Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole, a structural and analog of acetazolamide, show interesting carbonic anhydrase inhibitory properties, diuretic, and anticonvulsant action

Article abstract of DOI:10.3109/14756366.2015.1096270

Three salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole (Hats) were prepared and characterized by physico-chemical methods. The p-toluensulfonate, the methylsulfonate, and the chlorhydrate monohydrate salts of Hats were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) and as anticonvulsants and diuretics, since many CAIs are clinically used as pharmacological agents. The three Hats salts exhibited diuretic and anticonvulsant activities with little neurotoxicity. The human (h) isoforms hCA I, II, IV, VII, IX, and XII were inhibited in their micromolar range by these salts, whereas pathogenic beta and gamma CAs showed similar, weak inhibitory profiles.

Full text of DOI:10.3109/14756366.2015.1096270

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole,
a structural and analog of acetazolamide,
show interesting carbonic anhydrase inhibitory
properties, diuretic, and anticonvulsant action
Jorge R. A. Diaz, Gerardo Enrique Camí, Malva Liu-González, Daniel R. Vega,
Daniela Vullo, Américo Juárez, José C. Pedregosa & Claudiu T. Supuran
To cite this article: Jorge R. A. Diaz, Gerardo Enrique Camí, Malva Liu-González, Daniel R.
Vega, Daniela Vullo, Américo Juárez, José C. Pedregosa & Claudiu T. Supuran (2015): Salts of
5-amino-2-sulfonamide-1,3,4-thiadiazole, a structural and analog of acetazolamide, show
interesting carbonic anhydrase inhibitory properties, diuretic, and anticonvulsant action,
Journal of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2015.1096270
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1096270
Published online: 26 Oct 2015.
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Date: 27 October 2015, At: 04:16

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1096270
!
ORIGINAL ARTICLE
Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole, a structural and
analog of acetazolamide, show interesting carbonic anhydrase
inhibitory properties, diuretic, and anticonvulsant action
1
Jorge R. A. Diaz , Gerardo Enrique Camı , Malva Liu-Gonzalez , Daniel R. Vega , Daniela Vullo , Americo Juarez ,
1
2
3
4
5
´
Jose C. Pedregosa , and Claudiu T. Supuran
´
´
´
1
4
´
1
´
Area De Quımica General E Inorganica, Facultad De Quımica, Bioquımica Y Farmacia, CONICET PIP-6246, Universidad Nacional De San Luis, San
´
´
´
´
2
Luis, Argentina, S.C.S.I.E. (Unidad De Rx) – Facultad De Fısica, Universidad De Valencia, Valencia, Spain, Grupo Materia Condensada, GIyA,
3
´
4
GAIyANN, Comision Nacional De Energıa Atomica, San Martın, Buenos Aires, Argentina, Department of Neurofarba, University of Florence, Sesto
´
´
´
´
5
Fiorentino (Firenze), Italy, and Laboratorio De Farmacologıa, Departamento De Farmacia, Facultad De Quımica, Bioquımica Y Farmacia,
´
´
´
Universidad Nacional De San Luis, San Luis, Argentina
Abstract
Keywords
Three salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole (Hats) were prepared and characterized
by physico-chemical methods. The p-toluensulfonate, the methylsulfonate, and the chlorhy-
drate monohydrate salts of Hats were evaluated as carbonic anhydrase (CA, EC 4.2.1.1)
inhibitors (CAIs) and as anticonvulsants and diuretics, since many CAIs are clinically used as
pharmacological agents. The three Hats salts exhibited diuretic and anticonvulsant activities
with little neurotoxicity. The human (h) isoforms hCA I, II, IV, VII, IX, and XII were inhibited in
their micromolar range by these salts, whereas pathogenic beta and gamma CAs showed
similar, weak inhibitory profiles.
Anticonvulsant, carbonic anhydrase, diuretic,
sulfonamides, sulfonamide salts, thermal
stability
History
Received 14 August 2015
Revised 15 September 2015
Accepted 16 September 2015
Published online 22 October 2015
Introduction
notable increase in the CA inhibitory properties of metal
complexes of sulfonamides such as acetazolamide or its
deacetylated precursor, 5-amino-1,3,4-thiadiazole-2-sulfonamide
(Hats)23. Furthermore, continuing our studies on unsubstituted
heterocyclic sulfonamides and their metal complexes17–23, we
started to study the role of new salts of Hats with regard to their
biological activity. In this work, we report new salts of Hats with
p-toluenesulfonic acid, methylsulfonic acid, and the hydrochloride
monohydrate salts. We evaluated the CA inhibition and anticon-
vulsant/diuretic activities of these three sulfonamide salts.
