A novel proton transfer salt of 2-amino-6-sulfamoylbenzothiazole and its metal complexes: the evaluation of their inhibition effects on human cytosolic carbonic anhydrases

Article abstract of DOI:10.1080/14756366.2016.1247058

A novel proton transfer compound (SMHABT)⁺(HDPC)^? (1) obtained from 2-amino-6-sulfamoylbenzothiazole (SMABT) and 2,6-pyridinedicarboxylic acid (H₂DPC) and its Fe(III), Co(II), Ni(II) complexes (2–4), and Fe(II) complex of

Full text of DOI:10.1080/14756366.2016.1247058

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
A novel proton transfer salt of 2-amino-6-
sulfamoylbenzothiazole and its metal complexes:
the evaluation of their inhibition effects on human
cytosolic carbonic anhydrases
Zeynep Alkan Alkaya, Halil İlkimen, Cengiz Yenikaya, Yasemin Kaygısız,
Metin Bülbül, Tuncay Tunç & Musa Sarı
To cite this article: Zeynep Alkan Alkaya, Halil İlkimen, Cengiz Yenikaya, Yasemin Kaygısız,
Metin Bülbül, Tuncay Tunç & Musa Sarı (2017) A novel proton transfer salt of 2-amino-6-
sulfamoylbenzothiazole and its metal complexes: the evaluation of their inhibition effects on human
cytosolic carbonic anhydrases, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1,
231-239, DOI: 10.1080/14756366.2016.1247058
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1247058
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Group
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Date: 28 April 2017, At: 03:50

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 231–239
http://dx.doi.org/10.1080/14756366.2016.1247058
RESEARCH ARTICLE
A novel proton transfer salt of 2-amino-6-sulfamoylbenzothiazole and its metal
complexes: the evaluation of their inhibition effects on human cytosolic carbonic
anhydrases
a
b
b
c
d
€ € c
_
Zeynep Alkan Alkaya , Halil Ilkimen , Cengiz Yenikaya , Yasemin Kaygısız , Metin Bulbul , Tuncay Tunc¸ and
Musa Sarıe
b
aDepartment of Chemistry and Chemical Processing Technologies, Banaz Vocational School, Usak University, Us¸ak, Turkey; Department of
c
€
Chemistry, Faculty of Arts and Sciences, Dumlupinar University, Kutahya, Turkey; Department of Biochemistry, Faculty of Arts and Sciences,
d
€
Dumlupinar University, Kutahya Turkey; Department of Science Education, Faculty of Education, Aksaray University, Aksaray, Turkey;
eDepartment of Physics Education, Gazi University, Ankara, Turkey
ABSTRACT
ARTICLE HISTORY
A novel proton transfer compound (SMHABT)þ(HDPC)ꢀ (1) obtained from 2-amino-6-sulfamoylbenzothia-
zole (SMABT) and 2,6-pyridinedicarboxylic acid (H2DPC) and its Fe(III), Co(II), Ni(II) complexes (2–4), and
Fe(II) complex of SMABT (5) have been prepared and characterized by spectroscopic techniques.
Additionally, single crystal X-ray diffraction techniques were applied to complexes (2–4). All complexes
(2–4) have distorted octahedral conformations and the structure of 5 might be proposed as octahedral
according to spectral and analytical results. All compounds, including acetazolamide (AAZ) as the control
compound, were also evaluated for their in vitro inhibition effects on human hCA I and hCA II for their
hydratase and esterase activities. The synthesized compounds have remarkable inhibitory activities on hCA
I and hCA II. Especially, the inhibition potentials of the salt and the metal complexes (1–5) are comparable
with AAZ. Inhibition data have been analyzed by using a one-way analysis of variance for multiple compar-
isons (p < .0001).
Received 27 May 2016
Revised 19 September 2016
Accepted 20 September 2016
KEYWORDS
Carbonic anhydrase; metal
complex; proton transfer;
statistical analyses; 2-amino-
6-sulfamoylbenzothiazole;
2,6-pyridinedicarboxylic acid
Introduction
resulting from incompletion of the cycle and accumulating of the
liquid in the posterior chamber, and causing intraocular pressure
(IOP) elevation [16]. Glaucoma is a group of diseases characterized
by the gradual loss of visual field due to an elevation in intraocu-
lar pressure (IOP) [15,17,18] CA inhibitors (CAIs) decrease IOP by
reducing the rate of bicarbonate formation and thus the secretion
of the aqueous humor from ciliary body [18,19]. Some systemic
and topical CAIs used clinically have effective inhibition effects on
the enzyme, but they have also many side effects, such as weight
loss, fatigue, blurred vision, depression, metallic taste, and burning
of the eye [15,18,20,21]. This situation clearly demonstrates the
need for the development of new carbonic anhydrase inhibitors.
In the last years, 2-aminobenzothiazole derivatives and their
proton transfer salts and metal complexes have been studied as
carbonic anhydrase inhibitors by our group [22–25].
In this study, 2-amino-6-sulfamoylbenzothiazole (SMABT)
according to literature [26] and proton transfer compound
(SMHABT)þ(HDPC)ꢀ (1) and its Fe(III), Co(II), and Ni(II) complexes
(2–4) and Fe(II) complex of SMABT (5) have been prepared and
characterized by elemental, spectral (1H-NMR, 13C-NMR, IR and UV-
Vis), and thermal analyses, as well as using magnetic measurement
and molar conductivity techniques. Single crystal X-ray diffraction
techniques were applied to complexes 2–4. In order to compare
the inhibition data of newly synthesized compounds, 1–4, the sim-
ple metal complexes of H2DPC [27] and Fe(II) complexes of SMABT
The sulfonamide and benzothiazole derivatives constitute an
important class of drugs, with several types of pharmacological
agents possessing, among others, antibacterial, anticonvulsant,
antiinflammatory, antitubercular, therapeutic, protease, and car-
bonic anhydrase inhibitory effects [1,2]. Carbonic anhydrase (CA,
EC 4.2.1.1) is a metalloenzyme containing Zn2þ ion in its active
site to catalyze the simple but physiologically important reversible
reaction of carbon dioxide to bicarbonate ion and proton [3–6].
