Synthesis and characterization of Cu(II) complexes of 2-amino-6-sulfamoylbenzothiazole and their inhibition studies on carbonic anhydrase isoenzymes

Article abstract of DOI:10.1016/j.poly.2018.05.015

2-Amino-6-sulfamoylbenzothiazole (SMABT) and its proton transfer compound (HSMABT)⁺(HDPC)^? (1) with 2,6-pyridinedicarboxylic acid (H₂DPC), and their Cu(II) complexes (2 of SMABT, 3 and 4 of 1) have been prepared and characterized by spectroscopic techniques. Additionally, single crystal X-ray diffraction techniques were applied to all complexes. 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 complexes have remarkable inhibitory effects on hCA I and hCA II isoenzymes. The inhibition potentials of the proton transfer salt (1) and the metal complexes (2–4) are comparable with AAZ. Esterase K_i values of the compounds (1–4) are in the range of 0.089 ± 0.008 μM-0.149 ± 0.017 μM for hCA I and 0.046 ± 0.008 μM-0.085 ± 0.019 μM for hCA II. Inhibition data have been analyzed by using a one-way analysis of variance for multiple comparisons (p < 0.0001).

Full text of DOI:10.1016/j.poly.2018.05.015

Accepted Manuscript
Synthesis and Characterization of Cu(II) Complexes of 2-amino-6–sulfamoyl-
benzothiazole and Their Inhibition Studies on Carbonic Anhydrase Isoenzymes
Zeynep Alkan Alkaya, Halil İlkimen, Cengiz Yenikaya, Ekrem Tunca, Metin
Bülbül, Tuncay Tunç, Musa Sarı
PII:
S0277-5387(18)30253-5
https://doi.org/10.1016/j.poly.2018.05.015
POLY 13162
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
6 April 2018
6 May 2018
Please cite this article as: Z.A. Alkaya, H. İlkimen, C. Yenikaya, E. Tunca, M. Bülbül, T. Tunç, M. Sarı, Synthesis
and Characterization of Cu(II) Complexes of 2-amino-6–sulfamoylbenzothiazole and Their Inhibition Studies on
Carbonic Anhydrase Isoenzymes, Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.05.015
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Synthesis
and
Characterization
of
Cu(II)
Complexes
of
2-amino-
6-sulfamoylbenzothiazole and Their Inhibition Studies on Carbonic Anhydrase
Isoenzymes
Zeynep Alkan Alkayaa, Halil İlkimenb, Cengiz Yenikayab*, Ekrem Tuncac, Metin
Bülbülc, Tuncay Tunçd, Musa Sarıe
aDepartment of Chemistry and Chemical Processing Technologies, Banaz Vocational
School, Usak University, 64500 Banaz, Uşak, Turkey.
bDepartment of Chemistry, Faculty of Arts and Sciences, Dumlupınar University,
43100 Kütahya, Turkey.
cDepartment of Biochemistry, Faculty of Arts and Sciences, Dumlupınar University,
43100 Kütahya, Turkey.
dDepartment of Science Education, Faculty of Education, Aksaray University, 68100,
Aksaray, Turkey.
eDepartment of Physics Education, Gazi University, 06500 Ankara, Turkey.
*Corresponding Author: Cengiz Yenikaya
Department of Chemistry, Dumlupınar University, Kutahya Turkey
e-mail: cengiz.yenikaya@dumlupinar.edu.tr
1

Abstract:
2-Amino-6-sulfamoylbenzothiazole (SMABT) and its proton transfer compound
(HSMABT)+(HDPC)- (1) with 2,6-pyridinedicarboxylic acid (H2DPC), and their Cu(II)
complexes (2 of SMABT, 3 and 4 of 1) have been prepared and characterized by
spectroscopic techniques. Additionally, single crystal X-ray diffraction techniques were
applied to all complexes. 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 complexes have remarkable
inhibitory effects on hCA I and hCA II isoenzymes. The inhibition potentials of the proton
transfer salt (1) and the metal complexes (2-4) are comparable with AAZ. Esterase Ki values
of the compounds (1-4) are in the range of 0.089 0.008 µM-0.149 0.017 µM for hCA I and
0.046 0.008 µM-0.085 0.019 µM for hCA II. Inhibition data have been analyzed by using a
one-way analysis of variance for multiple comparisons (p < 0.0001).
Keywords: 2-amino-6-sulfamoylbenzothiazole, proton transfer, metal complex,
carbonic anhydrase, statistical analyses.
2