Sulfonamide inhibitors of the metalloenzyme carbonic anhydrase
(CA, EC 4.2.1.1), such as acetazolamide, have been used
clinically as diuretics, antiglaucoma, or anticonvulsant agents
for a long period1–9, whereas more recent drug design studies
have evidenced other CA inhibitors (CAIs) belonging to the
sulfonamide/sulfamate/sulfamide classes as molecules of interest
for developing novel therapies for obesity10,11 and cancer12–15
,
based on the selective inhibition of CA isozymes involved in such
pathologies, among which 15 such isoforms described so far in
humans1,16
.
Epilepsy is one of the most common serious neurologicaldi-
sorders characterized by recurrent seizures. Since several decades,
acetazolamide is used as an anticonvulsant agent in the treatment
ofepilepsy1,10,11. Despite the development of a rapid tolerance
consisting in diminished therapeutic efficacy after the initial
response of the patients, acetazolamide is still used in combin-
ation therapy with other antiepileptic drugs or inrefractory
epilepsies1.
Experimental
Salt formation
The synthesis of Hats was performed in two steps (Figure 1). In
the first step, the deacetylation of acetazolamide (AZA) to give
Hats was performed following the procedure reported in the
literature17. This product was characterized by its FTIR spectrum
resulting identical to that reported by Pedregosa et al.17,18. In the
second step, 100 mg of Hats (0.55 mmol) were suspended at room
temperature in 50 mL of absolute ethanol (99.5%, Merck,
Darmstadt, Germany) with permanent stirring. Then, 1.2 g of
p-toluenesulfonic acid (Aldrich, Seelze, Germany; 6.31 mmol);
0.45 mL of methylsulfonic acid (Aldrich, Seelze, Germany;
6.7 mmol); 0.45 mL of HCl (37%, Merck, Darmstadt, Germany;
5.5 mmol) were added maintaining the stirring during 2 h
at room temperature. White powders were obtained for each
We have obtained and determined the crystal structures of
sulfonamides incorporating the 1,3,4-thiadiazole ring17–23. The
results obtained by our group with these sulfonamides indicated a
´
Address for correspondence: Gerardo Enrique Camı, Area De Quımica
´
´
´
´
´
General E Inorganica, Facultad De Quımica, Bioquımica Y Farmacia,
CONICET PIP-6246, Universidad Nacional De San Luis, Chacabuco 917,
San Luis 5700, Argentina. E-mail: gecami88@gmail.com

2
J. R. A. Diaz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Figure 1. Preparation of 5-amino-2-sulfona-
mide-1,3,4-thiadiazole salts.
salt, 5-amino-1,3,4-thiadiazole-2-sulfonamide p-toluenesulfonate
To confirm these, melts or crystalline phase changes were used
(Hats.tosylate), 5-amino-1,3,4-thiadiazole-2-sulfonamide methyl- a Leitz Westlar heating microscope at this stage. The equipment
sulfonate (Hats.MeSO3H), and 5-amino-1,3,4-thiadiazole-2-sul- used for mass spectra was a VG AUTOSPEC. The spectral data
fonamide hydrochloride monohydrate (Hats.HCl). Colorless were obtained through of Electronic Impact (EI) at 70 eV and Fast
prismatic single crystals of Hats.tosylate, suitable for DRX Atom Bombardment (FAB). The values listed for each of the
studies, were obtained after 2 weeks at 15 ꢀC avoiding light compounds are expressed in unit of m/z.
exposure.
The reaction for obtaining Hats.tosylate occurred with a yield
of 95%. Elemental analysis for C2H5N4O2Sþ2 :CH3C6H4O3Sꢁ:
Found (calculated): C: 31.21% (30.95%); H: 2.29% (2.58%); N:
15.78% (16.05%); S: 28.53% (27.50%).
Methods
All animals used in experiments were handled in accordance
with international standard for the use and care of laboratory
animals. The corresponding protocols were approved by the
Universidad Nacional de San Luis, following the provisions of the
ANMAT29 6344/96.
