Synthesis of chiral pyrazolo[4,3-e][1,2,4]triazine sulfonamides with tyrosinase and urease inhibitory activity

Article abstract of DOI:10.1080/14756366.2016.1238362

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 tyrosinase and urease inhibitory activity. Evaluation of prepared derivatives demonstrated that compounds (8b) and (8j) are most potent mushroom tyrosinase inhibitors whereas all of the obtained compounds showed higher urease inhibitory activity than the standard thiourea. The compounds (8a), (8f) and (8i) exhibited excellent enzyme inhibitory activity with IC₅₀ 0.037, 0.044 and 0.042 μM, respectively, while IC₅₀ of thiourea is 20.9 μM.

Full text of DOI:10.1080/14756366.2016.1238362

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis of chiral pyrazolo[4,3-e][1,2,4]triazine
sulfonamides with tyrosinase and urease
inhibitory activity
Mariusz Mojzych, Paweł Tarasiuk, Katarzyna Kotwica-Mojzych, Muhammad
Rafiq, Sung-Yum Seo, Michał Nicewicz & Emilia Fornal
To cite this article: Mariusz Mojzych, Paweł Tarasiuk, Katarzyna Kotwica-Mojzych, Muhammad
Rafiq, Sung-Yum Seo, Michał Nicewicz & Emilia Fornal (2016): Synthesis of chiral pyrazolo[4,3-
e][1,2,4]triazine sulfonamides with tyrosinase and urease inhibitory activity, Journal of Enzyme
Inhibition and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1238362
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1238362
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Date: 30 October 2016, At: 15:02

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2016
http://dx.doi.org/10.1080/14756366.2016.1238362
RESEARCH ARTICLE
Synthesis of chiral pyrazolo[4,3-e][1,2,4]triazine sulfonamides with tyrosinase
and urease inhibitory activity
Mariusz Mojzycha,b, Paweł Tarasiuka, Katarzyna Kotwica-Mojzychc, Muhammad Rafiqd,e, Sung-Yum Seod,
Michał Nicewiczb and Emilia Fornalf
b
aDepartment of Physico-Chemical Hazards of Health and Ecology, Institute of Rural Health, Lublin, Poland; Department of Chemistry,
c
Siedlce University of Natural Sciences and Humanities, Siedlce, Poland; Department of Hematooncology and Bone Marrow Transplantation Unit,
d
e
Medical University of Lublin, Lublin, Poland; Department of Biology, Kongju National University, Gongju, Republic of Korea; Department of
f
Biochemistry and Biotechnology (Baghdad-ul-Jadeed Campus), The Islamia University of Bahawalpur, Pakistan; EMF Lab, Lublin, Poland
ABSTRACT
ARTICLE HISTORY
Received 24 July 2016
Revised 30 August 2016
Accepted 31 August 2016
Published online 20 October
2016
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 tyrosinase and urease inhibitory activ-
ity. Evaluation of prepared derivatives demonstrated that compounds (8b) and (8j) are most potent mush-
room tyrosinase inhibitors whereas all of the obtained compounds showed higher urease inhibitory
activity than the standard thiourea. The compounds (8a), (8f) and (8i) exhibited excellent enzyme inhibi-
tory activity with IC50 0.037, 0.044 and 0.042 lM, respectively, while IC50 of thiourea is 20.9 lM.
KEYWORDS
Pyrazolo[4,3-e][1,2,4]triazine;
sulfonamides; tyrosinase
inhibitors; urease inhibitors
Introduction
kidney stones, hepatic encephalopathy, hepatic coma urolithiasis
and urinary catheter encrustation21
.
Sulfonamides constitute an important class of drugs. They possess
various types of pharmacological activities such as antibacterial,
high-ceiling diuretic, hypoglycemic, antithyroid, anti-inflammatory
Our current work continuing our interest in the synthesis of
new sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine as
new inhibitors of tyrosinase and urease enzymes. The synthesis
pathway leading to the title compounds is depicted in Scheme 1.
and antiglaucoma1–9
.
Moreover, numerous sulfonamides were
found to act as tyrosinase and urease inhibitors10–12
.
Tyrosinase (EC 1.14.18.1) is a multifunctional copper containing
enzyme from oxidase family, widely distributed in animals, plants
and microorganisms13. It catalyzes two distinct reactions of mel-
anin synthesis, the hydroxylation of L-tyrosine by monophenolase
action and the oxidation of L-DOPA (3,4-dihydroxyphenylalanine)
to o-dopaquinone14. Tyrosinase catalyzes melanin biosynthesis
which plays a crucial role in protecting the skin from solar radia-
tions. Melanin can serve as a photoprotector and is responsible for
the pigmentation and color patterns of mammalian skin. It has
been reported that effective tyrosinase inhibitors could ameliorate
skin hyperpigmentation, neurodegenerative diseases, and could
potentially improve cancer remedies15,16. However, when exposed
to excessive ultraviolet light, melanin produced in excess causes
various skin disorders17.