Introduction
The sulfamoyl and benzothiazole derivatives constitute an important class of drugs,
with several types of pharmacological agents possessing, among others, antibacterial,
anticonvulsant, anti-inflammatory, antitubercular, therapeutic, protease, and carbonic
anhydrase inhibitory effects [1-3].
The metal complexes for 2,6-pyridinedicarboxylic acid (or dipicolinic acid, H2DPC)
and its deprotonated forms (HDPC- and DPC2-) show interesting structural features with
various coordination modes [4], stabilization of unusual oxidation states [5] and insulin-
mimetic effects [6,7]. H2DPC with Cu(II) ion commonly has one or two coordination modes
[4]. In one coordination mode, a single planar DPC ligand associates in the equatorial plane of
a metal cation with other ligands such as, H2O or pyridine-based heterocycles [4,8], which
occupy the remaining sites. This leads to the formation of square pyramidal [4,9-13] or
octahedral coordination geometry [4,10-12,14]. In the second coordination mode, two
perpendicularly coordinated planar dipicolinic molecules generate distorted octahedral
coordination geometry [4,9,10,14].
In this study, 2-amino-6-sulfamoylbenzothiazole (SMABT) [15] and proton transfer
compound (SMHABT)+(HDPC)- (1) [17] have been prepared according to literature. Their
Cu(II) complexes have been also prepared between SMABT and Cu(II) in order to give (2) as
a simple complex, and between 1 and Cu(II) to give 3 which is an ionic complex containing
(SMHABT)+ and [Cu(DPC)2]2- ions, and also 4 which is a nonionic mixed ligand complex as
{[Cu(DPC)(SMABT)(H2O)]2H2O}. The mixed ligand metal complexes have generally shown
better biological activities which are better than those of the simple ones [8-14]. They are
characterized by elemental, spectral (IR and Uv-Vis.) and thermal analyses, as by using
magnetic measurement and molar conductivity techniques. Single crystal X-ray diffraction
3

techniques were applied to all complexes (2-4). The simple Cu(II) complex of H2DPC [17]
has also been prepared in order to compare the inhibition data of all studied compounds.
In addition, we have investigated the potential use of these compounds as new
inhibitors of human carbonic anhydrase isoenzymes, hCA I and hCA II. Carbonic anhydrases
(CAs, EC 4.2.1.1) are ubiquitous metalloenzymes used to catalyze interconversion of carbon
dioxide and water to produce bicarbonate and proton, and are encoded by seven unrelated
gene families: α-, β-, γ-, δ-, ζ-, η-, and θ-CAs [18]. There are sixteen α-CA isoforms or CA-
related proteins identified in mammals [19]. Among the α-CAs, hCA I is major CA isoform,
and hCA II is also present in the human eye [20]. hCA II participates in aqueous humor
secretion [21], and the inhibition of this isoenzyme is extremely important in the treatment of
glaucoma, which is a group of diseases characterized by the gradual loss of visual field due to
an elevation in intraocular pressure (IOP) [21,22].
2. Experimental section
2.1. General methods and materials
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 and
Cu were detected with Perkin Elmer Optima 4300 DV ICP-OES. Crystallographic data were
recorded on a Bruker Kappa APEX II CCD area-dedector X-ray diffractometer. FT-IR spectra
were recorded in the 4000-400 cm-1 region with Bruker Optics, Vertex 70 FT-IR spectrometer
using ATR techniques. Thermal analyses were performed on Pelkin Elmer SII Exstar 6000
TG/DTA 6300 model using platinum crucible with 10 mg sample. Measurements were taken
in the static air within a 30-900 ºC temperature range. The UV–Vis spectra were obtained for
DMSO solution of the compounds (10-3 M) with a SHIMADZU UV-2550 spectrometer in the
range of 200–900 nm. Magnetic susceptibility measurements at room temperature were
4

