Synthesis of aminocyanopyrazoles via a multi-component reaction and anti-carbonic anhydrase inhibitory activity of their sulfamide derivatives against cytosolic and transmembrane isoforms

Article abstract of DOI:10.3109/14756366.2012.720573

A convenient protocol for the multicomponent reaction (MCRs) between malononitrile with an orthoester and hydrazine derivatives, under acid catalyst is described. A series of aminocyanopyrazoles 4 was prepared, isolated and characterized. These pyrazoles reacted with sodium nitrite followed by secondary amine reagent and with formic acid to lead pyrazolotriazines 6 and pyrazolopyrimidinones 7. Some of the aminopyrazoles were converted to the corresponding sulfamides by reaction with sulfamoyl chloride. The aminopyrazoles incorporating phenyl and tosyl moieties were tested as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the human (h) hCA I, II, IX and XII. Many of them showed low micromolar or submicromolar inhibition of these enzymes. The corresponding sulfamides were low nanomolar CA inhibitors.

Full text of DOI:10.3109/14756366.2012.720573

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(2): 343–349
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.720573
ReseaRch aRtIcle
Synthesis of aminocyanopyrazoles via a multi-component
reaction and anti-carbonic anhydrase inhibitory activity
of their sulfamide derivatives against cytosolic and
transmembrane isoforms
Fatma Allouche1, Fakher Chabchoub1, Fabrizio Carta2, and Claudiu T. Supuran2
1Laboratoire de Chimie Appliquée: Hétérocycles, Corps Gras et Polymères Faculté des Sciences de Sfax, 3018 Sfax,
Tunisie and 2University of Florence, dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto
Fiorentino, Italy
abstract
A convenient protocol for the multicomponent reaction (MCRs) between malononitrile with an orthoester and
hydrazine derivatives, under acid catalyst is described. A series of aminocyanopyrazoles 4 was prepared, isolated and
characterized. These pyrazoles reacted with sodium nitrite followed by secondary amine reagent and with formic
acid to lead pyrazolotriazines 6 and pyrazolopyrimidinones 7. Some of the aminopyrazoles were converted to the
corresponding sulfamides by reaction with sulfamoyl chloride. The aminopyrazoles incorporating phenyl and tosyl
moieties were tested as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the human (h) hCA I, II, IX
and XII. Many of them showed low micromolar or submicromolar inhibition of these enzymes. The corresponding
sulfamides were low nanomolar CA inhibitors.
Keywords: MCR aminocyanopyrazoles, pyrazolo[3,4-d][1,2,3]triazines, pyrazolo[4,3-d]pyrimidin-7-ones, carbonic
anhydrase inhibitor
Introduction
valuable MCR aminocyanopyrazoles and to study their
application by diazotation in hydrochloric acid and by
action of formic acid.
Due to the convenience and high degree of atom econ-
omy, multicomponent reactions (MCRs) have become
one of the most efficient tools for rapid scaffold construc-
tion and introduction of molecular diversity1,2. MCRs are
important synthetic tools that can be useful in the prepa-
ration of biologically active compounds3. In our search
for operationally simple, resource and cost-effective
processes, we have been investigating pyrazole MCRs.
eir classical syntheses in two steps were well described
in the literature4–6. In fact, pyrazoles constitute an impor-
tant family of compounds7,8 due to their applications
as antiviral9, as xanthine oxidase inhibitors10, as dual
MAO-B inhibitors and anti-inflammatory analgesics11
and have considerable chemical and pharmacological
importance12–15. In this work, our objective is to achieve
Results and discussion
Synthesis of 5-amino-4-cyano-1-substituted
pyrazoles 4
e first step of our strategy was the formation of MCR
5-amino-4-cyano-1- substituted pyrazoles 4. Several
works mention the synthesis of the 5-amino-4-cyano
pyrazoles4–6 prepared via a standard addition of hydrazine
derivatives to ketene ethoxymethylene compounds.
However, we were delighted to observe the sole formation
of pyrazoles 4 when introducing equivalent amounts
of malononitrile 1 with orthoester 2 and hydrazine
Address for Correspondence: Fatma Allouche, Laboratoire de Chimie Appliquée : Hétérocycles, Corps Gras et Polymères Faculté des
Sciences de Sfax, 3018 Sfax, Tunisie. Tel.: +216 26 299 719. Fax: +216 74 67 66 06. E-mail: fatmaallouch@yahoo.fr
(Received 06 August 2012; revised 07 August 2012; accepted 08 August 2012)
343

344 F. Allouche et al.
derivatives 3 in the presence of few drops of acetic acid in
the ethanol heated under reflux overnight. is procedure
gave satisfying results: phenyl hydrazines afforded the
pyrazole in good yields (Scheme 1).