This reaction also provides the equilibrium of some ions, such as
sodium ion, calcium ion, and chloride ion [7,8]. Therefore, carbonic
anhydrases take part in many physiological processes [9–11].
These isozymes are found in many organisms throughout the
phylogenetic tree [12]. Sixteen of these isozymes are expressed in
humans, and 13 of them are catalytically active [13,14]. Among
the human CAs, hCA I, and hCA II are cytosolic, and hCA II is
located in human eye, erythrocytes, gastrointestinal tract, and kid-
ney [15]. Bicarbonate ion, which is produced by carbonic anhy-
drases, has an important role in the formation of aqueous humor.
This fluid is produced in the eye’s posterior chamber and is trans-
ferred into anterior chamber through the space between lens and
iris. Later, aqueous humor moves through trabecular meshwork
and Schlemm’s canal. Thus, the cycle is completed, and the intra-
ocular pressure is kept in balance. But some structural defects,
such as blockage in trabecular meshwork and Schlemm’s canal, or
the smoother of the iris, prevent the flow of aqueous humor (5) have been prepared. Furthermore, we have investigated
€
Department of Chemistry, Dumlupinar University, 43100, Kutahya, Turkey
CONTACT Cengiz Yenikaya
cengiz.yenikaya@dpu.edu.tr
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

232
Z. A. ALKAYA ET AL.
the potential use of these compounds as new inhibitors of hCA H, 3.43%; N, 11.93%; S, 13.65%; Ni, 6.25%. Found: C, 35.80%; H,
isoenzymes in the treatment of glaucoma.
3.40%; N, 11.90%; S, 13.62%; Ni, 6.20%; for 5 (C21H29N9O11S6Fe): C,
30.32%; H, 3.51%; N, 15.16%; S, 23.13%; Fe, 9.71%. Found: C,
30.30%; H, 3.56%; N, 15.20%; S, 23.10%; Fe, 9.65%.
Experimental section
General methods and materials
X-ray data collection and structure refinement
All chemicals used were analytical reagents and were commercially
purchased from Aldrich. Elemental analyses for C, H, N, and S were
performed on Elementar Vario III EL (Hanau, Germany) and Fe, Co,
and Ni were detected with Perkin Elmer Optima 4300 DV ICP-OES
(Perkin Elmer Inc., Wellesley, MA). 1H and 13C-NMR spectra were
recorded with Bruker DPX FT NMR (500 MHz) spectrometer
(Karlsruhe, Germany) (SiMe4 as internal standard and 85% H3PO4 as
an external standard). FT-IR spectra were recorded in the
4000–400 cmꢀ1 region with Bruker Optics, Vertex 70 FT-IR spec-
trometer using ATR techniques (Ettlingen, Germany). Thermal analy-
ses were performed on Pelkin Elmer SII Exstar 6000 TG/DTA 6300
model (SII Nanotechnology, Japan) using platinum crucible with 10-
mg sample. Measurements were taken in the static air within a
30–900 ꢁC temperature range. The UV–Vis spectra were obtained
for DMSO solution of the compounds (10ꢀ3 M) with a SHIMADZU
UV-2550 spectrometer (Shimadzu Co, Kyoto, Japan) in the range of
200–900 nm. Magnetic susceptibility measurements at room tem-
perature were performed using a Sherwood Scientific Magway MSB
MK1 model magnetic balance (Sherwood Scientific Ltd, Cambridge,
UK) by the Gouy method using Hg[Co(SCN)4] as calibrant. The molar
conductance of the compounds were determined in water/ethanol
(1:1) and in DMSO (10ꢀ3 M) at room temperature using a WTW
Cond 315i/SET Model conductivity meter (Weilheim, Germany).
The crystal and instrumental parameters used in the unit-cell
determination, and data collection are summarized in Table 1 for
the compounds 2–4. Crystallographic data of 2–4 were recorded
on a Bruker Kappa APEX II CCD area-detector X-ray diffractometer
employing plane graphite monochromatized with MoKa radiation
(k ¼ 0.71073 Å), using x-2h scan mode. The empirical absorption
corrections were applied by multi-scan via Bruker, SADABS soft-
ware [28]. The structures were solved by the direct methods and
subsequently completed by difference Fourier recycling. All non-
hydrogen atoms were refined anisotropically using the full-matrix
least-squares techniques on F2. The SHELXS-97 and SHELXL-97 [29]
programs were used for all the calculations. The H atoms were
placed in idealized positions and constrained to ride on their par-
ent atoms with distances in the range of NꢀH ¼ 0.77(3)–1.03(8) Å,
CꢀH ¼ 0.93 Å and with Uiso(H) ¼ 1.2Ueq(C,N). Hydrogen atoms of
water molecules were located from difference Fourier maps and
refined with isotropic thermal parameters with a distance of
OꢀH ¼ 0.72(7)–1.10(11) Å and with Uiso(H) ¼ 1.2Ueq(O). The draw-
ings of molecules were accomplished with the help of ORTEP-3 for
Windows [30].
