endothelium denuded aortic rings with 30 mM of
compound (8a) for 30 min before either isoprenaline (0.01–
Chemistry
10 mM) was added.
The synthesis of target aza-analogues of sildenafil (8a–m) was
To define the mechanisms by which compound (8a) relaxes achieved by a convenient multiple procedure starting from 3-
vascular smooth muscle, another series of experiments were done methylsulfanyl-1,2,4-triazine (1) as shown in Scheme 1. First,
in aortic rings. The rings were exposed to various modulating using our previous established procedure9, the reaction of (1) with
agents such as L-NAME (10 mM), ODQ (1 mM), glibenclamide nitrobutane or nitroethane and KOH in DMSO at room tempera-
(1 mM) for 20 min, and then vascular relaxation was carried out by ture gave appropriate oximes (2ab) as main products. In the next
cumulative additions of compound (8a) at the plateau of the step, the readily available oximes (2ab) were transformed into
phenylephrine-induced contractions.
ketones (3ab) in good yield10, which reacted with methylhy-
drazine in the presence of acidic media according to standard
procedure to give suitable hydrazones (4ab) as key intermediates
for the preparation of 1H-pyrazolo[4,3-e][1,2,4]triazine deriva-
tives (5ab). The hydrazones (4ab) could be converted into
derivatives (5) under conventional heating (10% HCl, EtOH,
reflux, 1 h)11 or under solvent free reaction conditions according
to our previous published procedure12. Using Guillaumet and co-
workers method13 for the palladium-catalyzed cross-coupling
reaction of 3-methylsulfanyl-1,2,4-triazine with boronic acid
derivatives we have reacted 5-methylsulfanyl-1H-pyrazolo[4,3-
e][1,2,4]triazines (5ab) with 2-ethoxyphenylboronic acid in the
presence of copper (I) 3-methylsalicylate to obtain derivatives
(6ab) in excellent yield. Chlorosulfonylation reaction of com-
pounds (6ab) in neat chlorosulfonic acid at 0 ꢀC proceeded
smoothly and selectively at the 50-position of the phenyl ring
to give the desired products (7ab) in excellent yield.
Measurement of blood pressure and heart rate. Rats were
anesthetized with thiopental (60 mg/kg i.p.). The right carotid
artery was cannulated with polyethylene tube filled with heparin
in saline to facilitate pressure measurements using a Datamax
apparatus (Columbus Instruments). Compound (8a) was admin-
istered at a dose of 10 mg/kg i.p. after a 15 min stabilization
period at a volume equivalent to the 1 mL/kg.
Data analysis. The results are presented as the means S.E.M.
Statistically significant differences between groups were calcu-
lated using one-way analysis of variance (ANOVA) and the post-
hoc Dunnett’s multiple comparison test. The criterion for
significance was set at p50.05. Concentration–relaxation
curves were analysed using GraphPad Prism 4.0 software
(GraphPad Software Inc., San Diego, CA). Relaxations are
Scheme 1. Synthetic pathway to the sildenafil analogues (8a–m). Reagents and conditions: (a) CH3CH2CH2CH2NO2 (for R ¼C3H7) or CH3CH2NO2
(for R ¼CH3), KOH, DMSO, 2 h, 80–86%; (b) Na2S2O4, H2O/dioxane, rt, 12 h, 55–65%; (c) CH3NH–NH2, PTSA, EtOH, rt, 1 h, 50–55%; (d) method
A: 10% HCl, EtOH, reflux, 1 h, 58–61%; method B: PTSA, 140 ꢀC, 1 min, 61%; (e) ethoxyphenylboronic acid, Pd(PPh3)4, CuMeSal, THF, Ar, reflux,
overnight, 75–80%; (f); ClSO3H, 0 ꢀC to rt, 2 h, 75–95%; (g) appropriate amine, anhydrous MeCN, rt, overnight, 72–93%.

DOI: 10.3109/14756366.2015.1024674
Relaxant effects of selected sildenafil analogues
5
The chlorosulfonyl derivatives (7ab) were readily coupled with Sildenafil inhibited contraction induced by KCl depolarising
appropriate amines in acetonitrile at room temperature to produce solution by 85–90% with IC50 values of 7.0 and 14.3 mM in
the final sulfonamides (8a–m) as new chiral analogues of endothelium intact and endothelium denuded aorta, respectively,
sildenafil in high yield.
suggesting that its vasorelaxant effect is endothelium dependent.
