Synthesis, characterization and antiglaucoma activity of a novel proton transfer compound and a mixed-ligand Zn(II) complex

Article abstract of DOI:10.1016/j.bmc.2009.11.031

A novel proton transfer compound, pyridin-2-ylmethanaminium 2,4-dichloro-5-sulfamoylbenzoate (1), and a mixed-ligand Zn(II) complex, bis(2,4-dichloro-5-sulfamoylbenzoate)(2-aminomethylpyridine)aquazinc(II) monohydrate (2), have been synthesized from the same free ligands, which are 2,4-dichloro-5-sulfamoylbenzoic acid (Hsba) and 2-aminomethylpyridine (amp). They have been characterized by elemental, spectral (¹H NMR, IR and UV-vis.) and thermal analyses. Additionally, magnetic measurement and single crystal X-ray diffraction technique were applied to compound 2. In the complex, Zn(II) ion exhibits a distorted octahedral configuration coordinated by O1 and O1ⁱ atoms of two mono dentante sba anions and N1, N2, N2ⁱ atoms of bidentante amp anion and a water molecule (O1w). The free ligands Hsba and amp, and the products 1 and 2, and acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on human Carbonic Anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cell by affinity chromatography for their hydratase and esterase activities. The IC₅₀ values of products 1 and 2 for hydratase activity are 0.26 and 0.13 μM for hCA I and 0.30 and 0.15 μM for hCA II, respectively. The IC₅₀ values of the same inhibitors for esterase activity are 0.32 and 0.045 μM for hCA I and 0.29 and 0.23 μM for hCA II, respectively. In relation to esterase activities, the inhibition equilibrium constants (K_i) were also determined and found 0.25 and 0.058 μM on hCA I and 0.22 and 0.24 μM on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of newly synthesized compounds 1 and 2 to parent compounds Hsba and amp and to AAZ indicated that 1 and 2 have effective inhibitory activity on hCA I and II, and might be used potential inhibitors.

Full text of DOI:10.1016/j.bmc.2009.11.031

Bioorganic & Medicinal Chemistry 18 (2010) 930–938
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, characterization and antiglaucoma activity of a novel proton
transfer compound and a mixed-ligand Zn(II) complex
b
a
a
a
c
Cengiz Yenikaya a, , Musa Sarı , Metin Bülbül , Halil Ilkimen , Hülya Çelik , Orhan Büyükgüngör
_
*
a Department of Chemistry, Faculty of Arts and Sciences, Dumlupınar University, 43100 Kütahya, Turkey
b Department of Physics Education, Gazi University, 06500 Ankara, Turkey
c Department of Physics, Faculty of Arts and Sciences, Ondokuz Mayıs University, 55139 Kurupelit, Samsun, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel proton transfer compound, pyridin-2-ylmethanaminium 2,4-dichloro-5-sulfamoylbenzoate (1),
and mixed-ligand Zn(II) complex, bis(2,4-dichloro-5-sulfamoylbenzoate)(2-aminomethylpyri-
Received 28 August 2009
Revised 9 November 2009
Accepted 13 November 2009
Available online 20 November 2009
a
dine)aquazinc(II) monohydrate (2), have been synthesized from the same free ligands, which are 2,4-
dichloro-5-sulfamoylbenzoic acid (Hsba) and 2-aminomethylpyridine (amp). They have been character-
ized by elemental, spectral (1H NMR, IR and UV–vis.) and thermal analyses. Additionally, magnetic mea-
surement and single crystal X-ray diffraction technique were applied to compound 2. In the complex,
Zn(II) ion exhibits a distorted octahedral configuration coordinated by O1 and O1i atoms of two mono
dentante sba anions and N1, N2, N2i atoms of bidentante amp anion and a water molecule (O1w). The
free ligands Hsba and amp, and the products 1 and 2, and acetazolamide (AAZ) as the control compound,
were also evaluated for their in vitro inhibitor effects on human Carbonic Anhydrase isoenzymes (hCA I
and hCA II) purified from erythrocyte cell by affinity chromatography for their hydratase and esterase
Keywords:
2,4-Dichloro-5-sulfamoylbenzoic acid
Inhibition
2-Amino-3-methylpyridine
Proton transfer
Zn(II) complex
Glaucoma
Crystal structure