Urease (EC 3.5.1.5) is a nickel containing enzyme that catalyzes
the hydrolysis of urea to ammonia and carbon dioxide or carba-
mate by at least 1014 over the spontaneous reaction18. It is of our
interest to focus on the discovery of novel urease inhibitors as it is
already known that these compounds play a crucial part in the
therapies both human and plants disorders. In agriculture, high
urease activity during urea fertilization causes significant environ-
mental and economic problems by releasing abnormally large
amounts of ammonia into the atmosphere19,20. Moreover, it con-
Experimental
Chemistry
Materials and methods
All chemicals used were of reagent grade quality and were used
as received without any further purification and were obtained
from Sigma-Aldrich. Melting points were determined on a Mel-
Temp apparatus and are uncorrected. 1H- and 13C-NMR spectra
were recorded on a Varian spectrometer (400 MHz for 1H and
100 MHz for 13C). The chemical shift values are expressed in ppm
(part per million) with TMS as internal reference. The relative inte-
grals of peak areas agreed with those expected for the assigned
structures. Molecular weight of final compounds were assessed by
electrospray ionization mass spectrometry (ESI/MS) on a Agilent
Technologies 6538 UHD Accurate Mass Q-TOF LC/MS. Elemental
compositions are within 0.4% of the calculated values. For prep-
aration and spectroscopic data of compounds 2–7 and 8a, 8b, 8 h,
8i see literature22–24
.
Synthesis of sulfonamides 8a–j
A mixture of sulfonyl chloride 7 (100 mg, 0.29 mmol) and amine
tributes to the development of peptic ulcers, pyelonephritis, (100 mg, 1 mmol) in anhydrous acetonitrile (5 mL) was stirred
CONTACT Mariusz Mojzych
mmojzych@yahoo.com
Department of Chemistry, Siedlce University of Natural Sciences and Humanities, 3-go Maja 54, 08-110
Siedlce, Poland
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

2
M. MOJZYCH ET AL
H3C
NH
N
N
N
N
N
N
N
N
N
a
c
b
N
N
N
N
O
HON
SCH 3
SCH 3
SCH 3
SCH 3
H3C
4
CH3
3
CH3
2
1
d
H3C
N
N
H3C
H3C
N
N
H3C
N
N
N
N
N
N
g
N
N
f
e
N
O
N
O
N
O
N
N
N
N
N
SCH 3
H3C
H3C
H3C
H3C
O
S
O
O
S
O
R
Cl
7
8a-j
6
5
8a : R = ( S )-NHCH(CH3)CH2OH
8b : R = ( R)-NHCH(CH3)CH2OH
8c : R = ( S )-NHCH(CH2OH)CH(CH 3)CH3
8d : R = ( R)-NHCH(CH2OH)CH(CH 3)CH3
8e : R = ( S )-NHCH2CH(OH)CH 3
8f : R = ( R)-NHCH2CH(OH)CH 3
8g : R = (S )-NHCH2CH(OH)CH 2OH
8h : R = ( R)-NHCH2CH(OH)CH 2OH
8i : R = ( S )-NHCH(CH2OH)CH2CH(CH3)CH3
8j : R = ( R)-NHCH(CH2OH)CH2CH(CH3)CH3
Scheme 1. Synthetic pathway to the sulfonamides 8a–i. Reagents and conditions: (a) CH3CH2NO2, 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, 140ꢀC, 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%.
overnight at room temperature, and then the reaction mixture 132.1, 134.5, 142.1, 146.7, 158.4, 160.4. HRMS (ESI, m/z) calcd. for
was concentrated in vacuo to afford the crude sulfonamide, as a C17H22N6O4S [Mþ] 406.1423. Found [Mþ] 406.1426. Anal. calcd. for
yellow solid2,24. The residue was purified on silica gel using a mix- C17H22N6O4S: C, 50.23; H, 5.46; N, 20.68. Found: C, 49.89; H, 5.49;
ture of CH2Cl2:EtOH (25:1) as eluent to give the titled compounds N, 20.55.