Purification of carbonic anhydrase I and II isoenzymes from
human erythrocytes
Erythrocytes were purified from human blood. The blood samples
were centrifuged at 1500 rpm for 20 min, and plasma was
removed. Later, red cells were washed with isotonic solution (0.9%
NaCl), and the erythrocytes were hemolyzed with 1.5 volumes of
ice-cold water. Cell membranes were removed by centrifugation at
4 ꢁC, 20000 rpm for 30 min. The pH of hemolysate was adjusted to
8.7 with solid TRIS (tris(hydroxymethyl)aminomethane). The
Synthesis of 1 and metal complexes (2–5)
SMABT as the starting compound was synthesized from sulfanila-
mide (SA) and 4-thioureidobenzenesulfonamide (TBS) according to
literature [27]. 1H NMR and 13C NMR of the compound are given
in Supplementary Table S1 and other spectroscopic data can be
found in Supplementary Tables S2 and S4 and Supplementary
Figures S1 and S2.
R
hemolysate was applied to affinity column (SepharoseV4B-L-tyro-
A solution of SMABT (1.145 g, 5 mmol) in 25 mL ethanol was
added to the solution of H2DPC (0.836 g, 5 mmol) in 25 mL etha-
nol. The mixture was refluxed for 3 h and then was cooled to
room temperature. The reaction mixture was kept at room tem-
perature for 3 h to give white solid of 1 (1.783 g, 90% yield).
A solution of 1 mmol metal(II) salt [0.278 g FeSO4.7H2O or
0.249 g Co(CH3COO)2.4H2O or 0.248 g Ni(CH3COO)2.4H2O] in water
(10 mL) was added dropwise to the solution of 1 (0.519 g, 1 mmol)
for 2–4 or was added dropwise to the solution of SMABT (0.229 g,
1 mmol) for 5 in water/ethanol (1:1) (20 mL) with stirring at room
temperature for two hours to complete the reaction. On filtration
the reaction mixture, the solution was kept at room temperature
for 2 weeks to give yellow crystalline solid for 2 (0.275 g, %80
yield) or brown crystalline solid for 3 (0.399 g, %85 yield) or green
crystalline solid for 4 (0.329 g, %70 yield) or orange amorphous
solid for 5 (0.285 g, 62% yield) (Figure 1). The single crystals of
complexes 2–4 suitable for X-ray diffraction were separated and
washed with EtOH/water (1:1).
sine-p-aminobenzene sulfonamide) pre-equilibrated with 25.0 mM
TRIS-HCl/0.1 M Na2SO4 (pH 8.7). After extensive washing with a
solution of 25.0 mM TRIS-HCl/22.0 mM Na2SO4 (pH 8.7), the hCA I
and hCA II isoenzymes were eluted with the solution of 1.0 M
NaCl/25.0 mM Na2HPO4 (pH 6.3) and 0.1 M NaCH3COO/0.5 M
NaClO4 (pH 5.6), respectively [31]. For quantitative protein deter-
mination, the Bradford method was used with bovine serum albu-
min as standard [32]. Also, the purity control of the isoenzymes
was performed with SDS-PAGE after the purification [33].
Determination of hydratase and esterase activities of hCA I
and hCA II
The CO2 hydratase activity of the enzyme was determined at 0 ꢁC
in a veronal buffer (pH 8.15) with the pH-stat method as the indi-
cator and saturated carbon dioxide solution as the substrate in a
final volume of 4.2 mL. The time (in seconds) taken for the solution
to change from pH 8.15 to pH 6.50 was measured. The enzyme
unit (EU) is the enzyme amount that reduces the non-enzymatic
reaction time by 50%. The activity of an enzyme unit was calcu-
lated by using the equation ((t0ꢀtc)/tc), where t0 and tc are times
for pH change of the non-enzymatic and enzymatic reactions,
respectively [34].
Anal. Calcd. for 1 (C14H12N4O6S2): C, 42.42%; H, 3.05%; N,
14.13%; S, 16.18%. Found: C, 42.45%; H, 3.00%; N, 14.15%; S,
16.15%; for 2 (C21H22N5O14S2Fe): C, 36.64%; H, 3.22%; N, 10.17%; S,
9.32%; Fe, 8.11%. Found: C, 36.65%; H, 3.20%; N, 10.20%; S, 9.30%;
Fe, 8.10%; for 3 (C28H32N8O17S2Co): C, 35.78%; H, 3.43%; N,
11.92%; S, 13.65%; Co, 6.27%. Found: C, 35.75%; H, 3.40%; N,
Esterase activity was examined by following the change in the
11.95%; S, 13.67%; Co, 6.25%.; for 4 (C28H32N8O17S2Ni): C, 35.79%; absorbance at 348 nm of 4-nitrophenylacetate to 4-nitrophenolate

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
233
NH2
(a)
NH2
NH2
S
NH
S
N
Br2
KSCN
SO2NH2
H2NO2S
SMABT
SO2NH2
SA
TBS
O
(b)
NH2
O
NH2
HO
HO
O
S
N
S
NH
N
HN
O
H2NO2S
O
H2NO2S
O
SMABT
H2DPC
1
(c)
O
O
NH2
O
NH2
O
O
O
S
NH
S
NH
X H2O
Metal (II) Salt
N
N
M
HN
O
O
O
H2NO2S
O
O
H2NO2S
O
n
M = Fe(III), n = 1, X = 4
M = Co(II), n = 2, X = 5
M = Ni(II), n = 2, X = 5
(2),
(3),
(4).
1
(d)
H2NO2S
S
FeSO4.7H2O
NH2
[Fe(SMABT)3(OH)2(H2O)].2H2O
N
SMABT
Figure 1. Syntheses of all compounds (a for SMABT and b for 1 and c for 2–4 and d for 5).
5
ion over a period of 3 min at 25 ꢁC using a spectrophotometer and esterase activities of CA isoenzymes were examined in the
presence of various inhibitor concentrations. Regression analysis
graphs were drawn by plotting the percent enzyme activity versus
inhibitor concentration and IC50 values were calculated [17].