The diuretic effect was evaluated using male and female adult
Wistar rats weighing 220 g. The administration of solutions was
performed by oral gavage in a volume of 5 mL/100 g body weight
following the method proposed by Lipschitz et al.30. All animals
were deprived of water 12 h before the experiment having free
access to food. In addition to the urine volume, sodium excretion
and urinary potassium were recorded31 and the concentrations of
these ions were quantified in a flame spectrometer Metrolab
model 315 (Shimadzu Corporation, Kyoto, Japan).
The anticonvulsant activity of drugs was tested in mice
Rockland (weighing 25–30 g, administering the sulfonamides
intraperitoneally), against seizures induced by intraperitoneal
administration of nikethamide or picrotoxin as control drugs.
Through the ‘‘rota-rod test’’32, the neurological deficit was
evaluated, which is evidenced by the ability of the mouse to stay
in balance (on a cylinder rotating at 15 rpm) in three trials of
1 min each. Animals were distributed into three lots: negative
control (saline), positive control (phenobarbital), and experimen-
tal compounds (Hats.tosylate, Hats.MeSO3H, and Hats.HCl). The
compounds were administered intraperitoneally. The mice are
placed on the rota-rod bar and put into operation the apparatus.
Animals that do not fall off the bar in 5 min will be chosen for the
test. Thirty minutes after intraperitoneal administration, the
mice are placed on the moving bar and the number of falls in
5 min was registered. Then the analysis of the recorded data was
achieved.
X-ray data for Hats.tosylate were collected with an Enraf–Nonius
FR590 CCD area detector (Labx, Schwerzenbach, Switzerland)
using Collect24 and HKL Denzo–Scalepack25 software with
2
˚
graphite monochromated Mo-Ka (k ¼ 0.71073 A) at 293 K. The
structure was solved by direct methods using SHELXS-9726 and
all the non-hydrogen atoms were refined anisotropically by full-
matrix least-squares on F2 using SHELXL-9726. The H atoms
attached to N were found in a difference Fourier map, further
˚
idealized (N–H:0.86 A), and finally allowed to ride. All calcula-
tions, including Figures 1–3, were performed using WinGX27 and
Ortep-3 for windows28. The crystal structure of Hats tosylate has
been deposited in the CSD with reference number CCDC 730598.
These data can be obtained free of charge via www.ccdc.cam.
ac.uk/data_request/cif. The compound crystallizes forming color-
less triclinic prisms with the space group P1.
ꢁ
Elemental analysis (C, H, N, and S) was carried out with a
Carlo Erba EA 1108 microanalyser (Triad Scientific, Manasquan,
´
NJ) belonging to the Instituto de QuımicaFısica de Materiales,
MedioAmbiente y Energıa (INQUIMAE), Universidad de Buenos
´
´
Aires, Argentine; sulfanilamide was used as the standard.
Fourier transformed infrared (FT-IR) spectra were recorded on
´ ´
a Nicolet Protege 460 spectrometer (Conquer Scientific, San
Diego, CA) provided with a CsI beam splitter in the 4000–
250 cmꢁ1 range with 32 scans and spectral resolution of 4 cmꢁ1
using the KBr pellet technique.
,
Thermogravimetric (TGA) and differential thermal analysis
(DTA) curves were obtained with Shimadzu TGA-51
a
Thermal Analyzer (Shimadzu Corporation, Kyoto, Japan) and
DTA-50 Thermal Analyzer, using platinum pans, ꢁf1lowing air at
50 ml minꢁ1 and at a heating rate of 10 ꢀC min from room
temperature to 1000 ꢀC.

DOI: 10.3109/14756366.2015.1096270
Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole
Table 1. Crystal data of Hats.tosylate.
3
The ‘‘chimney’’ test33 is another in vivo test used to evaluate
the mouse motor coordination impairments. It is based on the time
that the rodent needs to traverse a tube of glass or clear acrylic
20 cm long in a span of 10 s. In this assay, the degree of muscular
relaxation in mice is evaluated. Animals were distributed into
three lots: negative control (saline), positive control (phenobar-
bital), and experimental (sulfonamides). The rota-rod test was
used to evaluate the activity of drugs that interfere with motor
coordination (Test of Dunham, 1957), while the ‘‘chimney’’ test
was used to estimate locomotor activity33. Neurotoxicity experi-
ments were carried out with adults Rockland albino mice
weighing 25–30 g. The doses of sulfonamides studied were 20,
50, and 90 mg/kg of mouse.