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-3-methylbutan-2-
yl)benzenesulfonamide (8c)
Yield 91%, mp 115–120 ꢀC. 1H-NMR (CDCl3) d: 0.83 (dd, 6H,
(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 (8a)
Yield 92%, mp 132–136 ꢀC. 1H-NMR (CDCl3) d: 1.09 (d, 3H, J1 ¼6.8 Hz, J2 ¼2.8 Hz), 1.34 (t, 3H, J ¼ 6.8 Hz), 1.82 (s, 1H,
J ¼ 6.4 Hz), 1.35 (t, 3H, J ¼ 6.8 Hz), 2.63 (bs, 1H, OH, exchanged with J ¼ 6.8 Hz), 2.69 (s, 3H), 3.07–3.11 (m, 1H), 3.52–3.55 (m, 2H), 4.17
D20), 2.70 (s, 3H), 3.39–3.46 (m, 2H), 3.56 (d, 1H, J ¼ 7.6 Hz), 4.19 (q, (q, 2H, J ¼ 6.8 Hz), 4.32 (s, 3H), 5.23 (d, 1H, J ¼ 8.4 Hz, NH,
2H, J ¼ 6.4 Hz), 4.31 (s, 3H), 5.11 (d, 1H, J ¼ 6.0 Hz, NH, exchanged exchanged with D2O), 7.11 (d, 1H, J ¼ 8.8 Hz), 7.96 (dd, 1H,
with D2O), 7.14 (d, 1H, J ¼ 9.2 Hz), 7.98 (dd, 1H, J1 ¼8.4 Hz, J1 ¼8.8 Hz, J2 ¼2.8 Hz), 8.27 (d, 1H, J ¼ 2.8 Hz). 13C-NMR (CDCl3) d:
J2 ¼2.4 Hz), 8.28 (d, 1H, J ¼ 2.4 Hz).13C-NMR (CDCl3) d: 11.0, 14.4, 11.0, 14.4, 18.5, 19.1, 29.6, 34.8, 60.9, 62.5, 64.9, 112.6, 126.7, 130.4,
17.9, 34.8, 51.5, 64.9, 65.9, 112.7, 126.9, 130.4, 131.3, 132.1, 134.5, 131.4, 132.4, 134.5, 142.1, 146.6, 158.4, 160.3. HRMS (ESI, m/z)
142.2, 146.7, 158.4, 160.4. HRMS (ESI, m/z) calcd. for C17H22N6O4S calcd. for C19H26N6O4S [Mþ] 434.1736 Found [Mþ] 434.1741. Anal.
[Mþ] 406.1423. Found [Mþ] 406.1427. Anal. calcd. for calcd. for C19H26N6O4S: C, 52.51; H, 6.03; N, 19.35. Found: C, 52.18;
C17H22N6O4S: C, 50.23; H, 5.46; N, 20.68. Found: C, 50.00; H, 5.49; H, 6.06; N, 19.22.
N, 20.50.
(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 (8d)
Yield 72%, mp 115–120 ꢀC. 1H-NMR (CDCl3) d: 0.84 (dd, 6H,
(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 (8b)
Yield 89%, mp 132–136 ꢀC. 1H-NMR (CDCl3) d: 1.09 (d, 3H, J1 ¼6.8 Hz, J2 ¼3.6 Hz), 1.35 (t, 3H, J ¼ 6.8 Hz), 1.82 (s, 1H,
J ¼ 6.4 Hz), 1.34 (t, 3H, J ¼ 6.8 Hz), 2.69 (s, 3H), 2.84 (bs, 1H, OH, J ¼ 6.8 Hz), 2.70 (s, 3H), 3.08–3.11 (m, 1H), 3.50–3.58 (m, 2H), 4.18
exchanged with D20), 3.38–3.45 (m, 2H), 3.55 (d, 1H, J ¼ 7.6 Hz), (q, 2H, J ¼ 6.8 Hz), 4.32 (s, 3H), 5.16 (d, 1H, J ¼ 8.8 Hz, NH,
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), 7.11 (d, 1H, J ¼ 8.8 Hz), 7.96 (dd, 1H,
exchanged with D2O), 7.13 (d, 1H, J ¼ 8.8 Hz), 7.98 (dd, 1H, J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.28 (d, 1H, J ¼ 2.4 Hz). 13C-NMR (CDCl3) d:
J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.27 (d, 1H, J ¼ 2.4 Hz). 13C-NMR (CDCl3) d: 11.0, 14.4, 18.5, 19.1, 29.6, 34.8, 60.9, 62.5, 64.9, 112.6, 126.8, 130.4,
11.0, 14.4, 17.8, 34.8, 51.5, 64.9, 65.9, 112.7, 126.9, 130.4, 131.3, 131.4, 132.4, 134.5, 142.2, 146.7, 158.4, 160.3. HRMS (ESI, m/z) calcd.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
for C19H26N6O4S [Mþ] 434.1736 Found [Mþ] 434.1741. Anal. calcd.
for C19H26N6O4S: C, 52.51; H, 6.03; N, 19.35. Found: C, 52.18; H,
6.10; N, 19.15.