To determine the Ki values as well as the inhibition type, three
different inhibitor concentrations giving 30%, 50%, and 70% inhib-
ition were selected. For each of these inhibitor concentrations,
enzyme activity was measured in the presence of various substrate
concentrations (0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, and 0.7 mM),
and the data were linearized with Lineweaver–Burk plot for Vmax
and the Ki determination. Enzyme activity was also measured in
(SHIMADZU UV 1700 PharmaSpec) according to the method
described in the literature [35]. The enzymatic reaction, in a total
volume of 3.0 mL, contained 1.4 mL of 0.05 M TRIS–SO4 buffer (pH
7.4), 1.0 mL of 3.0 mM 4-nitrophenylacetate, 0.5 mL H2O, and
0.1 mL enzyme solution. A reference measurement was obtained
by preparing the same cuvette without enzyme solution.
Determination of IC50 and Ki values of the compounds
To determine the IC50 values (the concentration of inhibitor produc- the presence of the same substrate concentrations but in the
absence of any inhibitor to determine the Vmax (Supplementary
Figure S3) [17].
ing a 50% inhibition of CA activity) of the simple ligands, proton
transfer salt, metal complexes, and acetazolamide (AAZ), hydratase,

234
Z. A. ALKAYA ET AL.
Table 1. Crystal data and structure refinement details for compounds 2–4.
2
3
4
Empirical formula
Formula weight
T[K]
C21H22N5O14S2Fe
688.41
C28H32N8O17S4Co
939.79
C28H32N8O17S4Ni
939.55
296(2)
296(2)
296(2)
Wavelength (Å)
Crystal system, space group
Unit cell dimensions (Å,
0.71073
Monoclinic, P21/c
0.71073
Triclinic, P1
0.71073
Triclinic, P1
ꢀ
ꢀ
ꢁ
)
a
b
c
a
b
c
13.287(3)
28.417(6)
7.5289(15)
9.2195(3)
14.4582(4)
14.6132(4)
98.6100(10)
106.4460(10)
91.2240(10)
1842.97(9)
2
9.118(4)
14.428(5)
14.614(5)
98.902(19)
106.27(2)
91.19(2)
1819.1(11)
2
0.852
102.199(4)
V (Å3)
Z
2778.6(10)
4
0.772
Absorbtion coefficient (mmꢀ1
)
0.780
1.694
Dcalc (mg/m3)
1.646
1.715
F(000)
1412
966
968
Crystal dimensions (mm)
0.32 ꢂ 0.11 ꢂ 0.064
1.43–28.83
ꢀ18ꢃ h ꢃ15
ꢀ38ꢃ k ꢃ30
ꢀ10ꢃ l ꢃ4
7755
0.70 ꢂ 0.29 ꢂ 0.29
1.43–28.56
ꢀ12ꢃ h ꢃ12
ꢀ19ꢃ k ꢃ19
ꢀ19ꢃ l ꢃ19
40682
0.36 ꢂ 0.22 ꢂ 0.18
1.43–28.46
ꢀ12ꢃ h ꢃ12
ꢀ19ꢃ k ꢃ19
ꢀ19ꢃ l ꢃ19
36415
h range for data collection (ꢁ)
Index ranges
Reflections collected
Independent reflections
Data/parameters
Max. and min. transmission
Final R indices [I ꢄ 2r(I)]
7265
9410
7932/579
9189
5418/428
0.955, 0.903
R1 ¼ 0.0583
wR2 ¼ 0.1107
R1 ¼ 0.1251
wR2 ¼ 0.179
1.203
6014/579
0.858, 0.799
R1 ¼ 0.0606
wR2 ¼ 0.160
R1 ¼ 0.1022
wR2 ¼ 0.212
0.997
0.798, 0.762
R1 ¼ 0.0374
wR2 ¼ 0.1052
R1 ¼ 0.0448
wR2 ¼ 0.1111
1.077
R indices (all data)
Goodness-of-fit on F2
Largest difference in peak and hole (e Åꢀ3
)
0.713, ꢀ0.544
0.539, ꢀ0.402
0.633, ꢀ0.568
Statistical analysis
closer to 180ꢁ [168.6(2)ꢁ, 178.57(6), and 178.67(12)ꢁ, respectively].
The dihedral angles for all complexes defined by the mean planes
of two DPC2ꢀ ligands are 105.02(18)ꢁ for 2, 103.47(6)ꢁ for 3, and
101.98(11)ꢁ for 4, showing that they fall almost perpendicular. The
M-N and M-O bond distances lie within expected range of
1.970(3)–2.086(5) Å and 2.004(5)–2.1992(15) Å, respectively (Table
2). In all essential details, the geometries of the molecules regard-
ing bond lengths and angles of the compounds are in good
agreement with the values observed in similar Fe(III) [22,23,25],
Co(II) [22,23], and Ni(II) [22,24,25] complexes.
All the presented data were confirmed by at least three independ-
ent experiments and are expressed as the mean SD. Data were
analyzed by using a one-way analysis of variance for multiple com-
parisons (SPSS 13.0, SPSS Inc., Chicago, IL). p < .0001 was consid-
ered to be statistically significant.
Results and discussion
Crystal structures of 2–4
The molecular structures of 2–4, with the atom labeling of sym-
metric units, are shown in Figures 2–4. The details of the crystal
structure solutions are summarized in Table 1, and the selected
bond lengths and angles are listed in Table 2.