Empirical formula
Formula weigh
Temperature
ꢁ
C18H18N8O10S6
698.76
293 (2) K
˚
0.71073 A
Crystal system
Space group
Parameters
Triclinic
P-1
ꢀ
˚
a ¼ 9.3220 (3) A, ꢂ ¼ 92.7390 (13)
ꢀ
˚
˚
b ¼ 10.8990 (4) A, ꢃ ¼ 101.5110 (16)
ꢀ
c ¼ 14.7830 (6) A, ꢄ ¼ 95.6290 (13)
3
˚
1461.15 (9) A
Volume
Z
Density (calc.)
Absorption coefficient
F (000)
2
1.588 g/cm3
0.533 mmꢁ1
716
An Applied Photophysics stopped-flow instrument was used
for assaying the CA catalyzed CO2 hydration activity34. Phenol
red (at 0.2 mM) was used as an indicator, working at the
absorbance maximum of 557 nm with 10 mM Hepes (pH 7.4) or
TRIS (pH 8.3) as buffers and 0.1 M NaClO4 (for maintaining
constant ionic strength), at 20 ꢀC, following the CA-catalyzed
CO2 hydration reaction for a period of 10–100 s (the uncatalyzed
reaction needs around 60–100 s in the assay conditions, whereas
Crystal dimension
y range
Range all indices
0.2 ꢂ 0.16 ꢂ 0.12 (mm)
1.41–27.43ꢀ
ꢁ12 ꢃ h ꢃ 12, ꢁ13 ꢃ k ꢃ 13,
ꢁ18 ꢃ I ꢃ 18
Collected reflections
Independent reflections
Integrity ꢅ (max.)
Refinement method
Data/restrictions/parameters
Adjusted on F2
Indices R (final) [I42ꢆ(I)]
Indices R (all data)
Max. difference peak
and hole
9081
5629 [R (int) ¼ 0,1021]
ꢅ ¼ 27.43: 84.4%
Full-matrix least-squares on F2
5629/0/401
the catalyzed ones are of around 6–10 s), as described earlier35–43
.
0.921
R1 ¼ 0.0760, wR2 ¼ 0.1705
The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters. The uncatalyzed rates
were determined in the same manner and subtracted from the total
observed rates. Enzyme concentrations in the assay system were
in the range of 10 nM for all the enzymes considered in the
present study, which have been obtained as recombinant proteins,
R1 ¼ 0.2487, wR2ꢁ¼30.2756
˚
0.617y–0.559 e.A
in-house, as reported earlier43–48
.
Results and discussion
ions in the asymmetric unit. The anion p-toluensulfonate and
cation H2ats+ are distributed in alternative parallel layers along
with the b-axis and stabilized by hydrogen bonds between the 2-
Characterization
In a previous work, we have reported the crystal structures and sulfonamido group and sulfonate ion, 5-amino and protonated N2
spectroscopic
characterization
of
Hats.MeSO3H
and (azole) group and sulfonate group (Table 2). The ꢀ-stacking
Hats.HCl22,23. Crystal and experimental data for Hats.tosylate between phenyl groups are reinforced by interactions between
are listed in Table 1. Figure 2 shows ORTEP diagram of the these groups and terminal methyl moieties, which stabilize the
studied compound, and Figure 3 shows the molecular packing. crystal lattice.
Hats.tosylate consists of two H2ats+ and two p-toluenesulfonate
Figure 2. ORTEP diagram of Hats.tosylate.

4
J. R. A. Diaz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Figure 3. Unit cell with hydrogen atoms of
Hats.tosylate.
Table 2. H-bonds interactions in Hats.tosylate crystal structure.
˚
Distance (A)
Donor–H
˚
˚
Distance (A) H . . .
Acceptor
Distance (A) Donor . . .
Acceptor
Angle (deg.) Donor–H . . .
Acceptor
Donor–H . . . Acceptor
N(1A)–H(1A1) . . . O(5B)a
N(1A)–H(1A2) . . . O(2B)a
N(2A)–H(2A) . . . O(4B)a
N(4A)–H(4A1) . . . O(3B)b
N(4A)–H(4A2) . . . O(5A)c
N(1B)–H(1B1) . . . O(3A)d
N(1B)–H(1B2) . . . O(5B)e
N(2B)–H(2B) . . . O(5A)d
N(4B)–H(4B1) . . . O(4B)f
N(4B)–H(4B2) . . . O(4A)c
CG . . . CG
0.860
0.860
0.860
0.861
0.860
0.860
0.860
0.860
0862
2.033
2.522
2.002
1.997
2.125
1.907
2.078
1.989
2.108
2.161
2.833
2.948
2.808
2.858
2.984
2.745
2.807
2.824
154.35
111.50
155.79
177.45
177.30
164.64
142.12
163.55
164.03
148.91
2.946
2.930
Slippage angle (deg.)