Carbonic anhydrase inhibitory action
Compounds 4a–f and 8a–c have been tested as inhibitors
of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the
human (h) hCA I, II, IX and XII (Table 1). e inhibition/
activation of CAs are well understood processes, with
most classes of inhibitors binding to the metal center28–34
,
Synthesis of pyrazolotriazines 6 and
pyrazolopyrimidinones 7
whereas activators bind at the entrance of the active
site cavity and participate in proton shuttling processes
between the metal ion – bound water molecule and the
environment35. It should be mentioned that recently other
inhibition mechanisms than the binding to the metal
center were reported for α-CAs, which do not directly
involve the metal ion from the enzyme active site. For
example polyamines bind to the enzyme by anchoring
to the zinc-coordinated water/hydroxide ion36, whereas
coumarins act as prodrugs and bind at the entrance of
the active site cavity, rather far away from the metal ion37–
39. e compounds 4 reported here contain an amino
moiety attached to the pyrazole ring which may bind to
the enzyme similar to the polyamine spermine, which is
anchored by means of one of its primary amino moieties
to the zinc-coordinated water molecule36. Furthermore,
some secondary/tertiary sulfonamides as those present
Taking advantage of the presence of the two functional
groups in 5-amino-4-cyano-1-substituted pyrazoles 4,
we prepared the 4-chlorotriazine by diazotation in hydro-
chloric acid16–20. In the literature amino cyano pyrazoles
undergo diazotization reaction with NaNO2 in a mixture
of HCl/AcOH and leads to the pyrazolotriazin-4-ones21.
To the best of our knowledge 5-substituted chloropyr-
azolotriazines 5a–c are not described in the literature.
e chlorine atom was easily substituted by a secondary
amine to afford compounds 6a–c. We note that the mass
spectra of triazines 5 and 6 showed a molecular mass
decrease by the mass of two nitrogen atoms. is is has
been mentioned in literature22.
e reaction of formic acid in the presence of H2SO4
on anthranilonitrile and analogs is well described in the
literature23–27. We used classical conditions to synthesize
compound 7. 5-amino-4-cyano-1-substituted pyrazoles
4 was refluxed with formic acid in the presence of H2SO4
under stirring providing pyrazolopyrimidinones 7 from
good to very good yields (Scheme 2). is protocol allows
to generalize the synthesis of pyrazolo pyrimidinones
and increase in some cases the yield of the reaction. e
1
structures of compounds 7 were determined by IR, H,
13C NMR spectra, mass spectroscopy and elemental anal-
ysis. ese data spectrum demonstrate in particularly the
disparities of CN group in the aminocyanopyrazoles.
Some of the amino-containing pyrazoles investigated
here were converted to the corresponding sulfamides by
reaction with sulfamoyl chloride. It is in fact well know
that the sulfamide function is a good zinc-binding group
for generating potent CA inhibitors28,29. Several such
compounds, of type 8a-8c were prepared in this way.
Scheme 2.
Table 1. hCA I, II, IX and XII I nhibition data with compounds
1a–1c and 4.
Ki (µM)*
Compound
hCA I
12.6
10.1
9.4
hCA II
6.21
hCA IX
0.34
hCA XII
0.49
4a
4b
4c
4d
4e
4f
7.14
0.29
0.36
5.37
0.27
0.44
5.4
0.79
1.24
3.12
5.9
1.13
0.75
0.49
3.1
0.82
0.47
0.15
1a
1b
1c
0.12
0.09
0.08
0.064
0.051
0.024
0.022
0.011
0.010
0.008
0.012
0.010
aMean from 3 different assays by a CO2 hydrase, stopped flow assay41.
Scheme 1.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of aminocyanopyrazoles 345
in derivatives 4d–f, were recently shown to be inhibitor,
probably binding at the entrance of the cavity, in the
coumarin-binding site40.
syringes techniques to transfer solutions. IR spectra were
determined in KBr on a JASCO FT-IR-420 spectrometer
which precision is of 2 cm−1 covering field 400–4000 cm−1.