(S)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-yl)-
4-ethoxy-N-(1-hydroxy-4-methylpentan-2-
yl)benzenesulfonamide (8i)
Yield 91%, mp 112–116 ꢀC. 1H-NMR (CDCl3) d: 0.73 (d, 3H,
J ¼ 6.8 Hz), 0.81 (d, 3H, J ¼ 6.8 Hz), 1.27–1.29 (m, 1H), 1.32–1.38 (m,
4H), 1.56–1.60 (m, 1H), 2.70 (s, 3H), 3.36–3.39 (m, 1H), 3.41–3.45
(m, 1H), 3.54–3.57 (m, 1H), 4.19 (q, 2H, J ¼ 6.8 Hz), 4.33 (s, 3H), 5.03
(d, 1H, J ¼ 8.0 Hz, NH, exchanged with D2O), 7.13 (d, 1H, J ¼ 8.8 Hz),
7.98 (dd, 1H, J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.30 (d, 1H, J ¼ 2.4 Hz). 13C-
NMR (CDCl3) d: 11.0, 14.4, 21.9, 22.7, 24.3, 34.8, 41.1, 53.8, 64.7,
64.9, 112.7, 127.0, 130.4, 131.4, 132.4, 134.5, 142.2, 146.7, 158.3,
160.4. HRMS (ESI, m/z) calcd. for C20H28N6O4S [Mþ] 448.1893
Found [Mþ] 448.1899. Anal. calcd. for C20H28N6O4S: C, 53.55; H,
6.29; N, 18.74. Found: C, 53.33; H, 6.39; N, 18.63.
(S)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]
triazin-5-yl)-4-ethoxy-N-(2-hydroxy-propyl)-
benzenesulfonamide (8e)
Yield 90%, mp 132–135 ꢀC. 1H-NMR (CDCl3) d: 1.13 (d, 3H,
J ¼ 6.4 Hz), 1.34 (t, 3H, J ¼ 6.8 Hz), 2.67 (bs, 1H, OH exchanged with
D2O), 2.70 (s, 3H), 2.79–2.86 (m, 1H), 3.05–3.11 (m, 1H), 3.86–3.94
(m, 1H), 4.17 (q, 2H, J ¼ 6.8 Hz), 4.32 (s, 3H), 5.35 (t, 1H, J ¼ 6.0 Hz,
NH, exchanged with D2O), 7.13 (d, 1H, J ¼ 8.8 Hz), 7.95 (dd, 1H,
J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.24 (d, 1H, J ¼ 2.4 Hz). 13C-NMR (CDCl3) d:
11.0, 14.4, 20.5, 34.8, 50.1, 64.9, 66.3, 112.8, 127.0, 130.4, 131.2,
131.3, 134.5, 142.1, 146.7, 158.4, 160.4. HRMS (ESI, m/z) calcd. for
C17H22N6O4S [Mþ] 406.1423 Found [Mþ] 406.1426. Anal. calcd. for
C17H22N6O4S: C, 50.23; H, 5.46; N, 20.68. Found: C, 49.89; H, 5.52;
N, 20.53.
(R)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-yl)-
4-ethoxy-N-(1-hydroxy-4-methylpentan-2-
yl)benzenesulfonamide (8j)
Yield 93%, mp 112–116 ꢀC. 1H-NMR (CDCl3) d: 0.70 (d, 3H,
J ¼ 6.4 Hz), 0.78 (d, 3H, J ¼ 6.4 Hz), 1.25–1.27 (m, 1H), 1.31–1.36 (m,
4H), 1.54–1.60 (m, 1H), 2.67 (s, 3H), 3.10 (bs, 1H, OH), 3.32–3.42 (m,
2H), 3.52–3.57 (m, 1H), 4.18 (q, 2H, J ¼ 6.8 Hz), 4.30 (s, 3H), 5.34 (d,
1H, J ¼ 8.0 Hz, NH, exchanged with D2O), 7.11 (d, 1H, J ¼ 8.8 Hz),
7.96 (dd, 1H, J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.27 (d, 1H, J ¼ 2.4 Hz). 13C-
NMR (CDCl3) d: 11.0, 14.4, 21.9, 22.7, 24.3, 34.8, 41.1, 53.8, 64.7,
64.9, 112.7, 127.0, 130.4, 131.4, 132.4, 134.5, 142.2, 146.7, 158.3,
160.4. HRMS (ESI, m/z) calcd. for C20H28N6O4S [Mþ] 448.1893
Found [Mþ] 448.1894. Anal. calcd. for C20H28N6O4S: C, 53.55; H,
6.29; N, 18.74. Found: C, 53.21; H, 6.33; N, 18.59.