For comparison, IBMX, relaxed the endothelium intact and
denuded aorta with IC50 values of 39.3 and 26.4 mM, respectively,
suggesting that its vasorelaxant effect is endothelium independent
(Table 1). Since compound (8a) turned out to be the most active,
with vasorelaxant properties similar to those of IBMX, it was
Pharmacology
The aim of this work was to assess vasorelaxant properties of
seven new structural analogues of sildenafil (marked with the
numbers: 8a, 8b, 8c, 8d, 8e, 8f and 8g) and to explain their
mechanism of action.
Influence on isolated rat thoracic aorta contracted by KCl
The vasorelaxant effects of selected sildenafil analogues,
sildenafil (a selective PDE5 inhibitor) and IBMX (non-selective
PDE inhibitor) in isolated aortic rings precontracted by KCl with
and without endothelium were investigated. Compounds 8a, 8b,
8c, 8f and 8g (1–300 mM) relaxed KCl (60 mM)-precontracted
endothelium intact aortic rings in a dose-dependent manner and
they were able to inhibit the contractile response completely
(Figure 2). The IC50 values were at the range of 48.9–178.1 mM
(Table 1). In endothelium denuded aorta cumulative concentra-
tions of compounds 8a, 8b, 8c, 8f and 8g (1–300 mM) produced
concentration-dependent vasorelaxations with IC50 values of
39.7–143.8 mM, indicating that the relaxant responses of these
agents are likely to be endothelium independent. Compounds (8d)
and (8e) relaxed the KCl depolarising solution precontracted
Figure 2. Effects of tested compounds on vascular tension in the
endothelium-intact, KCl (60 mM) precontracted aortic rings. The data are
vessels only by 52.4–65.6% in endothelium intact aorta and by the means of four experiments S.E.M.
37–48% in endothelium denuded aortic rings (Figure 2, Table 1).
Table 1. Vasorelaxant potencies of tested compounds on sustained contraction of aortic rings induced by KCl depolarising solution (60 mM).
E (+)
E (ꢂ)
Compound
R
R1
IC50 (mM) Maximal relaxation (at 300 mM) (%) IC50 (mM) Maximal relaxation (at 300 mM) (%)
n
8a
C3H7
48.9 3.l
178.1 25.2
128.1 11.3
–
100
39.7 5.6
143.8 55.0
100.0 5.2
–
100
4
N
N
N CH
3
3
8b
8c
8d
CH3
CH3
CH3
94.7
89.4
4
4
4
N CH
NH
94.8
52.4
90
N
37.4
H
N
CH
3
H
HO
8e
8f
CH3
CH3
–
65.6
96.7
–
48.2
92
4
4
H
N
H
CH
3
HO
120.0 4.7
108.3 9.5
H
N
H
OH
H C
CH
3
3
8g
CH3
118.0 3.9
98.9
100.2 4.8
93
4
H
N
OH
H
H C
CH
3
3
Sildenafil
IBMX
7.0 1.0
39.3 3.8
90.0
96.2
14.3 3.2
26.4 9.3
85
88.4
4
4
Aortic rings with functional endothelium (E +) and without endothelium (E ꢂ). The data are the means of four experiments S.E.M.

6
M. Mojzych et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 3. Vasorelaxant effects of compound (8a) on KCl and phenyl-
ephrine-induced contractions in isolated endothelium-intact E(+) and
denuded E(ꢂ) rat aorta. (˙) KCl E(+), (g) KCl E(ꢂ), (m) phenylephrine
E(+), (œ) phenylephrine E(ꢂ). The data are the means of 4–8
experiments S.E.M.
Figure 5. The vasorelaxant effect of compound (8a) on endothelium
intact rat aorta precontracted by phenylephrine (10 mM) in the absence
and presence of L-NAME (10 mM), ODQ (1 mM), glibenclamide (1 mM).
The data are the means of four experiments S.E.M.
Figure 4. Additive effects of compound (8a) and IBMX (1 mM) on rat
aortic rings precontracted by phenylephrine (10 mM). The data are the
means of four experiments S.E.M.
Figure 6. Effect of compound (8a) (30 mM) on isoprenaline-induced
vasorelaxation in endothelium denuded rat aorta precontracted by
phenylephrine (10 mM).
selected for further pharmacological research aiming to explain its
mode of action.