1
Data of Table 1 show that compounds 4 act as medium
potency, micromolar or submicromolar inhibitors of the
four investigated CA isoforms. Against the cytosolic hCA
I they showed activity with inhibition constants in the
range of 3.1–12.6 µM41–45. Sulfonamides 4d–f were more
active than the corresponding phenyl derivatives 4a–c.
e second cytosolic isoform, hCA II, the physiologically
dominant one, was better inhibited by compounds 4
compared to hCA I, with KIs in the range of 0.79–7.14 µM.
e structure activity relationship (SAR) was also similar
for the two isoforms.
e tumor-associated, transmembrane isoforms
hCA IX and XII were better inhibited by compounds 4
compared to the cytosolic ones mentioned above. us,
against hCA IX, compounds 4 showed inhibition in the
range of 0.27–1.24 µM, with the phenyl derivatives more
active than the sulfonamides in this case. For hCA XII the
inhibition constants were in the range of 0.15–3.12 µM
and the SAR more complicated as the best inhibitor was
the sulfonamide 4f, but the phenyl derivatives 4a–c also
showed quite significant inhibition (Table 1).
As these compounds may not bind to the Zn(II) ion
from the CA active site, we hypothesize that they inhibit
the enzyme either binding similar to spermine (anchor-
ing to the zinc-coordinated water molecule) or as the
coumarins, at the entrance of the active site cavity. is
hypothesis should be verified by solving the X-ray crystal
structure of adducts of these compounds with some CA
isoforms46.
e sulfamides 8 were much more potent inhibitors of
all CA isoforms compared to the previously mentioned
compounds, being low nanomolar inhibitors of all four
CA isoforms investigated here (Table 1). is is probably
due to their direct binding to the metal ion within the
enzyme active site28.
e spectra of NMR H and NMR 13C were recorded on
an AC Bruker 250 MHz or Bruker Advance III 400 MHz
spectrometers in CDCl3 or DMSO-d6 and the chemical
shifts are expressed in ppm. e multiplicities of the sig-
nals are indicated by the following abbreviations: s: sin-
glet, d: doublet, t: triplet, q: quadruplet, m: multiplet, brs:
broad singlet and the coupling constants are expressed
in Hz. e assignment of exchangeable protons (OH and
NH) was confirmed by the addition of D2O. e melting
points were determined in Electrothermal 9100 appara-
tus and are uncorrected. e reactions were monitored
by thin layer chromatography (TLC) using aluminium
sheets with silica gel 60 F254 (230–400 mesh ASTM) as the
stationary phase and ethylacetate/n-hexane or MeOH/
DCM were used as eluants. e mass spectrometer
was operated in EI mode at 70 eV and MS spectra were
recorded from m/z 50 to 650.
General procedure for synthesis of
aminocyanopyrazoles (4)
A solution of malononitrile 1 (33 mmol), orthoester 2 (34
mmol), hydrazine (33 mmol) 3 and few drops of acetic
acid in ethanol (30 mL) was heated under reflux over-
night. e product, which precipitates, was filtered and
recrystallized from ethanol.
5-Amino-1-phenyl-1H-pyrazolo-4-carbonitrile (4a)
Yield 83%, white solid; mp = 166°C (methanol); IR (cm−1):
1594, 1640, 2217 (CN), 2917, 3432, 3221, 1H NMR (δ ppm,
DMSO): 6.82 (2H, s, NH2), 7.40–7.55 (6H, m), 13C NMR
(δ ppm, DMSO): 144.60 (C-3), 73.48 (C-4), 152.74 (C-5),
114.17 (CN), 117.08–137.52 (C-arom).
5-Amino-3-methyl-1-phenyl-1H-pyrazolo-4-carbonitrile (4b)
Yield 85%, white solid; mp = 133°C (methanol); IR (cm−1)
1598, 1645, 2215 (CN), 3331, 3227, 1H NMR (δ ppm,
DMSO) 2.15 (3H, s, CH3), 6.66 (2H, s, NH2), 7.35–7.52 (5H,
m);13C NMR (δ ppm, DMSO) 11.28 (CH3), 74.23 (C-4),
115.57 (CN), 124.40–138.03 (C-arom), 150.56 (C-5),
151.96 (C-3); MS m = e/z 199 [M + 1]+, 100%.