(R)-3-(1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazin-5-yl)-
4-ethoxy-N-(2-hydroxy-propyl)-benzenesulfonamide (8f)
Yield 90%, mp 132–135 ꢀC. 1H-NMR (CDCl3) d: 1.13 (d, 3H,
J ¼ 6.4 Hz), 1.34 (t, 3H, J ¼ 6.8 Hz), 2.59 (bs, 1H, OH exchanged with
D2O), 2.70 (s, 3H), 2.79–2.86 (m, 1H), 3.05–3.11 (m, 1H), 3.86–3.94
(m, 1H), 4.18 (q, 2H, J ¼ 6.8 Hz), 4.32 (s, 3H), 5.29 (bs, 1H, NH,
exchanged with D2O), 7.13 (d, 1H, J ¼ 8.8 Hz), 7.95 (dd, 1H,
J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.24 (d, 1H, J ¼ 2.4 Hz). 13C-NMR (CDCl3) d:
11.0, 14.4, 20.6, 34.8, 50.1, 64.9, 66.4, 112.8, 127.0, 130.4, 131.3,
131.3, 134.5, 142.1, 146.8, 158.4, 160.5. HRMS (ESI, m/z) calcd. for
C17H22N6O4S [Mþ] 406.1423 Found [Mþ] 406.1425. Anal. calcd. for
C17H22N6O4S: C, 50.23; H, 5.46; N, 20.68. Found: C, 49.91; H, 5.56;
N, 20.48.
Pharmacology
Reagents
Mushroom tyrosinase (EC 1.14.18.1), urease from Jack bean (EC
3.5.1.5), L-DOPA (3,4-dihydroxyphenylalanine), Kojic acid, thio-
urea, EDTA, sodium nitroprusside and active chloride, were pur-
chased from Sigma (St. Louis, MO, USA). Stock solutions of the
reducing substrates were prepared in phosphate buffer (20 mM,
pH 6.8).
(S)-N-(2,3-dihydroxypropyl)-3-(1,3-dimethyl-1H-
pyrazolo[4,3-e][1,2,4]triazin-5-yl)-4-
ethoxybenzenesulfonamide (8g)
Yield 78%, mp 117–121 ꢀC. 1H-NMR (MeOH-d4) d: 1.29–1.33 (m,
5H), 2.68 (s, 3H), 2.85–2.90 (m, 1H), 3.04–3.08 (m, 1H), 3.49 (t, 2H,
J ¼ 6.0 Hz), 3.67 (m, 1H), 4.22 (q, 2H, J ¼ 6.8 Hz), 4.31 (s, 3H), 7.36
(d, 1H, J ¼ 8.8 Hz), 8.01 (dd, 1H, J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.17 (d, 1H,
J ¼ 2.4 Hz). 13C-NMR (DMSO) d: 11.1, 14.7, 35.0, 46.4, 63.9, 65.0,
70.7, 113.9, 127.1, 130.4, 130.6, 132.4, 134.4, 141.5, 147.0, 158.4,
159.9. HRMS (ESI, m/z) calcd. for C17H22N6O5S [Mþ] 422.1372
Found [Mþ] 406.1375. Anal. calcd. for C17H22N6O5S: C, 48.33; H,
5.25; N, 19.90. Found: C, 48.05; H, 5.29; N, 19.77.
Mushroom tyrosinase inhibition assay
The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.)
was used for in vitro bioassays as described previously with
some modifications.25,26 Briefly, 140 lL of phosphate buffer
(20 mM, pH 6.8), 20 lL of mushroom tyrosinase (30 U/mL) and
20 lL of the inhibitor solution were placed in the wells of a 96-
well micro plate. After pre-incubation for 10 min at room tem-
perature, 20 lL of L-DOPA (3,4-dihydroxyphenylalanine) (0.85 mM)
was added and the plate was further incubated at 25 ꢀC for
20 min. Subsequently the absorbance of dopachrome was meas-
ured at 475 nm using a micro plate reader (OPTIMax, Tunable).
Kojic acid was used as a reference inhibitor and for negative
tyrosinase inhibitor phosphate buffer was used instead of the
inhibitor solution. The extent of inhibition by the test com-
pounds was expressed as the percentage of concentration
necessary to achieve 50% inhibition (IC50). Each concentration
was analyzed in three independent experiments run in triplicate.
The IC50 values determined by the data analysis and graphing
software Origin 8.6, 64-bit (OriginLab Corporation, Northampton,
MA).
(R)-N-(2,3-dihydroxypropyl)-3-(1,3-dimethyl-1H-
pyrazolo[4,3-e][1,2,4]triazin-5-yl)-4-
ethoxybenzenesulfonamide (8h)
Yield 79%, mp 117–121 ꢀC. 1H-NMR (MeOH-d4) d: 1.29–1.33 (m,
5H), 2.69 (s, 3H), 2.85–2.90 (m, 1H), 3.04–3.08 (m, 1H), 3.49 (t, 2H,
J ¼ 6.0 Hz), 3.66 (m, 1H), 4.22 (q, 2H, J ¼ 6.8 Hz), 4.31 (s, 3H), 7.36
(d, 1H, J ¼ 8.8 Hz), 8.01 (dd, 1H, J1 ¼8.8 Hz, J2 ¼2.4 Hz), 8.17 (d, 1H,
J ¼ 2.4 Hz). 13C-NMR (MeOH-d4) d: 20.2, 23.8, 44.1, 55.5, 72.9, 74.1,
79.8, 123.0, 136.1, 139.4, 139.6, 141.5, 143.4, 150.5, 156.1, 167.4,
169.0. HRMS (ESI, m/z) calcd. for C17H22N6O5S [Mþ] 422.1372
Found [Mþ] 406.1376. Anal. calcd. for C17H22N6O5S: C, 48.33; H,
5.25; N, 19.89. Found: C, 48.14; H, 5.35; N, 19.77.