activities. The IC50 values of products 1 and 2 for hydratase activity are 0.26 and 0.13
l
M for hCA I
M for hCA II, respectively. The IC50 values of the same inhibitors for esterase activity
M for hCA I and 0.29 and 0.23 M for hCA II, respectively. In relation to esterase activ-
M on hCA
M on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of
and 0.30 and 0.15
are 0.32 and 0.045
l
l
l
ities, the inhibition equilibrium constants (Ki) were also determined and found 0.25 and 0.058
l
I and 0.22 and 0.24
l
newly synthesized compounds 1 and 2 to parent compounds Hsba and amp and to AAZ indicated that
1 and 2 have effective inhibitory activity on hCA I and II, and might be used potential inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
an elevation in intraocular pressure (IOP), and is the second leading
cause of blindness worldwide.14,15 Since CA inhibitors have been
The sulfonamides constitute an important class of drugs, with
several types of pharmacological agents possessing antibacterial,
antiglaucoma, diuretic, hypoglycemic, antithyroid, protease inhib-
itory and antitumor activity, among others.1–4 With the sulfona-
mides as lead structures, different classes of pharmacological
agents have been obtained, such as antibacterial sulfonamides that
inhibit the zinc enzyme Carbonic Anhydrase (CA; EC 4.2.1.1), in
which the zinc ion is the prosthetic group and is coordinated by
histidine side chains in the active site. CA catalyzes the reversible
hydration of carbon dioxide to bicarbonate and protons, a very
simple but critically important physiological reaction for organ-
isms.5–10 This enzyme has 16 different isoenzymes presently
known. Several of these isoenzymes (CA-II and CA-IV) are ex-
pressed in human eyes11–14 causing glaucoma, which is a group
of diseases characterized by the gradual loss of visual field due to
shown to reduce intraocular pressure exclusively by lowering the
aqueous humor flow, these compounds have been used for years
for the treatment of glaucoma.16,17
Although there are limited reported crystal structure studies on
Hsba derivatives,18,19 a huge number of crystal structures of a wide
range of metal complexes with aminopyridines were studied by
means of X-ray diffraction. Aminopyridines and their derivatives
in most cases act as monodentate ligands which coordinate the
metal ions through the nitrogen of the ring,20 though there are sev-
eral works on 2-aminomethylpyridine complexes in which the
amino group also participates in coordination.21–25 2-Aminometh-
ylpyridines serve as useful chelating ligands in a variety of inor-
ganic and organometallic applications.26,27 The high incidence of
pharmacological activity among heteroaromatic amines has also
stimulated many research efforts to prepare compounds of this
structural type.28,29
Zinc was chosen in this work, because zinc complexes have
antimicrobial effect against bacteria, fungi, and viruses, as well as
* Corresponding author.
E-mail address: yenikaya@dumlupinar.edu.tr (C. Yenikaya).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.031

C. Yenikaya et al. / Bioorg. Med. Chem. 18 (2010) 930–938
931
Table 1
inhibitory effects against carbonic anhydrases, carboxy peptidases,
thermolysin, and alcohol dehydrogenase.30–32
Crystal data and structure refinement details for compound 2
In this study, a novel proton transfer compound, namely pyridin-
2-ylmethanaminium 2,4-dichloro-5-sulfamoylbenzoate (1), and a
mixed-ligand Zn(II) complex, namely bis(2,4-dichloro-5-sul-
famoylbenzoate)(2-aminomethylpyridine)aquazinc(II) monohy-
drate (2), formulated as (Hamp)+(sba)ꢀ and [Zn(sba)2(amp)
(H2O)]ꢁH2O, respectively, have been synthesized and characterized
by elemental, spectral (1H NMR, IR and UV–vis) and thermal analy-
ses. Magnetic measurement and single crystal X-ray analysis of the
complex (2) were also reported. Furthermore, we have investigated
the potential use of these compounds as new inhibitors of hCA iso-
enzymes in the treatment of glaucoma.