The complex 2, (SMHABT)[Fe(DPC)2].4H2O, crystallizes in the
monoclinic P21/c space group. The structure of 2 consists of one
SMHABTþ cation, one [Fe(DPC)2]– anion, and four uncoordinated
1H and 13C NMR studies of 1
The 1H-NMR and 13C-NMR spectra of the compound 1 were
obtained in d6-DMSO with and without D2O at room temperature
using TMS as internal Standard (Supplementary Figures S4 and
S5). The 1H signals were assigned on the basis of chemical shifts,
multiplicities, intensities of the signals, and coupling constants.
Table 4 lists complete 1H-NMR and 13C-NMR spectra of the com-
pound 1.
water
molecules.
All
the
other
complexes,
(SMHABT)2[M(DPC)2].5H2O [M ¼ Co(II) for 3 and Ni(II) for 4], crystal-
In 1H NMR spectrum of 1 (Supplementary Figure S4a, Table 4),
ꢀ
lize in the triclinic P1 space group. The structures of 3 and 4 con-
sist of one [M(DPC)2]2ꢀ anion and two counter SMHABTþ cations the H4, H5, and H7 protons of the ring of (SMHABT)þ are doublet,
and five uncoordinated water molecules.
doublet–doublet, and doublet, respectively, with 1H intensity and
3
they are observed at 7.44 ppm (H4, JH4-H5 ¼8.46 Hz), 7.67 ppm
In complexes 2–4, the metal ion coordinates to four oxygen
atoms and two nitrogen atoms of two 2,6-pyridinedicarboxylate
ions resulted with a distorted octahedral conformation. Both carb-
oxylate oxygen atoms from DPC2ꢀ occupy the trans-apical posi-
(H5, 3JH5-H4 ¼8.46 Hz, 4JH5-H7 ¼1.96 Hz), and 8.12 ppm (H7,
4JH7-H5 ¼1.87 Hz). H16 proton of the (HDPC)- ring is doublet and
another doublet with 1H intensity and observed at 8.18 ppm
tions of the metal, and define low trans-angle value around metal (3JH16-H15
¼ 7.70 Hz and JH16-H15’
¼7.69 Hz). Although
3
or H15’
or H15
ion for all complexes within the range of 150.062(18)ꢁ–155.16(6)ꢁ the H16 seems to be symmetrical and is expected to give triplet,
for O1-M-O2 or O5-M-O6 which reveal a rather rigid structure of the tautomeric of H13 between O18 and O18’, H16 might be unsym-
such tri-dentate ligands [M ¼ Fe(III) for 2, Co(II) for 3 and Ni(II) for metrical and might give doublet with H15 and another doublet
4] (Table 2). In structures of 2–4, the N1-M-N2 trans-angles are with H15’ [36]. H15 and H15’ protons are observed at 8.25 ppm as

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
235
Figure 2. An ORTEP drawing of asymmetric unit of 2 with the atom-numbering scheme. Displacement ellipsoids are drawn at the 40% probability level.
Figure 3. An ORTEP drawing of asymmetric unit of 3 with the atom-numbering scheme. Displacement ellipsoids are drawn at the 40% probability level.
4
doublet–doublet with 2H intensity (3JH15-H16 ¼7.67 Hz, JH15- singlet with 2H intensity, and they are observed at 7.88 ppm and
7.20 ppm, respectively. These protons (H3, H11, H12 and H13) were
not observed in the 1H NMR spectrum obtained in d6-DMSO with
D2O due to deuterium exchange (Supplementary Figure S4b).
3
4
¼ 1.14 Hz or JH15’-H16 ¼7.67 Hz, JH15’-H15 ¼1.14 Hz) [36]. The
H15’
hydrogen atoms (H3 and H13) in compound 1 were not observed
in the 1H NMR spectrum. The H11 and H12 protons of NH2 are

236
Z. A. ALKAYA ET AL.
Figure 4. An ORTEP drawing of asymmetric unit of 4 with the atom-numbering scheme. Displacement ellipsoids are drawn at the 40% probability level.
Table 2. Selected bond distances [Å] and angles [ꢁ] of compounds 2–4.
Compound 2: (SMHABT)[Fe(DPC)2].4H2O
Table 3. Hydrogen bonds for compounds 2–4 (Å, ꢁ).