0.859
Centroid–centroid (A)
˚
˚
Centroid-Plane (A)
3.513
3.508
CGB . . . CGBh
4.410
4.291
37.2
25.7
CGA . . . CGBh
CGA: centroid of cyclic group C3A, C4A, C5A, C6A, C7A and C8A.
CGB: centroid of cyclic group C3B, C4B, C5B, C6B, C7B and C8B.
a: ꢁx + 1, ꢁy + 1, ꢁz; b: x + 1, y + 1, z; c: ꢁx + 1, ꢁy + 1, ꢁz + 1; d: x, y, z ꢁ 1; e: ꢁx, ꢁy + 1, ꢁz; f: x, y + 1, z; h: ꢁx + 1, ꢁy + 1, ꢁz + 1; i: ꢁx + 2, ꢁy,
ꢁz + 1.
The complete assignment of the vibrational modes of Hats has similar to those observed in Hats, Hats.MeSO3H and Hats.HCl.
been studied by our group23. Based on these studies and using The NH deformations are observed at higher frequencies in the
appropriate literature35, it has been possible to assign the salts regarding Hats. These facts confirm the implication of the
vibrational modes of Hats salts. In this work, the assignment of amino groups in the formation of hydrogen bonds to stabilize
vibrational modes of Hats.tosylate has been done by comparison the crystal structure. It is also interesting to note that the
with the other two above mentioned salts and Hats. Figure 4 corresponding modes ꢇas SO2 groups and CN appear at higher
shows the FTIR spectra of Hats.tosylate and Table 3 proposes frequencies than Hats, suggesting that these links are strengthened
assignation of vibrational modes.
with respect to Hats (Table 3). This fact indicates that the S–O and
Analyzing the vibrational modes mentioned above, it can be C–N bonds are strengthened. The stretching N–N appears at
seen that NH2 stretching appears at lower frequencies and NH2 similar frequencies in all cases.
deformation at higher frequencies in the three salts with respect to
The result of mass spectra shows that the molecular ions are
Hats. These facts are consistent with the respective crystal not visible due to its relative abundance which is lower than 5%
structures, where these groups are interacting with the hydrogen and mainly due to the ionic nature of the salts. In the three salts,
bridge formation, stabilizing the crystal structure, weakening the the fragment of m/z 180 (compounds Hats.tosylate and
NH bond. Four modes corresponding to NH stretching are Hats.MeSO3H) and m/z 181 (V) has a 100% relative abundance.
observed. The values observed at 3319 and 3246 cmꢁ1 correspond This fragment corresponds to Hats and protonated Hats. In
to the asymmetric and symmetric stretching of the –NH2 group addition, compound Hats.tosylate appears to be a fragment of m/z
attached to the carbon of the thiadiazole ring, at higher 172 (55% relative abundance) due to p-toluenesulfonic acid. The
frequencies in Hats.tosylate with respect to Hats.MeSO3H and Hats.MeSO3H shows a fragment at m/z 96 (40% relative
Hats.HCl, and these three frequencies are less than Hats (Table 3). abundance) corresponding to methylsulfonic acid. The mass
The other two values observed at 3177 and 3067 cmꢁ1 are very spectra of three salts are presented in the Supplementary material.

DOI: 10.3109/14756366.2015.1096270
Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole
5
Figure 4. FTIR spectrum of Hats.tosylate.
Table 3. Proposed assignment of the FTIR spectrum and a comparison of selected.
Infrared
Assignment
(a) Proposed assignment of Hats.tosylate vibrational modes.