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system, space group
Unit cell dimensions: (Å, °)
C20H18Cl4N4O10S2Zn
745.71
296(2)
0.71073
Monoclinic, P 21/m
a
b
c
b
7.8650(4)
18.9429(9)
9.1899(5)
89.839(4)
11369.16(12)
2
1.502
1.809
752
2.15–26.50
0.580 ꢂ 0.460 ꢂ 0.340
Integration
ꢀ9 6 h 6 9, ꢀ23 6 k 6 23,
ꢀ11 6 l 6 11
17,221
V (Å3)
Z
l
(mmꢀ1
)
Dcalcd (Mg mꢀ3
F(0 0 0)
)
2. Experimental section
h range (°)
Crystal size (mm)
Absorption correction
Limiting indices
2.1. General methods and materials
All chemicals used were analytical reagents and were commer-
cially purchased from Aldrich. Zn(CH3COO)2ꢁ2H2O, 2-aminometh-
ylpyridine and 2,4-dichloro-5-sulfamoylbenzoic acid were used
as received. Elemental analyses for C, H, N and S were performed
on Elementar Vario III EL and Zn was detected with Perkin Elmer
Optima 4300 DV ICP-OES. 1H NMR spectra were recorded with Bru-
ker DPX FT NMR (500 MHz) spectrometer (SiMe4 as internal stan-
dard 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 spectrometer using ATR techniques. Thermal analyses
were performed on SII Exstar 6000 TG/DTA 6300 model using plat-
inum crucible with 10 mg sample. Measurements were taken in
the static air, within 30–700 °C temperature range. The UV–vis
spectra were obtained for aqueous solutions of the compounds
(10ꢀ3 M) with a SHIMADZU UV-2550 spectrometer in the range
900–200 nm. Magnetic susceptibility measurement at room tem-
perature was performed using a Sherwood Scientific Magway
MSB MK1 model magnetic balance by the Gouy method using
Hg[Co(SCN)4] as calibrant. Molar conductance of the compound
was determined in DMSO (10ꢀ3 M) at room temperature using a
WTW Cond 315i/SET Model conductivity meter.
Reflections collected
Unique reflections
Observed reflections
Parameters refined
Refinement method
Rint
Final R indices [I P 2
R indices (all data)
Goodness of fit on F2
2920
2728
223
Full-matrix least-squares on F2
0.0298
r
(I)]
R1 = 0.0267, wR1 = 0.0664
R2 = 0.0292, wR2 = 0.0676
1.076
ꢀ0.467 and 0.427
Largest difference peak
and hole (e Åꢀ3
)
the compound 2. Crystallographic data of 2 were recorded on a
Stoe IPDS II CCD X-ray diffractometer employing plane graphite
monochromatized with Mo K
ꢀ 2h scan mode. Unit cell parameters were refined from the
a radiation (k = 0.71073 Å), using
x
setting angles of 25 centered reflections in the range of
2.15 6 h 6 26.50 for the complex compound. The structures were
solved by the direct methods and refined by full-matrix least-
squares techniques on F2 using the solution program SHELXS-97
and refined using SHELXL-97.33 The empirical absorption corrections
were applied by the integration method via X-RED software.34 All
non-hydrogen atoms were refined with anisotropic displacement
parameters and hydrogen atoms were included in their idealized
positions and refined isotropically except for the water molecules.
Hydrogen atoms of water molecules were located from difference
maps and refined with isotropic thermal parameters, with distance
of O–H = 0.813(13)–0.822(11) Å and with Uiso(H) = 1.2Ueq(O). The
crystal data and structure refinement details for compound 2 are
given in Table 1. ORTEP drawings35 of the complex compound with
40% probability displacement thermal ellipsoids and atom-label-
ling schemes are shown in Figures 1, 2 and 3.
2.2. Synthesis of (Hamp)+(sba)ꢀ (1) and
[Zn(sba)2(amp)(H2O)]ꢁH2O (2)
A solution of amp (0.54 g, 5 mmol) in 10 mL ethanol was added
to the solution of Hsba (1.35 g, 5 mmol) in 10 mL ethanol. The mix-
ture was refluxed for 3 h, and then was cooled to room tempera-
ture. The reaction mixture was kept at room temperature for 3 h
to give white solid of 1 (1.51 g, 80% yield).