performed using a Sherwood Scientific Magway MSB MK1 model magnetic balance by the
Gouy method using Hg[Co(SCN)4] as calibrant. The molar conductances of the compounds
were determined in water/ethanol (1:1) and in DMSO (10-3 M) at room temperature using a
WTW Cond 315i/SET Model conductivity meter.
2.2. Synthesis of metal complexes (2-4)
2-Amino-6-sulfamoylbenzothiazole (SMABT) [15] and proton transfer compound
(SMHABT)+(HDPC)- (1) [16] have been prepared according to literature (Figures S1-S5 and
Tables S1-S3)
A solution of 1 mmol (0.199 g) Cu(CH3COO)2.H2O in water (10 mL) was added
dropwise to the solution of 1 mmol (0.229 g) SMABT for 2 or to the solution of 1 mmol of
(0.396 g) 1 for 3 in water/ethanol (1:1) (20 mL) with stirring at room temperature for one day.
The reaction mixture was kept at room temperature for two weeks to give green crystalline
solid for 2 (0.331 g, 45% yield), green crystalline solid for 3 (0.106 g, %40 yield). Compound
4 was obtained from the mother liquor of 3 after one week as turquoise crystalline solid
(0.109 g, 45% yield) (Fig.1). The single crystals of all complexes suitable for X-ray
diffraction were separated and washed with EtOH/water (1:1).
Anal. Calcd. for 2 (C23H32N6O12S4Cu4): C, 32.89%; H, 3.84%; N, 10.01%; S, 15.27%;
Cu, 15.12%. Found: C, 32.85%; H, 3.80%; N, 10.05%; S, 15.30%; Cu, 15.15%; for 3
(C28H32N8O17S2Cu): C, 35.61%; H, 3.42%; N, 11.87%; S, 13.58%; Cu, 6.73%. Found: C,
35.65%; H, 3.40%; N, 11.85%; S, 13.60%; Cu, 6.75%.; for 4 (C28H26N8O15S4Cu2): C,
34.67%; H, 2.70%; N, 11.55%; S, 13.22%; Cu, 13.10%. Found: C, 34.70%; H, 2.70%; N,
11.60%; S, 13.60%; Cu, 13.15%.
The experimental studies of purification of carbonic anhydrase I and II isoenzymes
from human erythrocytes, determination of hydratase and esterase activities of hCA I and
5

hCA II, determination of IC50 and Ki values of the compounds and statistical analysis are
given in the Supplementary file [23-29].
2.3. X-ray data collection and structure refinement
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-dedector X-ray diffractometer
employing plane graphite monochromatized with MoKα radiation (λ = 0.71073 Å), using ω-
2θ scan mode. The empirical absorption corrections were applied by multi-scan via Bruker,
SADABS software [30]. 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 [31] programs were used for all the calculations. The H atoms were placed in
idealized positions and constrained to ride on their parent atoms with distances in the range of
N-H = 0.71(6)-0.92(9) Å, C-H = 0.93-0.96Å and with Uiso(H)= 1.2Ueq(C,N) or 1.5Ueq(C) for
methyl atoms. The H atoms of water molecules were located from in a difference Fourier
maps and were constrained at distance of O-H = 0.74(3)-0.83(4) Å and with Uiso(H) =
1.5Ueq(O). The drawings of molecules were accomplished with the help of ORTEP-3 for
Windows [32].
3. Results and discussion
3.1. Crystal structures of 2-4
The molecular structures of 2-4, with the atom labeling of symmetric units, are shown
in Figures 2-4, respectively. The details of the crystal structure solutions are summarized in
Table 1 and the selected bond lengths and angles are listed in Table 2.
6