In conclusion, we report a novel multicomponent
reaction of malononitrile with orthoesters and hydrazine
derivatives that leads to the aminocyanopyrazoles 4 with
good yields. ese pyrazoles are open for further trans-
formation due to an already existing amino and cyano
groups in their structures. ese pyrazoles react with
sodium nitrite followed by secondary amine reagent and
with formic acid to lead respectively to pyrazolotriazines
6 and pyrazolopyrimidinones 7. is new protocol was
employed for the rapid synthesis of compounds 5 and 6
and for the amelioration of yield of compounds 7. Many
of therse compounds showed interesting CA inhibitory
activity.
5-Amino-3-ethyl-1-phenyl-1H-pyrazolo-4-carbonitrile (4c)
Yield 82%, white solid; mp = 137°C (methanol); IR (cm−1)
1597, 1647, 2209 (CN), 3433, 3296; 1H NMR (δ ppm,
DMSO) 1.18 (3H, t, J = 7.1 Hz, CH3), 2.53 (2H, q, J = 7.1
Hz, CH2), 6.61 (2H, s, NH2), 7.35–7.53 (5H, m); 13C NMR (δ
ppm, DMSO) 12.88 (CH3), 21.13 (CH2), 73.22 (C-4), 115.47
(CN), 124.43–138.11 (C-arom), 152.13 (C-3), 155.58 (C-5);
MS: m = e/z 213 [M + 1]+, 100%, 158 [M-54]+, 5%.
Materials and methods
5-Amino-1-tosyl-1H-pyrazolo-4-carbonitrile (4d)
Anhydrous solvents and all reagents were purchased from
Sigma-Aldrich, Alfa Aesar and TCI. All reactions involving
air- or moisture-sensitive compounds were performed
under a nitrogen atmosphere using dried glassware and
Yield 70%, white solid; mp = 192°C (methanol); IR (cm−1)
1527, 1616, 2211 (CN), 3347, 3225; 1H NMR (δ ppm,
DMSO) 2.27 (3H, s, CH3), 7.11 (2H, s, NH2), 7.13–7.51
(5H, m); 13C NMR (δ ppm, DMSO) 21.28 (CH3), 75.51
© 2013 Informa UK, Ltd.

346 F. Allouche et al.
(C-4), 113.81 (CN), 138.58 (C-3), 145.55 (C-5), 124.66–
129.07 (C-arom).
112.01 (C-7a), 137.03 (C-5) 157.91 (C-4), 125.04-132.89
(C-arom), GC-MS: m/z (%) = 232 (100), 204 (43), 182 (51),
169 (15), 142 (30), C12H10ClN5 Calculated (%): C 55.50, H
3.88, N 26.97, Found (%) C 55.48, H 3.89, N 26.96.
5-Amino-3-methyl-1-tosyl-1H-pyrazolo-4-carbonitrile (4e)
Yield 72%, white solid; mp = 185°C (methanol); IR (cm−1)
1534, 1628, 2216 (CN), 3381, 3227; 1H NMR (δ ppm,
DMSO) 2.04 (3H, t, CH3), 2.39 (3H, s CH3), 7.63 (2H, s,
NH2), 7.47–7.87 (4H, m); 13C NMR (δ ppm, DMSO) 13.06
(CH3), 21.53 (CH3), 73.97 (C-4), 113.71 (CN), 127.89–
133.44 (C-arom), 146.80 (C-3), 154.87 (C-5).
General procedure for synthesis of
aminopyrazolotriazines (6)
e chloropyrazolotriazine 5a–c (1 mmol) and corre-
sponding amine (10 mL) were refluxed for 6 h. e mix-
ture was cooled at room temperature. When a precipitate
was formed, it was filtered, washed twice with 15 mL of
water and twice with 8 mL of diethyl ether, and dried at
room temperature overnight. e products were recryst-
allised in methanol to give products 6.
5-Amino-3-ethyl-1-tosyl-1H-pyrazolo-4-carbonitrile (4f)
Yield 74%, white solid; mp = 178°C (methanol); IR (cm−1)
1526, 1643, 2215 (CN), 3346, 3222; 1H NMR (δ ppm,
DMSO) 0.87 (3H, t, J = 7.0 Hz, CH3), 2.16 (2H, q, J = 7.0 Hz,
CH2), 2.38 (3H, s, CH3), 7.38 (2H, s, NH2) 7.38–7.68 (5H,
m); 13C NMR (δ ppm, DMSO) 10.81 (CH3), 21.52 (CH2),
23.30 (CH3), 73.50 (C-4), 114.32 (CN), 128.20–136.42
(C-arom), 143.49 (C-3), 144.26 (C-5).