A solution of Zn(CH3COO)2ꢁ2H2O (0.199 g, 1 mmol) in water
(10 mL) was added dropwise to the mixed solution of free ligands
(0.108 g, 1 mmol for amp and 0.54 g, 2 mmol for Hsba) in ethanol
(20 mL) with stirring at room temperature for 1 h. The reaction
mixture was kept at room temperature for two weeks to give white
crystalline solid (0.412 g, 55% yield). On recrystallisation, colorless
crystals of 2 suitable for X-ray diffraction were separated and
washed with water.
2.4. Purification of isoenzymes hCA-I and II from human
erythrocytes
In order to purify hCA-I and II isoenzymes, first, human blood
was centrifuged at 1500 rpm for 20 min, and after the removal of
the plasma, the erythrocytes were washed with an isotonic solu-
tion (0.9% NaCl). After that, the erythrocytes were lysed with 1.5
volume of ice-cold water. The lysate was centrifuged at
20,000 rpm for 30 min to remove cell membranes and non-lysed
cells. The pH of the supernatant was adjusted to 8.7 with tris and
was then loaded onto an affinity column containing Sepharose-
Anal. Calcd for 1 (C13H13Cl2N3O4S): C, 41.25; H, 3.45; N, 11.13; S,
8.48. Found: C, 41.28; H, 3.46; N, 11.11; S, 8.48; Anal. Calcd for 2
(C20H18Cl4N4O10S2Zn): C, 32.20; H, 2.40; N, 7.50; S, 8.58; Zn, 8.70.
Found: C, 32.21; H, 2.43; N, 7.51; S, 8.60; Zn, 8.77.
2.3. Crystal structure determination of 2 (C20H18Cl4N4O10S2Zn)
4B-L-tyrosine-p-aminobenzene sulfonamide as the binding group.
After extensive washing with 25 mM tris–HCl/22 mM Na2SO4 (pH
8.7), the hCA-I and II isoenzymes were eluted with 1.0 M NaCl/
The crystal and instrumental parameters used in the unit-cell
determination and data collection are summarized in Table 1 for

932
C. Yenikaya et al. / Bioorg. Med. Chem. 18 (2010) 930–938
25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH
5.6).36,37 The amount of purified protein was estimated by the
Bradford method38 and SDS–PAGE was carried out to determine
whether the elute contained the enzyme.39
2.5. Hydratase activity assay
Carbonic anhydrase activity was assayed by following the
hydration of CO2 according to the method described by Wilbur
and Anderson.40 CO2-hydratase activity as an enzyme unit (EU)
was calculated by using the equation ((t0 ꢀ tc)/tc) where t0 and tc
are the times for pH change of the nonenzymatic and the enzy-
matic reactions, respectively. IC50 values (the concentration of
inhibitor producing a 50% inhibition of CA activity) have been ob-
tained as in vitro for free ligands amp and Hsba, and the synthe-
sized compounds 1 and 2, and acetazolamide (AAZ) as the
control compound.
Figure 1. An ORTEP drawing of asymmetric unit of 2 with the atom-numbering
scheme. Displacement ellipsoids are drawn at the 40% probability level.
2.6. Esterase activity assay
Carbonic anhydrase activity was assayed by following the
change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to
4-nitrophenylate ion over a period of 3 min at 25 °C using a spec-
trophotometer (CHEBIOS UV–vis) according to the method de-
scribed in the literature.41,42 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 mL of 3 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. IC50 values
have been obtained as in vitro for free ligands amp and Hsba,
and the synthesized compounds 1 and 2, and AAZ as the control
compound.
2.7. Determination of Ki values
The method for determination of Ki values is described else-
where.43–47 According to this study, in its first part, IC50 values
have been obtained as in vitro. IC50 of the inhibitors (free ligands
amp and Hsba, and the synthesized compounds 1 and 2, and AAZ
as the control compound) were assayed by the hydrolysis of p-
nitrophenylacetate on esterase activities of CA isoenzymes in the
presence of various inhibitor concentrations. The absorbance was
determined at 348 nm after 3 min.43 Regression analysis graphs
were drawn by plotting inhibitor concentrations versus enzyme
activity by using Microsoft Excel Program.