Table 1. Crystal data and structure refinement details for compounds 2-4.
2
3
4
Empirical formula
Formula weight
C22H30Cu2N6O14S4
857.86
C28H32CuN8O17S4
944.41
C28H26Cu2N8O15S4
969.91
T[K]
296(2)
296(2)
296(2)
Wavelength (Å)
0.71073
0.71073
0.71073
Crystal system, space
group
Monoclinic, P21/ꢀ
Triclinic, P1
Triclinic, P1
Unit cell dimensions
(Å, º)
a
b
c
α
β
γ
8.0679(4)
8.2980(4)
13.0375(6)
104.927(2)
97.568(2)
90.597(2)
835.13(7)
1
9.245(5)
14.425(5)
14.574(5)
98.617(5)
106.710(5)
90.774(5)
1837.2(13)
2
14.7958(9)
20.4121(14)
11.9090(8)
104.300(2)
V (Å3)
Z
3485.2(4)
4
Absorbtion coefficient 1.596
0.910
1.546
(mm-1)
Dcalc (Mg /m3)
F(000)
1.595
438
1.707
970
1.849
1968
Crystal
dimensions 0.25x0.14x0.10
0.25x0.16x0.10
014x0.10x0.08
(mm)
θ
range for data 1.63-28.49
1.88-28.60
2.81-26.36
collection (º)
Index ranges
-10≤ h ≤10
-11≤ k ≤11
-17≤ l ≤17
15630
-12≤ h ≤12
-19≤ k ≤19
-19≤ l ≤19
34222
-18≤ h ≤18
-25≤ k ≤25
-14≤ l ≤14
77224
Reflections collected
Independent
4233
9401
9792
reflections
Data/parameters
3795/235
7708/595
7175/571
Max.
and
min. 0.765, 0.852
0.840, 0.913
0.881, 0.884
transmission
Final
[I≥2σ(I)]
R indices (all data)
R
indices R2 = 0.0300
wR2 = 0.0745
R2 = 0.0336
wR2 = 0.0935
R1 = 0.0431
wR1 = 0.0880
1.030
R2 = 0.0270
wR2 = 0.0637
R1 = 0.0364
wR1 = 0.0687
1.054
R1 = 0.0264
wR1 = 0.0720
Goodness-of-fit on F2 1.038
Largest difference in 0.424, -0.403
peak and hole (e Å-3)
0.368, -0.443
0.343, -0.393
7

Table 2. Selected bond distances [Å] and angles [°] of compounds 2-4.
Compound 2: [Cu2(SMABT)2(Ac)2].2H2O
Cu1-O3
Cu1-O4
1.9613(14) Cu1-O2
1.9708(14) Cu1-O1
1.9779(14)
1.9852(14)
Cu1-N1
2.1899(14)
2.6515(4)
Cu1-Cu1i
O1-Cu1-N1
O1-Cu1-Cu1i
O2-Cu1-O1
O2-Cu1-N1
O2-Cu1-Cu1i
98.89(6) O3-Cu1-O1
85.00(4) O3-Cu1-O2
167.53(6) O3-Cu1-O4
93.55(6) O3-Cu1-N1
82.56(4) O3-Cu1-Cu1i
90.39(6) O4-Cu1-O1
88.84(7) O4-Cu1-O2
167.49(6) O4-Cu1-N1
97.16(6) O4-Cu1-Cu1i
82.87(4) N1-Cu1-Cu1i
88.79(7)
89.28(7)
95.30(6)
84.62(4)
176.11(4)
Compound 3: (HSMABT)2[Cu(DPC)2].5H2O
Cu1-O1
Cu1-O2
2.2389(17) Cu1-O5
2.1632(17) Cu1-O6
2.2172(15)
2.1281(15)
Cu1-N1
Cu1-N2
1.9538(15)
1.9387(15)
N2 Cu1 N1
N2 Cu1 O6
N1 Cu1 O6
N2 Cu1 O2
N1 Cu1 O2
178.35(6)
79.22(5)
101.68(5)
102.77(6)
78.52(6)
O6 Cu1 O2
97.53(7)
76.47(5)
102.54(5)
155.47(5)
91.04(7)
N2 Cu1 O1
N1 Cu1 O1
O6 Cu1 O1
O2 Cu1 O1
O5 Cu1 O1
102.35(5)
76.31(5)
90.06(7)
154.70(5)
91.90(6)
N2 Cu1 O5
N1 Cu1 O5
O6 Cu1 O5
O2 Cu1 O5
Compound 4: [Cu(DPC)(SMABT)(H2O)][Cu(DPC)(SMABT)(H2O)].H2O
Cu1-O1
Cu1-O3
Cu1-N1
Cu1-N2
2.0454(14) Cu1-O1w
2.0286(15) Cu2-O7
1.9317(17) Cu2-O9
1.9980(17) Cu2-N5
2.2263(18)
2.0230(16)
2.0645(16)
1.9312(18)
Cu2-N6
Cu2-O2w
2.0069(17)
2.321(2)
N1 Cu1 N2
N1 Cu1 O1
N1 Cu1 O3
N2 Cu1 O3
N2 Cu1 O1
O3 Cu1 O1
170.64(7)
78.71(6)
79.33(6)
97.40(6)
102.70(6)
155.88(6)
N1 Cu1 O1w 97.34(7)
N2 Cu1 O1w 91.69(7)
O3 Cu1 O1w 95.51(7)
N5 Cu2 O9
N6 Cu2 O9
O7 Cu2 O9
N5 Cu2 O2w 91.27(7)
N6 Cu2 O2w 93.95(7)
O7 Cu2 O2w 93.82(8)
79.00(7)
105.28(7)
157.72(7)
N5 Cu2 N6
N5 Cu2 O7
N6 Cu2 O7
173.19(7)
79.09(7)
96.17(7)
Symmetry code: (i) –x+1, –y+1, –z+1
The complex 2 crystallizes in the triclinic P1 space group. The structure of 2 has a
crystallographic inversion centre. The symmetric unit of 2,
[Cu2(SMABT)2(CH3COO)4].2H2O, contains two SMABT molecules, four acetate anions as
bridges, two Cu(II) cations and two uncoordinated water molecules. The coordination
8