D-HꢅꢅꢅA
2
d(D-H)
d(HꢅꢅꢅA)
d(DꢅꢅꢅA)
<D-HꢅꢅꢅA
Fe1-N1
Fe1-N2
O1-Fe1-O2 150.62(18)
O1-Fe1-O5 90.02(19)
O1-Fe1-O6 95.6(2)
N1-Fe1-N2 168.6(2)
2.086(5)
2.068(5)
Fe1-O1
Fe1-O2
O1-Fe1-N2 115.50(19)
O5-Fe1-O2 94.58(19)
O6-Fe1-O2 94.14(19)
O1-Fe1-N1 75.76(18)
2.034(5)
2.059(4)
Fe1-O5
Fe1-O6
O2-Fe1-N1 74.96(17)
O2-Fe1-N2 93.71(17)
O5-Fe1-N1 103.62(18)
O5-Fe1-N2 75.68(18)
2.023(4)
2.004(5)
N3-H3B-O4W
0.86
2.08
2.899(12)
3.129(8)
3.093(9)
2.919(9)
158.1
O1W-H14-O1
O1W-H14-O4
O3W-H32-O2
1.00(16)
1.00(16)
0.72(9)
2.17(16)
2.28(15)
2.21(10)
161(12)
138(12)
166(10)
3
4
Compound 3: (SMHABT)2[Co(DPC)2].5H2O
N4-H4’-O6
O2w-H2A-O4
0.86
0.77(4)
1.86
2.60(4)
2.667(2)
3.260(4)
155.1
146(3)
Co1-N1
2.0239(14) Co1-O1
2.1992(15) Co1-O5
2.1148(14) Co1-O6
2.1251(14)
2.1809(14)
Co1-N2
2.0277(14) Co1-O2
N1-Co1-N2 178.57(6)
N1-Co1-O1 74.81(5)
N1-Co1-O2 77.21(6)
N1-Co1-O5 103.47(6)
N1-Co1-O6 104.45(5)
N2-Co1-O1 103.82(5)
O1-Co1-O5 92.53(6)
N3-H31-O2W
N3-H32-O1W
N4-H4-O6
0.86
0.86
0.86
0.831(10)
2.03
2.02
1.89
2.50(3)
2.830(5)
2.811(6)
2.627(4)
3.238(6)
155.4
152.0
142.2
149(5)
N2-Co1-O2 104.12(6)
N2-Co1-O5 76.92(6)
N2-Co1-O6 75.10(5)
O1-Co1-O2 151.64(5)
O1-Co1-O6 92.11(6)
O2-Co1-O5 98.43(6)
O2-Co1-O6 90.37(6)
O5-Co1-O6 151.94(5)
O2W-H2B-O7
Compound 4: (SMHABT)2[Ni(DPC)2].5H2O
Ni1-N1
1.973(3)
Ni1-O1
2.151(3)
2.089(3)
Ni1-O5
Ni1-O6
2.094(3)
2.161(3)
FT-IR measurements
Ni1-N2
1.970(3)
Ni1-O2
N2-Ni1-N1 178.67(12)
N2-Ni1-O2 101.98(11)
N1-Ni1-O2 79.04(11)
N2-Ni1-O5 78.59(11)
N1-Ni1-O5 102.20(11)
O2-Ni1-O5 95.30(11)
N2-Ni1-O1 103.13(11)
N1-Ni1-O1 75.82(11)
O2-Ni1-O1 154.83(10)
O5-Ni1-O1 91.26(11)
N2-Ni1-O6 76.67(11)
N1-Ni1-O6 102.49(11)
O2-Ni1-O6 91.95(11)
O5-Ni1-O6 155.16(10)
O1-Ni1-O6 92.19(11)
The infrared spectral data of the starting compounds (SMABT and
H2DPC) and compounds 1–5 are given in Supplementary Table S2.
In the high frequency region, weak bands 3067–3098 cmꢀ1 are
attributed to the stretching vibrations of aromatic C-H for all com-
pounds. There are also broad absorption bands at
3502–3529 cmꢀ1, which are attributed to the ꢀ(OH) vibrations of
water molecules for compounds 2–5. The relatively weak and
broad bands at 2508–2737 cmꢀ1 are attributed to the ꢀ(Nþ-H)
vibration for 1–4 [37]. The absorption bands at 3423, 3324, 3293,
and 3237 cmꢀ1 of NH2 group of SMABT are slightly shifted from
those found for 1 (3433, 3424, 3385, and 3229 cmꢀ1), for 2 (3398,
Intermolecular in the complexes, (2–4) hydrogen bonds play important roles in
stabilizing the crystal structures. The ranges of the D-H … A angles and those of
the H … A and D … A distances indicate the presence of strong and weak hydro-
gen bondings in the structures 2–4 (Table 3).
13C-NMR spectrum of 1 exhibits 13 resonances (Table 4,
Supplementary Figure S5). Seven peaks of this spectrum [148 ppm
(C2), 119 ppm (C4), 132 ppm (C5), 156 ppm (C6), 124 ppm (C7),
137 ppm (C8), and 140 ppm (C9)] could be assigned to the carbons
of (SMHABT)þ moiety of compound 1. Four peaks at 167 ppm
(C14), 128 ppm (C15), 118 ppm (C16), and 170 ppm (C17) could be
assigned to the carbons of (HDPC)ꢀ ring moiety.
3355, 3337, and 3326 cmꢀ1), for
3 (3369, 3341, 3327, and
3297 cmꢀ1), for 4 (3425, 3360, 3337, and 3235 cmꢀ1), and for 5
(3422, 3319, 3297, and 3235 cmꢀ1) due to the weak intermolecular
interactions. The carboxylate groups for compounds H2DPC and
1–4 exhibit strong carbonyl bands in the region of
1705–1456 cmꢀ1. These bands are reflected by IR spectrum of the
The room temperature NMR spectra for compound 1 indi-
cate clearly the formation of the proton transfer compound asymmetric (ꢀas) and symmetric (ꢀs) stretching vibrations at 1701
with 1:1 ratio of SMABT and H2DPC (Supplementary Figures S4 and 1456 cmꢀ1 for H2DPC, 1705 and 1465 cmꢀ1 for 1, 1661 and
1474 cmꢀ1 for 2, 1654 and 1475 cmꢀ1 for 3, and 1632 and
and S5).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
Table 4. 1H-NMR and 13C-NMR chemical shifts (ppm) with coupling constants and assignments for compound 1.