Denuded rat aorta was pretreated with compound (8a) and its
response to isoprenaline, a cAMP-dependent vasodilator was
studied. Compound (8a) (30 mM) significantly increased the
potency of isoprenaline (Figure 6).
Influence on isolated rat thoracic aorta contracted by
phenylephrine
Compound (8a) (0.1–300 mM) produced a concentration depend-
ent inhibition of the contractile response induced by another
stimulatory agent, phenylephrine. In phenylephrine-induced con-
tractions the relaxation was again endothelium independent, with
the IC50 values of 27.8 mM for endothelium intact and 25.3 mM for
endothelium denuded aorta.
In the contractions induced by both stimulatory agents (KCl or
phenylephrine), with intact or denuded endothelium-ring, the
relaxant effect of compound (8a) was higher in phenylephrine
than in KCl-induced contraction (Figure 3).
The effects of compound (8a) were also investigated after
pretreatment with PDE nonselective inhibitor IBMX.
Vasorelaxation induced by compound (8a) had an additive
effect in the presence of IBMX (Figure 4).
In endothelium intact rat aorta, the vasorelaxant effect of
compound (8a) was reduced by pretreatment with the soluble
guanylyl cyclase (sGC) inhibitor ODQ and the KATP channel
blocker glibenclamide. Nevertheless, the vasorelaxation of com-
pound (8a) was not affected by the nitric oxide synthase (NOS)
inhibitor L-NAME (Figure 5).
Influence on the blood pressure and heart rate
The effects on the blood pressure and heart rate of compound (8a)
and sildenafil were determined after i.p. administration at the dose
of 10 mg/kg. Saline was used as a control without drug treatment.
Compound (8a) reduced blood pressure slightly but signifi-
cantly by 8% (systolic blood pressure) and 7% (diastolic blood
pressure) in 60–70 min after its administration (Figure 7). For
comparison, sildenafil initially reduced systolic and diastolic
pressure by 6% and 15%, respectively, but the pressure returned to
the baseline value within next couple of minutes after adminis-
tration (Figure 7). Neither sildenafil nor compound (8a)
influenced the heart rate, however, compound (8a) increased
myocardial contractility index (LV (dP/dt) max/LVP) in 80 min
after administration.
Discussion
In the cardiovascular system, modulation of cyclic nucleotides is
involved in regulation of vascular smooth muscles function and

DOI: 10.3109/14756366.2015.1024674
Relaxant effects of selected sildenafil analogues
7
Figure 7. The effect of compound (8a) and
sildenafil on blood pressure in anesthetized
rats at a dose of 10 mg/kg i.p. The data are the
means of 5–6 experiments S.E.M.
Statistical analyses were performed using a
one-way ANOVA test, post hoc Dunnet test;
*p50.05; **p50.02.
agents that inhibit PDE and increase cAMP and cGMP levels phenylephrine is mainly dependent on intracellular stores of
produce vasodilatation14.
calcium ions, so the lower concentration of PDE inhibitor is
Compounds 8a, 8b, 8c, 8f and 8g produced concentration sufficient14.
dependent aortic relaxation against the contraction induced by
We have demonstrated that the combination of compound (8a)
KCl depolarising solution in endothelium intact and denuded and IBMX, a non-selective PDE inhibitor, had an additive effect
aortic rings. PDE inhibitors that increase cyclic nucleotides on vasorelaxation what suggest that vasodilator effect of com-
(cAMP i cGMP) produce vasodilatation by activating the pound (8a) may be mediated by PDE inhibition.
appropriate protein kinases and decreasing intracellular cal-
In order to assess the involvement of endothelium-dependent
cium14. Our results showed that the tested compounds (except signalling in compound (8a) induced relaxation, the effect of
for derivative (8a), which acts as potent PDE inhibitor) were less L-NAME, a non-selective NOS inhibitor, was tested. Our results
active than reference compounds, sildenafil and IBMX. showed that compound (8a) induced vasorelaxation was not
Compound (8a) was the most active, with IC50 values comparable inhibited by L-NAME, what confirms the effects observed in
with that one of IBMX. It may be related to its chemical structure preliminary test and indicates that the endothelium-dependent
and similarity to sildenafil15. Other compounds and different NO–sGC–cGMP pathway may not be implicated in compound
sildenafil chemical modifications turned to be less active (8a) vasorelaxant properties. However, ODQ, a selective inhibitor
regarding their vasorelaxant properties.