environment of the two Cu(II) ions (Cu1 and Cu1i) can be described as distorted octahedron
with each Cu(II) atom being surrounded by four µ2-bridging bidentate acetate ligands in the
basal plane and one SMABT molecule in one axial position. In the structure, each acetate
molecules form distorted rectangle planes [Cu1-O3, O3-O4, O4-Cu1i, Cu1-Cu1i] around each
metal ion (the bond lengths for Cu-O between 1.9613(14) and 1.9852(14) Å), while the Cu(II)
ions are displaced by 0.428(4) Å from these planes towards the N atoms (Cu-N 2.1899(14)
Å). The N1-Cu1-Cu1i trans-angle is much closer to 180° (176.11(4)°). Finally, the sixth
coordination comes from Cu1-Cu1i metallic bond (2.6515(4) Å) (Table 2).
The complex 3 crystallizes in the triclinic P1 space group. The structures of 3,
(SMHABT)2[Cu(DPC)2].5H2O, consist of one [Cu(DPC)2]2- anion and two counter
SMHABT+ cations, and five uncoordinated water molecules. In complex 3, the Cu(II) ion is
coordinated by four oxygen atoms (O1, O2, O5 and O6) and two nitrogen atoms (N1 and N2)
of two DPC2- ions resulting with a distorted octahedral conformation. Both carboxylate
oxygen atoms from DPC2- occupy the trans-apical positions of the Cu(II) coordination
polyhedron with bond lengths [Cu1-O1 = 2.2389(17) Å, Cu1-O5 = 2.2172(15) Å, Cu1-O2 =
2.1632(17) Å and Cu1-O6 = 2.1281(15) Å], and define low trans-angle value around Cu(II)
ion as 154.70(5)° for O2-Cu1-O1 and 155.47(5)° for O6-Cu1-O5 which reveals a rather rigid
structure of such tri-dentate ligands. In contrast, the N1-Cu1-N2 trans-angle is much closer to
180° (178.35(6)°) and the dihedral angle defined by the mean planes of two DPC ligands is
102.35(5)° showing that they fall almost perpendicular.
The complex 4 crystallizes in the monoclinic P21/c space group. The symmetric unit
of 4, [Cu(DPC)(SMABT)(H2O)][Cu(DPC)(SMABT)(H2O)].H2O, consists of two independent
and similar molecules, each containing one coordinated DPC2- ion, one coordinated SMABT
molecule, one coordinated water molecule and one Cu(II) ion, and additionally one
uncoordinated water molecule for both independent centers (Figure 4). Each Cu2+ ions is
9

coordinated by two N atoms, one from DPC (N1 for Cu1 and N5 for Cu2) and one from
SMABT ring (N2 for Cu1 and N6 for Cu2), by two carboxylate O atoms from DPC (O1 and
O3 for Cu1 and O7 and O9 for Cu2) and with the axial site occupied by the one coordinated
water molecule (O1w for Cu1 and O2w for Cu2) (Fig. 4). Thus the coordination environment
of Cu2+ ions (for both molecule) have distorted square pyramid geometries with the structural
index τ, 0.25 for Cu1 and 0.26 for Cu2 [33]. Two N atoms from two different ligands, DPC
and SMABT, occupy equatorial positions (Fig. 4 and Table 2). The Cu-Owater bond distances,
arising from coordination of the water molecule to the central metal ion, are 2.2263(18) Å for
Cu1-O1w and 2.321(2) Å for Cu2-O2w, which are much longer than the Cu-O (carboxylate)
bond distances (Table 2).
In all essential details, the geometries of the complexes (2-4) regarding bond lengths
and angles are in good agreement with the values observed in similar Cu(II) complexes
[8-11,13,34,35]. Cu-Cu metal bond length for 2 is also comparable with a very similar
structure [34-36].
Hydrogen bonds for the complexes 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 hydrogen bondings in the structures 3 and 4 (Table
3).
Table 3. Hydrogen bonds for compounds 3 and 4 (Å, °)
D-H…A
d(D-H)
d(H…A)
d(D…A)
<D-H…A
3
N3-H3B-O4w
N4-H4-O1
N7-H7-O5
O2w-H2C-O2
0.79(3)
0.86
0.86
0.840(10) 2.03(2)
0.823(10) 1.992(12)
2.19(3)
1.84
1.88
2.894(3)
2.643(2)
2.616(2)
2.849(3)
2.802(3)
149(2)
154.6
143.2
163(6)
168(3)
O3w-H3C-O2w
4
N3-H31-O3
N7-H7B-O7
0.82(3)
0.86(3)
1.95(3)
1.86(3)
2.683(3)
2.652(3)
149(3)
151(3)
10