4-Morpholino-7-phenyl-7H-pyrazolo[3,4-d][1,2,3]triazine (6a)
Yield 79% mp = 170°C [methanol]; IR (cm−1) 1556 (C=N),
1
1453 (C=N), 1422 (N=N); NMR H (δ ppm, DMSO) 3.15
(4H, t, J = 7.1 Hz, CH2), 3.64 (4H, t, J = 7.1 Hz, CH2), 7.34-
7.68 ppm (5H, m), 7.75 (1H, s, H5); 13C NMR (δ ppm,
DMSO) 51.10 (CH2), 68.12 (CH2), 115.15 (C-7a), 93.01
(C-4a), 156.63 (C-5), 159.87 (C-4), 124.54 (C-arom);
GC-MS m/z (%) 255 (100), 197 (23), 119 (66), C14H14N6O
Calculated (%): C 59.50, H 5.00, N 29.77, O 5.67 Found (%)
C 59.52, H 5.03, N 29.78, O 5.65.
General procedure for synthesis of
chloropyrazolotriazines (5)
A solution of sodium nitrite (11.4 mmol) in water (7mL)
was added over 15 min to a suspension of the foregoing
5-amino-4-cyano-1-substituted pyrazole (8.1 mmol)
at 0-5°C in concentrated hydrochloric acid (16mL).
e resulting mixture was stirred at 0°C for a further
40 min and then allowed to stand at room temperature
overnight. e reaction mixture was quenched in water
(100 mL). e precipitate was washed twice with 15mL of
water, and dried under room temperature, recrystallized
in methanol.
5-Methyl-4-morpholino-7-phenyl-7H-pyrazolo[3,4-d][1,2,3]
triazine (6b)
Yield: 75%. mp = 159°C [methanol]. IR (cm−1): 1560 (C=N),
1
1467 (C=N), 1431 (N=N); NMR H (δ ppm, DMSO): 2.28
(3H, s, CH3), 3.17 (4H, t, J = 7.1 Hz, CH2), 3.66 (4H, t, J =
7.1 Hz, CH2); 7.35-7.62 ppm (5H, m); 13C NMR (δ ppm,
DMSO) 13.31 (CH3), 50.53 (CH2), 66.63 (CH2), 82.18
(C-4a), 115.39 (C-7a), 152.41 (C-5), 153.80 (C-4), 124.17–
139.23 (C-arom); GC-MS m/z (%) 269 (100), 211 (53), 133
(22), C15H16N6O: Calculated(%): C 60.80, H 5.44, N 28.36,
O 5.40 Found (%): C 60.82, H 5.45, N 28.34, O 5.42.
4-Chloro-7-phenyl-7H-pyrazolo[3,4-d][1,2,3]triazine(5a)
Yield 79%; mp = 170°C [methanol]; IR (cm−1) 1590 (C=N);
1
NMR H (δ ppm, DMSO) 7.75 (1H, s, H5), 7.23 ppm (5H,
s); 13C NMR (δ ppm, DMSO) 93.01 (C-4a), 131.15 (C-7a),
136.63 (C-5), 159.87 (C-4), 124.54–129.33 (C-arom);
GC-MS: m/z (%) 204 (100), 154 (24), 141 (17); C10H6ClN5
Calculated (%): C 51.85, H 2.61, N 30.23, Found (%) C
51.79, H 2.60, N 30.25.
5-Ethyl-4-morpholino-7-phenyl-7H-pyrazolo[3,4-d][1,2,3]
triazine (6c)
Yield 76%; mp = 170°C [methanol]; IR (cm−1) 1590 (C=N),
1
1526 (C=N), 1460 (N=N); NMR H (δ ppm, DMSO) 1.30
4-Chloro-5-methyl-7-phenyl-7H-pyrazolo[3,4-d][1,2,3]
triazine(5b)
(3H, t, J = 7.1 Hz, CH3), 2.72 (2H, q, J = 7.3 Hz, CH2),
3.18 (4H, t, J = 7.3 Hz, CH2), 3.66 (4H, t, J = 7.3 Hz, CH2),
7.33-7.63 ppm (5H, m); 13C NMR (δ ppm, DMSO) 12.86
(CH3), 21.28 (CH2), 50.18 (CH2), 66.32 (CH2), 80.71 (C-4a),
115.12 (C-7a), 153.52 (C-5), 157.26 (C-4), 123.85–138.89
(C-arom); GC-MS: m/z(%) 283 (100), 213 (25), 147 (36).