3319 (s)
3246 (s)
3177 (m)
ꢇas (C)NH2
ꢇs (C)NH2
ꢇas (S)NH2
3067 (m)
2984 (w)
2952 (w)
ꢇs (S)NH2 + ꢇ CH phenyl + ꢇ NH (ring)
ꢇ CH phenyl + ꢇ CH methyl
ꢇ CH methyl
1930–1912–1820–1802 (w)
1651 (s)
1596 (s)
Phenyl group disustituted
ꢈ(C)NH2
ꢈ(S)NH2
1551 y 1532 (m)
1495 (w), 1448 (w)
1383(s)
1241(s), 1208 (s)
1182(s)
1158(s)
ꢇ C–N
ꢇ C–C (ring) + ꢇ N–C
SO2 + ꢇas SO3H (IR); dip CH
ꢈ CH methyl
ꢇ
as
ꢇ N–N (ring)
ꢈ C–H
1141(s)
1122(s)
ꢇs SO2 + ꢇs SO3H
ꢈ CH methyl
1060 (m)
1032 (s)
1006 (s)
940 (m)
928 (m) y 813 (s)
798 (m)
710 (s)
685 (s)
643(m)
Rocking (S)NH2
ꢈ ring
ꢈ CH methyl
M S–N sulfonamide + wagging (S)NH2
D CH methyl
ꢇ C–S (ring) + d phenyl ring
d CH methyl + doop arC-H
ꢇ C–S + d phenyl ring
Ring torsion
622 (m) y 590 (s)
562 (s)
500 (s)
452 (m)
411(w)
401(w)
ꢇ C–S
Red modes
Wagging (C)NH2 + d SO2
Red modes
Rocking SO2
d SO2 + dip N-C (ring)
Assignment
Hats (cmꢁ1
)
Hats.tosylate (cmꢁ1
)
Hats.MeSO3H (cmꢁ1
)
Hats.HCl (cmꢁ1
)
(b) Comparison of selected vibrational modes for compounds Hats and its salts.
vas (C)NH2
vs (C)NH2
vas SO2
vs SO2
v NH2
v CN
v NN
3430
3325
1340
1140
1605
3320
3245
1385
1140
1650
3255
3240
1375
1145
1645
3280
3190
1375
1140
1640
1580–1500
1175
1595–1530
1180
1595–1525
1200
1585–1530
1185
ꢇ, stretching; ꢈ, deformation; ip., in plane; oop., out of plane; s, strong; m, medium; w, weak. All values are approximate
and rounded for ease in comparing them.

6
J. R. A. Diaz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Figure
Hats.tosylate.
5.
ATG-ATD
diagram
of
A very rich thermal behavior was observed in the studied
compounds. Hats.tosylate is stable up to 120 ꢀC. Starting at
120 ꢀC associated to an endothermic process, a change of the
crystalline phase occurred. This fact was observed in a heating
microscope stage. Compound Hats.MeSO3H too changed its
crystalline phase below 100 ꢀC. Compound Hats.HCl decomposed
at 120 ꢀC (onset 118 ꢀC) with loss of water of crystallization.
Immediately before decomposition, Hats.tosylate and
Hats.MeSO3H showed
[Hats.tosylate:
212 ꢀC
a
melting point near 200 ꢀC
(onset Hats.MeSO3H
203 ꢀC);
200 ꢀC (onset 196 ꢀC)]. Hats.HCl anhydrous melted at 194 ꢀC
(on set 187 ꢀC). Subsequently the three salts decomposed in many
endothermic and exothermic steps until complete calcinations
occur at 700 ꢀC (Hats.tosylate), 925 ꢀC (Hats.MeSO3H), and
600 ꢀC (Hats.HCl). Figure 5 shows thermal diagrams (TGA-DTA)
of Hats.tosylate. Thermal diagrams of compounds Hats.MeSO3H
and Hats.HCl are presented in the Supplementary material.
Figure 6. Diuresis of Hats salts (mL of urine versus time). From left to
right (each set of columns): control, Aza, furosemide, p-toluensulfonate
Hats, Hats methylsulfonate, Hats hydrochloride monohydrate.
comparisons used the Turkey test. Differences below a probability
level of 5% (p50.05) were considered statistically significant. To
carry out this study, we used the program GraphPad Prim
(GraphPad Software Inc., La Jolla, CA)40.