4
M. MOJZYCH ET AL
triazine (1) as shown in Scheme 1. Briefly, in the first step the reac-
tion of 1 with nitroethane in KOH/DMSO mixture at room tem-
perature gave appropriate oxime 2 as a main product22, which
was readily transformed into ketone 3 in good yield23. Compound
3 was subjected to the reaction with methylhydrazine in the pres-
ence of acidic media according to standard procedure to give suit-
able hydrazone 4 as key intermediate for the preparation of
Urease inhibition assay
The urease activity was determined by measuring amount of
ammonia produced with indophenols method described by
Weatherburn27. The reaction mixtures, comprising 20 lL of enzyme
(Jack bean urease, 5 U/mL) and 20 lL of test compounds in 50 lL
buffer (100 mM urea, 0.01 M K2HPO4, 1 mM EDTA and 0.01 M LiCl2,
pH 8.2), were incubated for 30 min at 37 ꢀC in 96-well plate.
Briefly, 50 lL each of phenol reagents (1%, w/v phenol and
0.005%, w/v sodium nitroprusside) and 50 lL of alkali reagent
(0.5%, w/v NaOH and 0.1% active chloride NaOCl) were added to
each well. The absorbance at 625 nm was measured after 10 min,
using a microplate reader (OPTIMax, Tunable). All reactions were
performed in triplicate. The urease inhibition activities were calcu-
lated according to the following formula:
1H-pyrazolo[4,3-e][1,2,4]triazine derivative 5. The hydrazone
4
could be converted into derivative 5 under conventional heating
(10% HCl, EtOH, reflux, 1 h)31 or under solvent free reaction condi-
tions according to our previous published procedure32. Using
Guillaumet and co-worker’s method33 for the palladium-catalyzed
cross-coupling reaction of 3-methylsulfanyl-1,2,4-triazine with bor-
onic acid derivatives we have reacted 5-methylsulfanyl-1H-pyra-
zolo[4,3-e][1,2,4]triazines 5 with 2-ethoxyphenylboronic acid in the
presence of copper (I) 3-methylsalicylate to obtain derivative 6 in
excellent yield. Chlorosulfonylation reaction of compound 6 in
neat chlorosulfonic acid at 0 ꢀC proceeded smoothly and select-
ively at the 5’-position of the phenyl ring to give the desired prod-
uct 7 in excellent yield. The chlorosulfonyl derivative 7 was readily
coupled with appropriate pure enantiomeric amines in acetonitrile
at room temperature to produce the final chiral sulfonamides 8a–i
in high yield.
À
Á
Urease inhibition activity ð%Þ ¼ ODcontrol ꢁ ODsample ꢂ 100 =ODcontrol
Where ODcontrol and ODsample represents the optical densities in
the absence and presence of sample, respectively. Thiourea was
used as the standard inhibitor for urease.
Kinetic study
Lineweaver–Burk plots of 1/absorbance versus 1/L-DOPA and
1/absorbance versus 1/Urea were used to determine the type of
enzyme inhibition. A series of experiments were performed to
Pharmacology
determine the inhibition kinetics by following method18,28,29
.
Different inhibitor concentrations of compound 8b with 0, 15, 30,
61, 123 and 247 lM, 8j with 0, 14, 28 and 55.8 lM, respectively
were used in case of mushroom tyrosinase inhibition. Substrate
L-DOPA concentration was between 0.062 and 2 mM in all tyrosin-
ase kinetic study. Pre-incubation and measurement time was the
same as discussed in mushroom tyrosinase inhibition assay proto-
col. Maximal initial velocity was determined from initial linear por-
tion of absorbance up to five minutes after addition of enzyme at
a 30 s interval. The inhibition type on the enzyme was assayed by
Lineweaver–Burk plots of inverse of velocities (1/V) versus inverse
of substrate concentration 1/[S] mM ꢁ 1, and the inhibition con-
stant Ki was determined by two methods, by secondary replot of
1/V (y-intercept of Lineweaver–Burk plot) versus inhibitor concen-
trations and by Dixon plot of inverse of velocities (1/V) versus
inhibitor concentrations.