Figure 2. An ORTEP drawing of symmetric unit of 2 with the atom-numbering
scheme. Displacement ellipsoids are drawn at the 40% probability level.
one sba anion, one amp anion, in which methylene carbon (C13)
has one hydrogen atom and amine nitrogen (N2) has half hydrogen
atom, one Zn(II) cation, half coordinated water molecule and half
uncoordinated water molecule (Fig. 1).
In the second part of the study, the concentrations of inhibitors
which give results 30%, 50%, and 70% inhibition on isoenzymes
were determined at five different substrate concentrations
In the symmetric unit (Fig. 2), the coordination environment of
the Zn(II) ion has a distorted octahedral configuration coordinated
by O1 and O1i atoms of two mono dentante sba anions and N1, N2,
N2i atoms of bidentante amp anion and a water molecule (O1w).
One of the H atoms on methylene carbon (C13) of amp molecule
belongs to neighbor symmetric unit, while amine nitrogen (N2)
in the amp molecule bears one H atom. The other H atom of –
NH2 group of free amp molecule was probably lost during complex
formation in order to balance the charge in the complex (2). Two
sba oxygen atoms (O1, O1i) and two amp nitrogen atoms (N1,
N2) comprise equatorial plane, while water oxygen atom (O1w)
and amp nitrogen atoms (N1) occupy the axial positions [O1w–
Zn–N1 = 164.88(9)°]. The Zn–O bond distances range from
1.9926(13) to 2.191(2) Å, and Zn–N1 and Zn–N2 bond distances
are 2.072(3) and 2.161(2) Å, respectively. These values are in good
agreement in the literature.48–52 The Zn–O1w bond distance, aris-
ing from coordination of the water molecule to the central metal
ion, is 2.191(2) Å, which is much longer than the Zn–O (carboxyl-
ate) bond distances.
(2.4 lM, 4.8 lM, and 7.3 lM). At each of these inhibitor concentra-
tions (30%, 50%, and 70%), enzyme activity was then measured in
the presence of various substrate concentrations given above and
the data were linearized with Lineweaver–Burk plot in order to ob-
tain Ki value (Fig. 4).
3. Results and discussion
3.1. Crystal structure of [Zn(sba)2(amp)(H2O)]ꢁH2O (2)
The molecular structure of [Zn(sba)2(amp)(H2O)]ꢁH2O (2), with
the atom labeling of asymmetric unit and symmetric unit of 2,
are shown in Figures 1 and 2, respectively. The crystal packing dia-
gram of 2 is shown in Figure 3. The details of the crystal structure
solution are summarized in Table 1. The selected bond lengths and
angles are listed in Table 2. The compound crystallizes in the
monoclinic P 21/m space group. The asymmetric unit contains

C. Yenikaya et al. / Bioorg. Med. Chem. 18 (2010) 930–938
933
structure ( Figs. 1 and 3). The parameters for hydrogen bonding
interaction in the complex are as follows: Hꢁ ꢁ ꢁO2w 2.21(2) Å,
N2ꢁ ꢁ ꢁO2w 2.932(4) Å, N2–H2ꢁ ꢁ ꢁO2w 143.2(11)°.
3.2. FT-IR measurements
In the high frequency region, weak bands between 3100 and
3037 and between 3037 and 2850 cmꢀ1 are attributed to the
stretching
respectively. There is a broad absorption band at 2900 cmꢀ1 attrib-
uted to the (OH) vibrations of carboxylate group of Hsba. This
m(C–H) vibrations of aromatic and methylene groups,
m
band is not observed in compound 1 due to proton transfer. There
are also broad absorption bands at 3495 and 2623 cmꢀ1 attributed
to the m(OH) vibrations of coordinated and uncoordinated water
molecules, respectively, in the complex compound 2. NH2 vibra-
tions at 3364 and 3289 cmꢀ1 in amp are shifted to lower frequen-
cies in compound 1 as 2821, 2740, 2707 cmꢀ1 due to proton
transfer to amine group.53 An absorption band was arisen at
3321 cmꢀ1, which was assigned to NH group of amp in IR spectrum
of the complex compound (2). NH2 vibrations of sulfonamides in
free Hsba (3425, 3278 cmꢀ1), in compounds 1 (3340, 3159 cmꢀ1
)
and 2 (3365, 3174 cmꢀ1) are observed with similar pattern. The
absorption bands for compounds 1 and 2 have shifted 80 cmꢀ1 in
average to lower frequencies due to molecular interaction in solid
state. The strong absorption bands for SO2 groups in Hsba in com-
pounds 1 and 2 are observed at the region of 1400–1070 cmꢀ1 with
similar profiles and almost similar vibrations.54 The strong C@O
vibration at 1684 cmꢀ1 of Hsba is shifted to 1621 and 1624 cmꢀ1
for compounds 1 and 2, respectively, indicating the role of carbox-
ylic acid group of free Hsba on the structures of the compounds
Figure 3. Crystal packing diagram of 2.