237
11
H N
O
6
H
N
O
O
7
1
S
18'
18
2
S
10
8
13
17
14
17'
12
NH
O
O
O
14'
15'
2
2
5
9
N
H
15
3
4
16
H3
–
C2
148 ppm
119 ppm
132 ppm
156 ppm
124 ppm
137 ppm
140 ppm
167 ppm
128 ppm
118 ppm
170 ppm
H4
7.44 (1H, d) [3JH4-H5 ¼ 8.46 Hz]
C4
C5
C6
C7
C8
C9
H5
7.67 (1H, dxd) [3JH5-H4 ¼ 8.46 Hz, JH5-H7 ¼ 1.96 Hz]
8.12 (1H, d) [4JH7-H5 ¼ 1.87 Hz]
7.88 (2H, s)
4
H7
H11
H12
H13
H15
H16
7.20 (2H, s)
–
8.25 (2H, dxd) [3JH15-H16¼7.67 Hz, JH15-H15’¼ 1.14 Hz]
C14 C14’
4
,
8.18 (1H, d þ d) [3JH16-H15/15’¼7.70 Hz and JH16-H15’/15¼7.69 Hz]
C15, C15’
3
C16
C17 C17’
,
1467 cmꢀ1 for 4. The differences (Dt) between the asymmetric 3, 776 nm for 4, and 585 for 5. The d–d transition for complex 2
containing Fe(III) ion with d5 has not been observed.
and symmetric stretches of the carboxylate groups of 2–4 are 187,
The room temperature magnetic moment of the metal com-
plexes is of 5.94 BM for 2, 3.82 BM for 3, 2.78 BM for 4, and 4.85
BM for 5 per metal ion, indicating the presence of five (Fe3þ, d5),
three (Co2þ, d7), two (Ni2þ, d8), and four (Fe2þ, d6) unpaired
electrons.
179, and 165, respectively, which suggest monodentate binding of
the carboxylate group to the metal ion in all complexes [38]. The
strong absorption bands in the region of 1637–1445 cmꢀ1 are
attributed to the ꢀ(C¼N) and ꢀ(C¼C) vibrations for all compounds.
The vibrations of the ꢀ(S¼O) are also observed in the
1430–1097 cmꢀ1 region for compounds SMABT and 1–5. The C–O
vibrations data for all compounds except SMABT are between
1375 and 1048 cmꢀ1 as expected. The ring wagging vibrations of
the pyridine groups are also observed in the 801–706 cmꢀ1 region
for compounds H2DPC and 1–5. The weak bands at 459–436 cmꢀ1
and 591–575 cmꢀ1 are from the M–N and M–O vibrations of the
compounds 2–5.
The molar conductivity data in DMSO are of 76.5 Xꢀ1 cm2
molꢀ1 for 2, 49.5 Xꢀ1 cm2 molꢀ1 for 3, 50.8 Xꢀ1 cm2 molꢀ1 for 4,
and 3.2 Xꢀ1 cm2 molꢀ1 for 5, indicating that the complex 2 is
ionic with a 1:1 ratio; the complexes 3 and 4 are ionic with a 2:1
ratio, and 5 is nonionic complex [39].
In vitro inhibition studies
In the present study, the inhibition effects on the hydratase and
esterase activities of hCA I and hCA II isozymes of the proton
transfer salt (1), its metal complexes (2–4), the starting compounds
(SA, TBS, SMABT, H2DPC), Fe(II) complex of SMABT (5), the metal
complexes of H2DPC, and the control compound AAZ have been
investigated. H2DPC and its metal complexes have not inhibited
the hydratase and esterase activities of hCA I and hCA II isoforms.
Hydratase activities of the isozymes have been inhibited by all
synthesized compounds. The proton transfer salt and metal com-
plexes have more potent inhibition effects than the starting com-
pounds. However, 1–5 are slightly weaker inhibitors than AAZ.
Only 4 has almost the same inhibition effect with AAZ (Table 5).
The inhibition effect of SMABT is weaker than the proton transfer
salt (1). Metal complexes of 1 are more powerful inhibitors than 1
for the hydratase activities of hCA I and hCA II. The structures of
2–4 are similar but their inhibition effects are different. Compound
4 is a more potent inhibitor than 2, 3, and 5 are. These differences
in the inhibitory effects could be derived from different metal ions
in the complexes for 2 and 3. However, it can be considered that
the difference of the inhibitory effect of 5 is due to the differences
in molecular structure. When the inhibition profiles of the synthe-
sized compounds on the hydratase activity of isozymes are consid-
ered, hCA II has been inhibited almost twice stronger than hCA I.
Inhibition levels of the compounds for the hydratase activities of
hCA I and hCA II are in the order of AAZ ꢇ 4 > 3 > 5 > 2 >
1 > SMABT > TBS > SA.
Thermal analyses of all complexes
Supplementary Figures S6–S9 show the TG-DTG and DTA curves
of compounds 2–5, and the thermal analyses results are given in
Supplementary Table S3.
For the compounds 2–5, the first stage, an endothermic peak
corresponds to the loss of water molecules, namely four moles for
2, five moles for 3, five moles for 4, and two moles for 5. The exo-
thermic second stage is consistent to the loss of following units,
that is, C5H5NO2S for 2, C14H16N6O4S4 for 3, C6H8N2O4S2 for 4, and
C21H21N7O6S6 for 5. The following units are also decomposed exo-
thermically in the third stage: C16H8N4O8S for 2, C15H6N2O8 for 3,
C22H14N6O8S2 for 4, and N2H2O2 for 5. The final decomposition
products are FeO for 2, CoO for 3, NiO for 4, and FeO for 5, and
they are identified by IR spectroscopy (Supplementary Table S3).
UV/vis spectrum, magnetic susceptibility, and molar conductivity
The electronic spectra of compounds 1–5 and the free ligands
SMABT and H2DPC were recorded in DMSO solution with 1 ꢂ 10ꢀ3
mol Lꢀ1 concentrations at room temperature (Supplementary
ꢆ
Table S4 and Figure S10). The characteristic p–p transitions in the
spectrum of 1 are of 305 nm and 290 nm, 310 nm and 303 nm for
2, 288 nm and 271 nm for 3, 286 nm and 268 nm for 4, and
305 nm and 290 nm for 5, with the same profiles as the free
ligands SMABT (305 nm and 290 nm) and H2DPC (303 nm). The
The esterase activities of the isozymes are similar to the inhib-
bands for the d–d transitions in DMSO are observed at 796 nm for ition of the hydratase activities. But a significant difference can be

238
Z. A. ALKAYA ET AL.
Table 5. The inhibition data and Ki values of hCA I and hCA II isozymes for hydratase and esterase activity.