3.2. FT-IR measurements
The infrared spectral data of the starting compounds (SMABT, H2DPC and 1) and
compounds 2-4 are given in Table S3. In the high frequency region, weak bands 3098-3067
cm-1 are attributed to the stretching vibrations of aromatic C-H for all compounds. There are
also broad absorption bands at 3504-3424 cm-1, which are attributed to the ν(OH) vibrations
of water molecules for compounds 2-4. The relatively weak and broad bands at 2752 and
2522 cm-1 are attributed to the ν(N+-H) vibration for 3 [37]. The absorption bands at 3423,
3324, 3293 and 3237 cm-1 of NH2 group of SMABT are slightly shifted from those found for
1 (3433, 3424, 3385 and 3229 cm-1), for 2 (3422, 3358, 3229 and 3228 cm-1), for 3 (3408,
3385, 3325 and 3244 cm-1) and for 4 (3358, 3327, 3292 and 3237 cm-1) 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 cm-1. These bands are reflected by IR
spectrum of the asymmetric (νas) and symmetric (νs) stretching vibrations at 1701 and 1456
cm-1 for H2DPC, 1705 and 1465 cm-1 for 1, 1647 and 1422 cm-1 for 3, 1648 and 1452 cm-1 for
4 from DPC2- ions, 1590 and 1450 cm-1 for 2 from Ac- ion. The differences (∆υ) between the
asymmetric and symmetric stretches of the carboxylate groups of 2-4 are 140, 195, and 196,
respectively, which suggest bridging for 2 and monodentate for 3 and 4, binding of the
carboxylate group to the metal ion [38]. The strong absorption bands in the region of 1637-
1445 cm-1 are attributed to the ν(C=N) and ν(C=C) vibrations for all compounds. The
vibrations of the ν(S=O) are also observed in the 1432-1098 cm-1 region for compounds
SMABT and 1-4. The C–O vibrations data for all compounds except SMABT are between
1363 and 1069 cm-1 as expected. The ring wagging vibrations of the pyridine groups are also
observed in the 801-702 cm-1 region for compounds H2DPC and 1, 3 and 4. The weak bands
11

at 459-436 cm-1 and 585-574 cm-1 are from the M–N and M–O vibrations of the compounds
2-4.
3.3. Thermal analyses of all complexes
Figures S6-S8 show the TG-DTG and DTA curves of the compounds 2-4, and the
thermal analyses results are given in Table S4.
For the compound 2, the first endothermic peak corresponds to the loss of two moles
of water. The endothermic second stage is consistent to the loss of the four moles of acetate
ions. The third stage, an exothermic peak, agrees to the loss of the C12H10N2O4S2 unit and the
C2H4N4S2 unit is decomposed exothermically in the following stage. The final decomposition
product is CuO identified by IR spectroscopy.
For the compounds 3 and 4, the first stage, an endothermic peak corresponds to the
loss of water molecules, namely five moles for 3 and three moles for 4. The exothermic
second stage is consistent with the loss of the following units C12H10N2O4S4 for 3 and
C10H10N2O4S2 for 4. The units are also decomposed exothermically in the third stage:
C16H12N6O8 for 3 and C18H16N6O9S2 for 4. The final decomposition products are CuO for 3
and 4, and they are identified by IR spectroscopy.
3.4. UV/vis Spectrum, magnetic susceptibility and molar conductivity
The electronic spectra of compounds 1-4 and the free ligands SMABT and H2DPC
were recorded in DMSO solution with 1x10-3 molL-1 concentrations at room temperature
(Table S5, Figure S9). The characteristic π- π* transitions in the spectrum of 1 are of 305 nm
and 290 nm, 288 nm and 271 nm for 2, 304 nm and 290 nm for 3, and 307 nm and 298 nm for
4, with the same profiles as the free ligands SMABT (305 nm and 290 nm) and H2DPC (303
12