C16H18N6O: Calculated (%): C 61.92, H 5.85, N 27.08, O
5.16 Found (%): C 61.90, H 5.83, N 27.10, O 5.17.
Yield 75% mp = 159°C [methanol]; IR (cm−1) 1586 (C=N);
1H NMR (δ ppm, DMSO) 2.38 (3H, s, CH3), 7.26–7.64 ppm
(5H, m); 13C NMR (δ ppm, DMSO) 13.52 (CH3), 94.54
(C-4a), 112.26 (C-7a), 137.38 (C-5), 153.08 (C-4), 120.01-
133.11 (C-arom); GC-MS: m/z (%)218 (100), 168 (36), 155
(14); C11H8ClN5 Calculated (%): C 53.78, H 3.28, N 28.51,
Found (%) C 53.79, H 3.30, N 28.53.
General procedure for synthesis of
pyrazolopyrimidinones (7)
5-amino-4-cyano-1-substituted pyrazole 4 (10 mmol)
was added portionwise over 1 h to a mildly refluxing
formic acid solution (20 mL) containing concentrated
sulphuric acid (1.2 mL). After an additional 30 min,
the solution was cooled to 0°C and poured on crushed
4-Chloro-5-ethyl-7-phenyl-7H-pyrazolo[3,4-d][1,2,3]
triazine(5c)
Yield 76%; mp = 179°C [methanol]; IR (cm−1) 1587 (C=N);
1
NMR H (δ ppm, DMSO) 1.35 (3H, t, J = 7.1 Hz, CH3),
2.80 (2H, q, J = 7.1 Hz, CH2), 7.51 ppm (5H, s); 13C NMR
(δ ppm, DMSO) 12.74 (CH3), 21.52 (CH2), 93.13 (C-4a),
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of aminocyanopyrazoles 347
ice. e resulting precipitate was collected by filtration,
washed with water, and dried to give pyrazolopyrimidi-
nones 7.
and the solution was stirred at r.t. until starting material
was consumed (TLC monitoring). en the solution was
quenched with slush and extracted with ethyl acetate
(3 × 20 mL). e combined organic layers were washed
with H2O (4 × 20 mL), brine (3 × 20 mL) dried over Na2SO4,
filtered and concentrated in vacuo to give a sticky residue
that was purified to afford the desired sulfamides 8a–c as
white solids.
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (7a)
Yield 82% mp = 164°C [methanol]; IR (cm−1) 1663 (C
1
= O), 1583 (C=N), 1545 (C=N), 1500 (C=C); NMR H (δ
ppm, DMSO) 7.34-7.65 (5H, m);7.72 (1H, s, H3); 7.75 (1H,
s, H6); 8.01 ppm (1H, s, NH); 13C NMR (δ ppm, DMSO):
93.01 (C-3a), 131.15 (C-7a), 136.63 (C-3), 142.00 (C-6),
159.87 (C-4), 124.54–129.33 (C-arom); GC-MS: m/z (%)
213 (100), 168 (28), 102 (58); C11H8N4O: Calculated (%): C
62.26, H 3.80, N 26.40, O 7.54 Found (%): C 62.28, H 3.81,
N 26.42, O 7.55.
Synthesis of 1-phenyl-5-sulphamido-1H-pyrazole-4-
carbonitrile (8a)
5-Amino-1-phenyl-1H-pyrazole-4-carbonitrile
4a
(0.05 g, 1.0 eq) was treated according to the general pro-
cedure previously described. e obtained residue was
purified by silica gel column chromatography eluting
with 50% ethyl acetate/n-hexane to afford 8a as a white
solid (Figure 1).
1-Phenyl-5-sulphamido-1H-pyrazole-4-carbonitrile
8a: 54% yield; silica gel TLC Rf 0.09 (50% ethyl acetate/n-
hexane); δH (400 MHz, DMSO-d6) 9.60 (1H, brs, exchange
with D2O, NH), 7.60 (8H, m, Ar-H, 3-H, SO2NH2), 7.42 (2H,
s, exchange with D2O, SO2NH2); δC (100 MHz, DMSO-d6)
155.5, 154.1, 141.3, 130.2, 125.0, 120.0, 117.0, 79.8.