Urease inhibition was measured by varying the concentration
of urea in the presence of different concentrations (0, 0.125, 0.25
and 0.5 lM) of compound 8a. Inhibitory constant (Ki) was deter-
mined as the intersection on the X-axis of the secondary replot of
1/V (y-intercept of Lineweaver–Burk plot) versus inhibitor concen-
trations and by Dixon plot of inverse of velocities (1/V) versus
inhibitor concentrations. Briefly the urea concentration was
changed from 3.12 to 100 mM for urease kinetics studies and
remaining procedure was same as describes in urease inhibition
assay protocol. Urease activity was determined by measuring
ammonia production using the indophenol method as reported
previously30. The results (change in absorbance per min) were
processed by using SoftMaxPro software(Molecular Devices
Corporation Sunnyvale, CA) and a Dixon plot was constructed by
plotting (1/V) versus inhibitor concentration [I].
Effect of inhibitors on tyrosinase and urease
Tyrosinase assay All chiral sulfonamides 8a–j are structurally simi-
lar and showed comparable inhibitory effects on mushroom tyro-
sinase [IC50 in the range of 27.9–40.17 lM]. Weaker inhibitors were
derivatives 8i (IC50 ¼40.17 lM), 8e (IC50 ¼39.75 lM), and 8c
(IC50 ¼39.00 lM) with (S)-(þ)-leucinol, (S)-(þ)-1-amino-3-propanol
and (S)-(þ)-2-amino-3-methyl-1-butanol moieties in sulfonamide
group. The most active inhibitor in the tested group was com-
pound 8j (IC50 ¼27.90 lM) which contains (R)-(ꢁ)-leucinol substitu-
ent (Table 1).
Tyrosinase contains one binuclear copper complex in the cata-
lytic center and the catechol hydroxyl groups of the substrate are
suggested to be bound to the copper atoms during the enzymatic
oxidation process. Phenolic hydroxyl groups are generally capable
to coordinate copper atoms causing inhibition of the enzyme in
competition to the catechol substrate. However, our previous
study10 showed that inhibitors with NH group in the sulfonamide
part (as piperazinyl, homopiperazinyl) are more active as they
reduce the enzyme activity of tyrosinase to 29.5%–33.5%. It was
suggested, that NH or free NH2 group are involved in the inhib-
ition effect of tyrosinase activity. Our suggestion was confirmed by
Table 1. The inhibitory effects of compounds 8a–8j on mushroom tyrosinase and
urease.
Compounds code
Mushroom tyrosinase IC50 (lM)
Urease IC50 (lM)
8a
8b
8c
8d
8e
8f
8g
8h
38.46 0.42
30.76 3.56
39.00 1.34
38.15 1.68
39.75 1.92
34.72 2.01
36.49 1.75
32.46 1.87
40.17 1.09
27.9 2.43
0.037 0.003
0.084 0.020
0.078 0.020
0.064 0.020
0.076 0.007
0.044 0.130
0.063 0.016
0.080 0.014
0.042 0.012
0.080 0.015
–
Results and discussion
8i
8j
Chemistry
Kojic Acid
Thio-Urea
16.69 2.8
–
20.7 0.45
The synthesis of target sulfonamides 8a–i was achieved by a con-
Bolded values represent the most active derivatives.
venient multiple procedure starting from 3-methylsulfanyl-1,2,4-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
the activity of some inhibitor with two NH groups that reduced 0.037 and 0.042 lM, respectively. In case of compound (8a) the
enzyme activity of tyrosinase to 20.2%.
presence of 2-hydroxy-1-methyl ethaneamine moiety with
S configuration at sulfonamide group has central importance in
urease inhibitory activity in the group of tested derivatives. This
moiety in compound (8a) is responsible for its most potent urease
inhibitory activity. On the other hand, isomeric derivative (8b) with
R-configuration in sulfonamide part showed the lowest activity in
the group of obtained compounds although it exhibited higher
urease inhibitory activity than standard thiourea.
The inhibition of the compound 8j may arise with strong par-
ticipation OH group due to its conformation. However, other
tested derivatives with OH were only weak tyrosinase inhibitors.
Therefore, we suggest that primary binding to one copper atom
should be strengthened by additional interactions between the
inhibitor e.g. p electrons of the ring and another copper atom or
the amino acid residues of the enzyme’s active site to accomplish
the inhibition.
Mechanism underlying inhibitory effect of compounds (8a), (8b)
and (8j)
Since (8b) and (8j) against mushroom tyrosinase and (8a) against
urease enzyme were the most potent inhibitors, we therefore fur-
ther study mechanism underlying their inhibitory effect. The kinetic
studies of the enzyme by the Lineweaver–Burk plot of (1/V) versus
Urease assay The synthesized pirazolotriazine-sulfonamides (8a–j)
have been evaluated for their inhibitory effects on urease. All of
the synthesized compounds showed better inhibitory activity with
IC50 ranging from 0.037 to 0.084 lM than thiourea with IC50 of
20.7 lM. The structures of amino alcohols in sulfonamide group
were varied to study their role in enzyme inhibitory activity. The
excellent activity was shown by derivatives (8a) and (8i) with IC50 1/[S] in the presence of different concentrations of inhibitors (8b),
Figure 1. a) Lineweaver–Burk plots for the inhibition of mushroom tyrosinase in the presence of compound (8b). Concentrations of (8b) were 0, 15, 30, 61, 123 and
247lM, respectively. Substrate L-DOPA Concentrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. b) The secondary replot of the Lineweaver–Burk plot, 1/V
(y-intercept) of a) versus various concentrations of (8b). c) The Dixon plot of the reciprocal of the initial velocities versus various concentrations of compound (8b) at
fixed substrate concentration.