of sGC markedly reduced the relaxation induced by compound
PDE1 and PDE3 play the important functional role in rat aorta (8a). This indicates that vasorelaxant effect of compound (8a)
smooth muscle, whereas in vascular endothelium PDE5 and PDE4 depends on sGC–cGMP signalling, at least partially. sGC and
isoforms are relevant14,16. It has been proven that the activity of cGMP are located not only in endothelium but also in vascular
the tested compounds in the contractions induced by KCl is smooth muscle cells and also PDE isoforms are differentially
endothelium independent. On the basis of our experiment we may distributed both in endothelial and vascular smooth muscle
state that their vasorelaxant effect is not only due to PDE5 cells16,18,19. In vascular smooth muscle, PDE1 (hydrolyzes cGMP
inhibition. If they had acted as selective PDE5 inhibitors, their and cAMP) and PDE3 (hydrolyzes cAMP preferentially but not
vasorelaxant activity would have been endothelium dependent, exclusively) isoenzymes play an important role as modulators of
what was observed in a case of sildenafil. In endothelium-devoid rat aorta contractility. PDE4 and PDE5 isoforms have been also
arteries the relaxant response to sildenafil was partially inhibited. found but in the absence of endothelium their functional role is
Sildenafil vasorelaxant properties result from PDE5 inhibition not relevant14. On the basis of our results we can assume that
and depend on endothelium and nitric oxide (NO)—soluble compound (8a) may be a degradating cGMP PDE isoforms
guanylyl cyclase (sGC)—cyclic guanosine monophosphate inhibitor, particularly PDE1 and PDE3.
pathway (cGMP). NO activation of the sGC results in an increase
In this work, we have also demonstrated that a KATP channel
in intracellular cGMP levels, which elicits cGMP-dependent (an ATP-sensitive potassium channel) blocker glibenclamide
protein kinase (PKG) signalling. PKG inhibits Ca2+ influx, significantly inhibited the vasorelaxant effect of compound (8a).
augments Ca2+ sequestration and decrease the sensitivity of K+ channels play a major role in the regulation of the resting
contractile elements to Ca2+. Selective PDE5 inhibition increase membrane potential and modulate vascular smooth muscle tone.
cGMP breakdown reduction and potentiate the vasorelaxant Endothelium-derived hyperpolarizing factor (EDHF) activates the
effect17.
K+ channels, and the subsequent K+ efflux causes hyperpolariza-
Since compound (8a) showed the highest activity in the model tion closing voltage-operated Ca2+ channels and decreasing Ca2+
of contraction induced by KCl, the screening assay, it was selected entrance that promotes muscle relaxation. Endothelial and smooth
for further pharmacological studies.
muscle cells express ATP-sensitive and Ca2+ activated K+
In the model of vasoconstriction induced by phenylephrine the channels. It is suggested that activation of KATP channels may
vasorelaxant properties of compound (8a) were endothelium also release endothelium-derived NO or EDHF20. Therefore, we
independent, similar to results obtained from the model of can state that compound (8a) induced vasorelaxation might
contraction induced by KCl. Comparing the effects of compound partially occur via activation of KATP channels.
(8a) in both model of vasoconstrictions, the IC50 values in the
contraction induced by phenylephrine were lower than the IC50 compound (8a) depends on cAMP, we determined its modulating
values in the contraction induced by KCl. This may be due to the effect on
In order to check whether the vasodilatory activity of
a
cAMP-dependent vasodilator. Compound (8a)
fact that high K+ solution induces a cell membrane depolarization enhanced the vasorelaxant effect of a cAMP-dependent vasodila-
and calcium influx from the outside, so in order to achieve tor, isoprenaline. This may suggest that compound (8a) is a cAMP
vasorelaxation higher concentrations of PDE inhibitor are degrading PDE inhibitor, presumably PDE3, which dominate in
required. In contrast, the model of vasoconstriction induced by vascular smooth muscle cells.

8
M. Mojzych et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
3. Cote RH. Characteristics of photoreceptor PDE (PDE6): similarities
and differences to PDE5. Int J Impotence Res 2004;16:S28–33.
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analogues, selective phosphodiesterase 5 inhibitors. Bioorg Med
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phenylhydrazones of 5-acyl-1,2,4-triazines. Heterocycles 2000;53:
2175–85.