Biological assays
Diuretic effect
The diuretic effect is attributed to the ability of these salts to
inhibit the renal CA1,4. This effect is demonstrated by the
alkalinity of the urine excreted. However, it has been suggested
that the inhibition of sodium reabsorption in different parts of the
nephron also contribute to this mechanism of action. To verify this
effect, six batches were prepared with eight animals each. Batch 1
(negative control) received saline physiological solution; batch 2
(positive control) received acetazolamide (20 mg/kg); batch 3
(reference) received furosemide (Sigma Ch. Co., St. Louis, MO)
(10 mg/kg); batch 4 received compound III (70 mg/kg); batch 5
received compound IV (70 mg/kg), and batch 6 received com-
pound V (70 mg/kg). Urine fractions were collected each hour for
a period of 5 h after the administration of each drug. The urinary
excretion volume was calculated using the following relationship:
Diuresis has two components: an increment of the urine
volume (water excretion) and a net loss of solutes (electrolytes)31.
Figure 6 shows comparative diuretic effect of Hats salts with
respect to acetazolamide and furosemide. Hats.MeSO3H pos-
sesses a similar effect which Hats.HCl as to the excretion of
electrolytes in the urine. However, the removal of liquid is
important in the first 60 min of the test, subsequently removing
liquid is similar to the batch control. Hats.MeSO3H exhibited a
significant diuretic effect during the test. Diuresis was significant
for the first 75 min of the test with respect to the control.
Hats.tosylate also presented a diuretic effect, which was main-
tained throughout the test. Spectrophotometric data of urinary
ions concentration yielded the results as exhibited in Table 4.
Anticonvulsant activity
collected volume
UVE ¼
ꢂ 10
volume administered
In a first assay, physiological saline solution (negative control)
was administered intraperitoneally to eight mice per group,
whereas compounds Hats.tosylate, Hats.MeSO3H, Hats.HCl, and
AZA at doses of 20, 50, and 90 mg/kg of live animal, in volumes
where UVE is the urinary volume excreted.
The recorded values were expressed as means SEM. It
applies statistical analysis one-way ANOVA and subsequent

DOI: 10.3109/14756366.2015.1096270
Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole
7
Table 4. Biological assays.
(a) Concentration of urinary ions observed
Control (saline solution):
Reference (furosemide):
Hats.tosylate:
Hats.MeSO3H:
Hats.HCl:
Na+: 0.93 mg/L
Na+: 2.85 mg/L
Na+: 4.9 mg/L
Na+: 5.8 mg/L
Na+: 6.4 mg/L
K+: 3.18 mg/L
K+: 5.4 mg/L
K+: 6.8 mg/L
K+: 8.6 mg/L
K+: 8.3 mg/L
(b) Effect of Hats salts and acetazolamide on nikethamide and picrotoxine-induced convulsions in mice
% Protection
Treatments
Dose (mg/kg)
Picrotoxine
Nikethamide
Saline
Hats
Hats.tosylate
Hats.MeSO3H
Hats.HCl
AZA
–
0
72*
38
53
44
0
79*
58
75
56
90
90
90
90
90
82**
96**
(c) Effect of Hats salts on locomotor activity of mice (rota-rod test)
Treatment
Dose (mg/mL)
No. of falls in 15 min
Saline
—
10
90
90
90
1.2 0.8
5.5 0.4
2.0 0.5***
2.2 0.9***
2.6 0.5***
Phenobarbital
Hats.tosylate
Hats.MeSO3H
Hats.HCl
(d) Effect of Hats salts on locomotor activity of mice (Chimney assay)
Treatment
Dose (mM/kg)
Time delayed on climbing (min.)
Saline
–
6
8
6
7
6
Hats.tosylate
Hats.MeSO3H
Hats.HCl
90
90
90
10
5***
7***
4***
Phenobarbital
–
*p50.05; **p50.01 in comparison with saline control. ***p50.05 in comparison with phenobarbital lot.
Table 5. Inhibition of human (h) isoforms hCA I – XII with hats salts and
AZA as standard drug.
of 0.1 mL per 30 g mouse, were administered to other animals.
After 30 min of administration of the test compounds, niketha-
mide was injected by the same route at 0.1 mL (dose 20 mg/kg). It
was observed that a dose of 20 mg/kg of test compound is not
fully effective for convulsions protection; convulsions were
observed in one or two mice per group. However, at higher
doses (50 and 90 mg/kg of test compound), the convulsions
protection was effective, since none of the mice suffered
convulsions.