nm). The bands for the d-d transitions in DMSO are observed at 760 nm for 2, 752 nm for 3,
and 748 for 5.
The room temperature magnetic moment of the metal complexes is of 1.70 BM for 2,
1.67 BM for 3, and 1.64 BM for 4 per metal ion, indicating the presence of one unpaired
electron (Cu2+, d9) for all compounds.
The molar conductivity data in DMSO are of 4.5 Ω-1cm2mol-1 for 2, 51.2 Ω-1cm2 mol-1
for 3, and 3.6 Ω-1cm2 mol-1 for 4, indicating that the complexes 2 and 4 are non-ionic, and the
complex 3 is ionic with a 2:1 ratio [39].
3.5. In vitro inhibition studies
In this study the inhibition effects of proton transfer salt (1) and its metal complexes (3
and 4), and metal complex of SMABT (2) on the hydratase and esterase activities of human
cytosolic carbonic anhydrases, hCA I and hCA II, have been investigated as in vitro.
Inhibition potentials of these compounds were compared with starting compounds (SA, TBS,
SMABT and H2DPC) and reference compound, AAZ. No inhibitory effect of H2DPC on the
enzyme was observed. In contrast, other starting compounds, proton transfer salt and metal
complexes inhibited both hydratase and esterase activities of isozymes. It is clearly observed
that the synthesized compounds are more selective towards clinically important isoform, hCA
II (Table 4).
When the inhibition of hydratase activity is examined, it is observed that the proton
transfer salt and metal complexes are more potent inhibitors than starting compounds. This is
especially pronounced for 2-4 compounds, and even the inhibition potencies of compounds 3
and 4 are comparable to AAZ. The proton transfer salt (1) formed between H2DPC and
SMABT increased the inhibition effect of SMABT and the coordination structure of this
compound with Cu (II) maximizes its inhibition effect. In metal complexes, the number of
13

SMABT ligands is more than one and they can interact more strongly with the active site of
the enzyme resulting excess inhibition effect of the metal complexes. It is remarkable that the
inhibitory effect on the hydratase activity of compounds 3 and 4 is about 25-fold higher than
that of the SMABT.
Table 4. The inhibition data and Ki values of hCA I and hCA II isozymes for hydratase and
esterase activity.
Hidrataz IC50 (µM)a,b
hCA I hCA II
Esteraz IC50 (µM) a,b
hCA I hCA II
Ki (µM) a,b
hCA I hCA II
Bileşik
AAZc
SA
TBS
0.390 0.008 0.200 0.005 0.420 0.004 0.310 0.008 0.260 0.003 0.140 0.005
30.44 0.008 5.67 0.003 28.14 0.012 5.36 0.005 26.32 0.009 4.14 0.011
7.846 0.005 4.334 0.021 0.499 0.010 0.234 0.006 0.268 0.007 0.114 0.003
No
Inhibition
No
No
Inhibition
No
No
Inhibition
No
No
Inhibition
No
No
Inhibition
No
No
Inhibition
No
H2DPC
CuDPC
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
6.348 0.012 3.876 0.014 0.567 0.008 0.322 0.006 0.306 0.004 0.162 0.010
4.075 0.018 1.998 0.009 0.258 0.017 0.133 0.015 0.149 0.017 0.085 0.019
1.724 0.006 0.716 0.006 0.201 0.022 0.121 0.012 0.098 0.015 0.046 0.008
0.245 0.012 0.132 0.008 0.245 0.003 0.112 0.009 0.099 0.006 0.053 0.008
0.267 0.009 0.140 0.004 0.267 0.009 0.125 0.012 0.089 0.008 0.048 0.009
SMABT
1
2
3
4
aMean standard error, from three different assays.
bp < 0.0001 for all analysis.
cAAZ was used as reference compound.
While the inhibition profile of the esterase activity of the compounds resembles the
inhibition of hydratase activity, sharp inhibition differences among the studies compounds for
the inhibition of hydratase activity are not found for the inhibition of esterase activity.
However, the synthesized compounds (1-4) exhibit about 2-3 times more potent inhibitory
properties than the SMABT compound. This increase in the inhibition of esterase activity
among the studied compounds can be attributed to the formation of the proton transfer salt
and the formation of coordination with Cu(II) which is analogous to the inhibition of
hydratase activity. In addition, another remarkable phenomenon in the inhibition of esterase
activity is that the differences between the inhibition potencies of the compounds 1-4 are not
14