Summarizing the biofunctional studies, in the case of
compound (8a) we may suspect that its vasorelaxant response
depends on cyclic nucleotides pathway since the pretreatment
with the sGC inhibitor ODQ attenuated the vasorelaxant response
and also, compound (8a) enhanced the vasorelaxant response to
cAMP-dependent vasodilator isoprenaline. However, compound
(8a) activates not only the sGC/cGMP and adenylyl cyclase (AC)/
cAMP pathways, but also may activate K+ channels. The
vasorelaxant mechanism of compound (8a), sildenafil analogue,
through sGC/cGMP and AC/cAMP pathways is probably a result
of its non selective PDE inhibitory activity. Compound (8a)
induced vasorelaxation is endothelium independent and is not
only due to PDE4 and PDE5 inhibitory activity, since these
subtypes of PDE have been found mainly in endothelium14.
In in vivo study, compound (8a) moderately decreased the
systolic and diastolic blood pressure and increased heart con-
tractility. In rat heart, PDE2, PDE3 and PDE4 exist16,18 and their
inhibition by compound (8a) may increase cyclic nucleotides
concentration and as a result compound (8a) produces vasodila-
tation and increases heart contractility.
In conclusion, the studies of seven new sildenafil analogues
showed their moderate and endothelium-independent vasorelax-
ant properties. The strongest, comparable with IBMX effect was
shown for compound (8a), being the closest structural sildenafil
analogue. Compound (8a) may be a non-selective PDE inhibitor,
and may inhibit isozymes degrading both cAMP and cGMP. Its
vasorelaxant activity may also involve K+ channel activation.
12. Mojzych M, Rykowski A. Transformations of phenylhydrazones of
5-acyl-1,2,4-triazines to pyrazolo[4,3-e][1,2,4]triazines or 4-cyano-
pyrazole. J Heterocycl Chem 2007;44:1003–7.
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substituted-1,2,4-triazines. Synlett 2002;3:447–50.
14. Noguera MA, Ivorra MD, Lugnier C, D’Ocon P. Role of cyclic
nucleotide phosphodiesterase isoenzymes in contractile responses of
denuded rat aorta related to various Ca2+ sources. Naunyn
Schmiedebergs Arch Pharmacol 2001;363:612–19.
15. Mojzych M, Dolashki A, Voelter W. Synthesis of pyrazolo[4,3-
e][1,2,4]triazine sulfonamides, novel Sildenafil analogues with
tyrosinase inhibitory activity. Bioorg Med Chem 2014;22:6616–24.
16. Lugnier C. Cyclic nucleotide phosphodiesterase (PDE) superfamily:
a new target for the development of specific therapeutic agents.
Pharmacol Therap 2006;109:366–98.
Conclusions
We have described facile and efficient method for the preparation
of new 1H-pyrazolo[4,3-e][1,2,4]triazine sulfonamides from
simple available starting materials. Our biological study with
sildenafil analogues indicated one compound (8a), which
exhibited micromolar and endothelium-independent vasorelaxant
properties in the rat aorta. The compound is undergoing further
modification of the structure to increase the relaxation potency.
Declaration of interest
´
17. Salom JB, Burguete MC, Perez-Asensio FJ, et al. Relaxant effect of
The authors declare no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
sildenafil in the rabbit basilar artery. Vascul Pharmacol 2006;44:
10–16.
18. Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases:
molecular regulation to clinical use. Pharmacol Rev 2006;58:
488–520.
19. Lo YC, Tsou HH, Lin RJ, et al. Endothelium-dependent and
-independent vasorelaxation by a theophylline derivative MCPT:
roles of cyclic nucleotides, potassium channel opening and
phosphodiesterase inhibition. Life Sci 2005;76:931–44.
20. Wu BN, Chen IC, Lin RJ, et al. Aortic smooth muscle relaxants
KMUP-3 and KMUP-4, two nitrophenylpiperazine derivatives of
xanthine, display cGMP-enhancing activity: roles of endothelium,
phosphodiesterase, and K+ channel. J Cardiovasc Pharmacol 2005;
46:600–8.
This research was partially funded by the National Science Centre,
Poland (grant NN405 092340). The authors gratefully acknowledge use of
the mass spectrometry services and facilities of the Center for
Interdisciplinary Research of The John Paul II Catholic University of
Lublin, Lublin, Poland, funded by POPW.01.03.00-06-003/09-00.
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