KI(nM)
hCA
I
hCA
II
hCA
IV
hCA
VII
hCA
IX
hCA
XII
Compound
Hats.tosylate
Hats.MeSO3H
Hats.HCl
3400
8300
932
320
327
365
12
2740
3630
2270
74
1980
4225
623
255
570
307
25
570
570
421
AZA
250
2.5
5.70
In a second assay, a dose of compound of 90 mg/100 g of
animal weigh was given in a volume of 0.1 mL per 10 g mouse,
intraperitoneally (using eight mice per group). After 30 min of
administration, test compounds were injected by the same route of used. For the negative control group (saline solution), one fall
picrotoxine 0.1 mL per 30 g mouse (20 mg/100 g body weight). (mean) had been recorded during the first 15 min. In the positive
In the control batch, we observed that in animals treated with control group, injected with phenobarbital (10 mg/kg), five falls
picrotoxine the convulsions appeared after 4 min post-adminis- were observed. In the third group previously injected with
tration. The animals injected with compounds Hats.tosylate and compound Hats.tosylate (90 mg/kg), two falls were observed
Hats.MeSO3H were protected from convulsions during 20–25 min during the first 15 min. In the fourth group, injected with
post-administration, whereas after longer periods of time, clonic compound Hats.MeSO3H (90 mg/kg), two falls were observed
convulsions appeared.
during the first 15 min. Finally, in the fifth group, injected with
The animals injected with compound Hats.HCl were pro- compound Hats.HCl (90 mg/kg), two falls during the first 15 min
tected from convulsions during the 20 min post-administration. were observed. The results showed no adverse effects on motor
Tonic convulsions appear thereafter, but the animals survived. coordination with the tested drugs compared with control animals
Acetazolamide showed a weaker protective effect than Hats salts (p40.05, one-way ANOVA and subsequent Turkey test) at the
since convulsions occurred within 15 min post-administration doses here assayed. Table 4 shows these results.
(Table 4).
Effect of Hats salts on locomotor activity of mice.
Chimney test
Neurotoxicity (rota-rod test)
The rota-rod assay has been used to examine in vivo neurotoxicity Five groups of eight animals each were employed. Animals used
of the sulfonamide salts. Five groups of eight animals each were in the first group (saline solution, negative control) showed a

8
J. R. A. Diaz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Table 6. Inhibition of PgiCA from porphyromona sgingivalis; and Can2,
from cryptococcus neoformans with hats salts investigated in this paper.
niketamide and picrotoxin. The sulfonamide salts were most
effective as anticonvulsants in the niketamide than in the
picrotoxin-induced convulsions assay. These facts suggest that
they are effective to protect against mild convulsions. The tested
compounds showed no effects of neurotoxicity in the full range of
effective doses in the study by the rota-rod test and chimney test.
Hats salts inhibited human, bacterial, and fungal CAs. These facts
led us to study more deeply these compounds as potential
antiinfective agents. This work stimulates further study for their
possible pharmacological applications.
KI (mM)
Compound
Can2
PgiCAbeta
PgiCAgamma
Hats.tosylate
Hats.MeSO3H
Hats.HCl
7.82
8.62
5.24
3.60
5.87
4.16
0.915
2.01
1.95
delay of 6 s to pass through the glass tube. The group injected with
phenobarbital (positive control) did not perform the test within the
considered time. The group intraperitoneally injected with
compound Hats.tosylate (0.1 mL/15 g mouse) led to a delay of
8 s. The group intraperitoneally injected with compound
Hats.MeSO3H (0.1 mL/15 g mouse) led to a delay of 6 s. The
group intraperitoneally injected the compound Hats.HCl (0.1 mL/
15 g mouse) led to a delay of 7 s. The results show that there is no
Acknowledgements
The authors thank Dr. C. Ardanaz (UNSL) for mass spectra facilities.
They also thank S.C.S.I.E. (X-ray section) of University of Valencia for
provision of the X-ray crystallographic facilities.
Declaration of interest
sedation or ataxia drug tested, compared with those animals of This work is supported by CONICET-PIP 6246 and UNSL. J. C. P. and D.
R. V. are members of CONICET.
the control batch (p40.05, one-way ANOVA and subsequent
Turkey test).
CA inhibition
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phenols and thiophenols, the enzymes were incubated for 10–
15 min with test compounds for allowing the formation of the
enzyme-inhibitor adduct41–45,49,50. Working under similar condi-
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drug allows the stabilization of the adduct through additional
interactions in which Hats cannot participate. This is consistent
with the X-ray crystal structures, in which the deprotonated
sulfonamido group appears bound to the Zn(II) ion from the
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Supplementary material available online
Supplementary Figure S1–S7