3-Methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(7b)
Yield: 86%. mp = 159°C [methanol]. IR (cm−1): 1665 (C=O);
1
1580 (C=N); 1541 (C=N); 1506 (C=C); NMR H (δ ppm,
DMSO): 2.25 (3H, s, CH3), 7.34-7.68 (5H, m), 7.72 (1H, s,
H6); 8.00 ppm (1H, s, NH); 13C NMR (δ ppm, DMSO) 13.76
(CH3), 93.00 (C-3a), 135.55 (C-3), 135.55 (C-7a), 139.76
(C-6), 160.30 (C-4), 124.54–129.33 (C-arom); GC-MS:
m/z (%) 227 (100), 200 (36), 182 (12), 168 (50), 131 (33),
116 (44), 104 (20).
Synthesis of 3-methyl-1-phenyl-5-sulphamido-1H-pyrazole-4-
carbonitrile 8b
3-Ethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (7c)
5-Amino-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile
4b (0.05 g, 1.0 eq) was treated according to the general
procedure previously described. e obtained residue
was purified by silica gel column chromatography elut-
ing with 70% ethyl acetate/n-hexane to afford 8b as a
white solid (Figure 2).
3-Methyl-1-phenyl-5-sulphamido-1H-pyrazole-4-
carbonitrile 8b: 48% yield; silica gel TLC Rf 0.11 (50%
ethyl acetate/n-hexane); δH (400 MHz, DMSO-d6) 9.99
(1H, brs, exchange with D2O, NH), 7.53 (5H, m, Ar-H),
7.42 (2H, s, exchange with D2O, SO2NH2), 2.18 (3H, s
Yield 90%; mp = 168°C [methanol]; IR (cm−1) 1665 (C =
1
O), 1580 (C = N), 1541 (C = N), 1506 (C=C); NMR H (δ
ppm, DMSO) 3.15 (3H, t, J = 7.1 Hz, CH3); 3.64 (2H, q, J =
7.1 Hz, CH2); 7.34–7.60 (5H, m, CH = C); 7.78 (1H, s, H6);
8.05 ppm (1H, s, NH); 13C NMR (δ ppm, DMSO): C2 93.01
(C-3a); C3131.15 (C-7a); C1 136.63 (C-3); C5 145.76 (C-6);
C4 159.87 (C-4); 124.54–129.33 (C-arom); GC-MS m/z (%)
= 241 (100), 186 (60), 141 (45), C13H12N4O: Calculated (%):
C 64.99, H 5.03, N 23.32, O 6.66 Found (%): C 64.97; H
5.00; N 23.35; O 6.65.
General procedure for the synthesis of compounds
(8a–c)47
Scheme 3 shows the preparation of sulfamides 8a–c.
Freshly prepared sulfamoyl chloride (2.0 eq) was added to
a 2.0 M solution of 5-amino-1-phenyl-1H-pyrazole-4-car-
bonitrile 4a–c in dry DMA under a nitrogen atmosphere
Scheme 3. Preparation of sulfamides 8a–c.
Figure 1.
© 2013 Informa UK, Ltd.

348 F. Allouche et al.
α-CAs, and 8.4, for β-CAs) as buffer, and 20 mM Na2SO4
(for maintaining constant the ionic strength), following
the initial rates of the CA-catalyzed CO2 hydration reac-
tion for a period of 10–100 s43. e CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the
kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5–10% of the reac-
tion have been used for determining the initial veloc-
ity. e uncatalyzed rates were determined in the same
manner and subtracted from the total observed rates.
Stock solutions of inhibitor (0.1 mM) were prepared in
distilled-deionized water and dilutions up to 0.01 nM
were done thereafter with the assay buffer. Inhibitor and
enzyme solutions were preincubated together for 15 min
at room temperature prior to assay, in order to allow for
the formation of the E-I complex. e inhibition con-
stants were obtained by non-linear least-squares meth-
ods using PRISM 3, as reported earlier42, and represent
the mean from at least three different determinations. All
CA isofoms were recombinant ones obtained in house as
Figure 2.
reported earlier43–45
.
Declaration of interest
e authors declare no conflict of interest.
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