so great. The most prominent feature of our study of inhibition of esterase activity is that
compounds 1-4 are more potent inhibitors than the AAZ compound.
The inhibition equilibrium constants (Ki values) of the compounds have been
determined by using esterase activity measurements. The Ki constants of the compounds 1-4
indicate that these compounds have stronger inhibitory effects than the AAZ compound
(Table 4). These results are important for designing potent inhibitors of hCA I and hCA II,
which are cytosolic isoforms. The inhibitor compounds synthesized within the scope of the
study can be modified to develop both more potent and more selective compounds against the
hCA II isoenzyme.
4. Conclusion
In the present work, three newly Cu(II) complexes (2-4) were prepared for the first
time. The complexes 2 and 3 crystallize in the triclinic P1 space group, whereas the complex
4 crystallizes in the monoclinic P21/c space group. In the complex 2, two Cu(II) ions are
connected to each other with a Cu-Cu bond and with four bridged acetate ions. Each Cu(II)
ions has also Cu-N bond from SMABT to give distorted octahedral geometry around Cu(II)
ions. In complex 3, the Cu(II) ion coordinates to four oxygen atoms and two nitrogen atoms
of two DPC2- ions resulted with a distorted octahedral conformation. The complex 4 consists
of two independent and different cationic Cu(II) sites. Cu(II) atoms are coordinated by two N
atoms, one from DPC2- and one from SMABT ring, two carboxylate O atoms from DPC and
one water molecule to give distorted square pyramidal structures. Intermolecular N-H…O and
O-H…O hydrogen bonds and π-π stacking interactions seem to be effective in the
stabilization of the crystal structure. Elemental analyses and all measurements show good
agreement with the structures.
15

The newly synthesized compounds (1-4) in the present study possess significant
inhibition effect on hCA I and on hCA II for hydratase and esterase activities. The inhibitory
effects of the compounds on CA isoenzymes are comparable to those of the AAZ compound,
and they should be subject to further inhibition in vivo tests.
5. Supplementary data
CCDC 1559299 (2), CCDC 1559300 (3), and CCDC 1559301 (4) contain
supplementary crystallographic data for this paper. This data can be obtained free of charge
via http://www.ccdc.cam.ac.uk (the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax (+44 1123 336 033; or email: deposit@ccdc.cam.ac.uk).
Acknowledgments
The authors acknowledge the support provided by Dumlupınar University Research Fund
(grant No. 2014/18). In addition, the authors acknowledge Aksaray University of Science and
Technology Application and Research Center, for the use of the Bruker SMART BREEZE
CCD diffractometer (purchased under grant No. 2010K120480 of the State of Planning
Organization).
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Tables and Figures
Table 1. Crystal data and structure refinement details for compounds 2-4.
Table 2. Selected bond distances [Å] and angles [°] of compounds 2-4.
Table 3. Hydrogen bonds for compounds 3 and 4 (Å, °)
Figure 1. Syntheses of all compounds (a for 2 and b for 3 and c for 4).
Figure 2. The molecular structure of (2), with atom labels and 50% probability displacement
ellipsoids for non-H atoms [Symmetry code: (i) -x + 1, -y + 1, -z + 1].
Figure 3. The molecular structure of (3), with atom labels and 50% probability displacement
ellipsoids for non-H atoms.
Figure 4. The molecular structure of (4), with atom labels and 50% probability displacement
ellipsoids for non-H atoms.
19

(a)
(b)
(c)
Figure 1. Syntheses of all compounds (a for 2 and b for 3 and c for 4)

Newly synthesized Cu(II) complexes of 2-amino-6-sulfamoylbenzothiazole (2-4) have
effective inhibitory activity on hCA I and II. 2 and 3 crystallize in the triclinic P space group
with distorted octahedral conformations. 4 crystallizes in the monoclinic P2/1c space group
and has distorted square pyramidal structure.
20