Hydratase IC50 (lM)a,b Esterase IC50 (lM)a,b
hCA I
Ki (lM)a,b
Compound
hCA I
hCA II
hCA II
hCA I
hCA II
AAZ
SA
TBS
H2DPC
MDPC
SMABT
1
2
3
4
5
0.390 0.008
30.44 0.008
7.846 0.005
No inhibition
No inhibition
6.348 0.012
4.075 0.018
2.533 0.023
1.601 0.008
0.397 0.011
1.751 0.013
0.200 0.005
5.670 0.003
4.334 0.021
No inhibition
No inhibition
3.876 0.014
1.998 0.009
1.009 0.013
0.703 0.014
0.212 0.006
0.887 0.011
0.420 0.004
28.14 0.012
0.499 0.010
No inhibition
No inhibition
0.567 0.008
0.258 0.017
0.409 0.021
0.066 0.004
0.136 0.003
0.149 0.008
0.310 0.008
5.360 0.005
0.234 0.006
No inhibition
No inhibition
0.322 0.006
0.133 0.015
0.189 0.005
0.041 0.003
0.054 0.001
0.067 0.008
0.260 0.003
26.32 0.009
0.268 0.007
No inhibition
No inhibition
0.306 0.004
0.149 0.017
0.192 0.019
0.028 0.002
0.043 0.012
0.068 0.014
0.140 0.005
4.140 0.011
0.114 0.003
No inhibition
No inhibition
0.162 0.010
0.085 0.019
0.101 0.005
0.013 0.006
0.021 0.004
0.035 0.013
AAZ was used as reference compound.
M ¼ Fe(III), Co(II), and Ni(II).
aMean standard error, from three different assays.
bp < 0.0001 for all analysis.
recognized immediately in the inhibition profiles of esterase activ- In complexes 2–4, the metal ion coordinates to four oxygen atoms
ities of hCA I and hCA II as it follows: all of the newly synthesized
compounds (1–5) are more powerful inhibitors than AAZ is. Only
in terms of the inhibition of the esterase activity of hCA I, com-
pound 2 has nearly same inhibition potential as AAZ. Also, the
esterase activities of hCA I and hCA II were inhibited stronger than
the hydratase activities of the isozymes. Another difference when
compared to the inhibition profile of hydratase activity is that 2
has acted as weaker esterase inhibitor than the proton transfer
salt (1). The inhibition effects of 1 are twice higher than SMABT.
The inhibitory effects of the metal complexes (2–5) were in the
range of 0.066 0.004 mM–0.409 0.021 mM for hCA I, and
0.041 0.003 mM–0.189 0.005 mM for hCA II. Compound 3 has the
most effective inhibitor effect on the esterase activities of the iso-
zymes. The inhibition potentials of 4 and 5 are close to each
other. Similar to hydratase activity inhibition results, it is shown
that hCA II has been inhibited twice stronger than hCA I, except
for complex 3 which is 1.5fold. Inhibition levels of the compounds
are in the order of 3 > 4 > 5 > 1 > 2 > AAZ > TBS > SMABT > SA
for hCA I, and of 3 > 4 > 5 > 1 > 2 > TBS > AAZ > SMABT > SA for
hCA II, respectively.
and two nitrogen atoms of two 2,6-pyridinedicarboxylate ions
resulted with a distorted octahedral conformation. Intermolecular
N-HꢅꢅꢅO and O-HꢅꢅꢅO hydrogen bonds and p–p stacking interac-
tions seem to be effective in the stabilization of the crystal struc-
ture. Elemental analyses and all measurements show good
agreement with the structures. The structure of 5 might be pro-
posed as octahedral according to results of elemental, spectral,
magnetic measurement, molar conductivity, and thermal analyses.
These five novel compounds possess significant inhibition
effect on hCA I and on hCA II for hydratase and esterase activities
with nearly nanomolar level. These results suggest that further
inhibition studies are worthwhile in order to obtain correlation in
such compounds and derivatives of such compounds should be
subject to further inhibition in vivo tests. The order of the inhib-
ition effects increasing through starting compounds and proton
transfer salt and the complexes of these compounds might be
due to the structural changes leading to an impressive difference
of activity.
Ki values of the compounds have been determined by using
esterase activity measurements (Table 5). Ki values clearly show
that the compounds have remarkable inhibition effects on hCA I
and hCA II isozymes for esterase activities. Especially 3–5 have
effective inhibition constants with nearly nanomolar level. The
inhibition potentials of these compounds (3–5) are 10 times, 4
times, and 6 times higher than AAZ, respectively. These results are
quite remarkable in terms of further studies.
In our previous studies, we investigated the inhibition poten-
tials of the proton transfer salts and the metal complexes derived
from 2-aminobenzothiazole [23], 2-amino-6-methylbenzothiazole
[22], 2-amino-6-methoxybenzothiazole [24], and 2-amino-6-chloro-
benzothiazole [25]. These compounds have also shown remarkable
inhibitory effects despite the fact that they do not contain sulfona-
mide moiety. In this study, the inhibitory effects of the compounds
are further improved by using 2-amino-6-sulfamoylbenzothiazole.
To summarize, carbonic anhydrase (hCA I and hCA II) inhibition
profiles of non-sulfamoyl benzothiazole and sulfamoyl benzothia-
zole derivatives are imparted to the literature.
Disclosure statement
The authors declare no conflict of interest. The authors are solely
responsible for the content and results presented in this paper.
Funding
This work was supported by the Dumlupinar University Research
Fund [(grant No. 2014/18)].
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