Figure 2. a) Lineweaver–Burk plots for the inhibition of mushroom tyrosinase in the presence of compound (8j). Concentrations of (8j) were 0, 14, 28 and 55.8 lM,
respectively. Substrate L-DOPA Concentrations were 0.062, 0.125, 0.25, 0.5, 1 and 2 mM, respectively. b) The Dixon plot of the reciprocal of initial velocities versus
various concentrations of compound (8j) at fixed substrate concentration.

6
M. MOJZYCH ET AL
Figure 3. a) Double reciprocal Lineweaver–Burk plots for the inhibition of Jack bean urease in the presence of compound (8a). Concentrations of (8a) were 0, 0.125,
0.25 and 0.5 lM, respectively. Substrate urea concentrations were 3.12, 6.25, 12.5, 25, 50 and 100mM, respectively. b) The secondary replot of the Lineweaver–Burk
plot, 1/V (y-intercept) of a) versus various concentrations of (8a). c) The Dixon plot of the reciprocal of the initial velocities versus various concentrations of compound
(8a) at fixed substrate concentration.
Conclusions
Table 2. Inhibitory effect of compound 8b and 8j on mushroom tyrosinase activ-
ity and of 8a on urease activity.
We have described facile and efficient method for the preparation
of new chiral 1H-pyrazolo[4,3-e][1,2,4]triazine sulfonamides from
simple available starting materials. The sulfonamides (8a–j) have
been synthesized to validate their role in tyrosine and urease
inhibitory activity. The most potent inhibitory activity against tyro-
sinase was displayed by compounds (8b) and (8j) with IC50 30.76
and 27.90 lM, respectively. All of the obtained derivatives showed
higher urease inhibitory activity than the standard thiourea. The
kinetic analysis exhibited that compounds (8b) is noncompetitive
inhibitor while (8j) is a mixed type inhibitor of tyrosinase and (8a)
is a mixed type inhibitor of urease. According to the systematic
investigation it could be deduced that pyrazolotriazine sulfona-
mides are a promising urease inhibitors for treatment of the ure-
ase related diseases.
Enzyme
Mushroom tyrosinase
Ki (lM) Type of inhibition
Urease
Compound
Ki (lM)
Type of inhibition
8a
8b
8j
–
40
20
–
0.01
–
–
Mixed type
Noncompetitive
Mixed type
–
–
Ki is enzyme inhibition constant.
– is not determined.
(8j) and (8a) which gave a series of straight lines as shown in
Figures 1–3(a). Furthermore, inhibition constant (Ki) were deter-
mined to gain insightful pathway by secondary replots and Dixon
plots as shown in Figures 1–3(b,c). The results showed that com-
pound (8b) behaved as a noncompetitive inhibitor (Figure 1), which
means that it could combine with both the free enzyme and the
enzyme-substrate complex. Thus, the interaction between (8b) and
the enzyme was independent from the interaction between the
substrate and the enzyme, and the inhibitor did not change sub-
strate-enzyme affinity. With increasing concentrations, a series of
lines was revealed with a common intercept on the 1/[S] axis, but
with different slopes. On the other hand in Dixon plot (8b) was con-
sistent with the characteristic patterns of noncompetitive inhibition
with Ki value 40 lM and compound (8j) show mixed type inhibition
with Ki value 20 lM as shown in Figure 2(a,b). In case of compound
(8a) whose kinetic mechanism was studied against urease, by
increasing the concentration of substrate (urea) gave family of
straight lines, all of which intersected within the second quadrant.
The analysis showed that Vmax decreased with increasing Km in the
presence of increasing concentrations of (8a) This behavior of com-
pound (8a), indicated that it is a mixed type inhibitor with respect
to the substrate urea with Ki value 0.01 lM as shown in Figure
3(a–c). The results of inhibition type and inhibition constants are
summarized in Table 2.
Acknowledgements
This research was partially funded by the National Science Center,
Poland (grant NN405 092340). The authors wish to acknowledge
The Children’s Memorial Health Institute, Warsaw, Poland for the
access to Q-TOF LC/MS; mass spectrometer purchase was sup-
ported by European POIG.02.01.00-14-059/09 project.
Disclosure statement
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
Funding
National Science Centre, Poland, 10.13039/501100004281 [grant
NN405 092340].

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