prepared in this study.55 The
m(C–O) vibration of Hsba is also
0.12
shifted from 1169 cmꢀ1 to 1116 and 1123 cmꢀ1 in 1 and 2, as indi-
cated in.56,57 The strong absorption bands at the region of 1400–
0.09
0.06
0.03
0
1590 cmꢀ1 are attributed to the
m(C@N) and m(C@C) vibrations in
free ligands, amp and Hsba, and compounds 1 and 2.58 The ring
wagging vibrations of the pyridine groups are also observed at
751 and 625 cmꢀ1 for amp, 770 and 628 cmꢀ1 for compound 1,
and 799 and 687 cmꢀ1 for compound 2. The weak bands in the re-
gion of 573 and 520 cmꢀ1 are responsible for the Zn–N and Zn–O
vibrations, respectively, of the complex 2.
-0.5
0
0.5
1
1.5
3.3. 1H NMR studies of (Hamp)+(sba)ꢀ (1) and
[Zn(sba)2(amp)(H2O)]ꢁH2O (2)
1/[S]x103 (M-1)
-0.03
Figure 4. Ki graph obtained from in vitro studies for compound 1 on hCA-II.
The 1H NMR spectra of the compounds (1 and 2) were obtained
in DMSO-d6 at room temperature using TMS as internal standard.
Table 3 lists complete 1H NMR assignments for the compounds 1
and 2. The 1H signals were assigned on the basis of chemical shifts,
multiplicities, intensity of the signals and coupling constants. The
CH2 protons (H8) are easily distinguishable as singlets, and they
are observed at 4.19 ppm for 1 and 4.16 ppm for 2. H5 and H6 pro-
tons of the amp rings are triplets and they are observed at
7.39 ppm (3JH5-H4,6 = 6.15 Hz) and 7.83 ppm (3JH6–H5,7 = 7.72 Hz) in
compound 1, respectively, and at 8.00 ppm (3JH5–H4,6 = 7.61 Hz)
and 7.51 ppm (3JH6–H5,7 = 6.36 Hz, one of the bands of the triplet
is thought to be under neighbor doublet due to the different cou-
pling constants for these sets of peaks, see Figure 6) in compound
2, respectively. In addition, H4 and H7 protons of the amp rings are
doublets as expected and arisen at 7.43 ppm (3JH4–H5 = 7.82 Hz)
and 8.57 ppm (3JH7–H6 = 4.65 Hz) in compound 1, respectively,
and at 8.61 ppm (3JH4–H5 = 6.36 Hz) and 7.54 ppm (3JH7–
H6 = 8.07 Hz) in compound 2, respectively. The 1H NMR spectrum
Table 2
Selected bond distances (Å) and angles (°) for 2
C2–S1
1.7793(18)
1.7239(19)
1.7288(18)
2.161(2)
2.072(3)
2.072(3)
Zn1–O1
Zn1–O1i
S1–O3
S1–O4
S1–N3
1.9926(13)
1.9926(13)
1.4295(16)
1.4314(16)
1.5836(18)
2.191(2)
C3–Cl1
C5–Cl2
Zn1–N1
Zn1–N2
Zn1–N2i
N2–N2i
Zn1–O1w
1.011(8)
O1–Zn1–N1
O1–Zn1–N2
O1–Zn1–O1w
O3–S1–O4
97.77(6)
145.38(11)
92.17(6)
119.26(10)
107.84(10)
107.28(10)
97.49(9)
N1–Zn1–N2
N1–Zn1–O1w
N2–Zn1–O1w
N2–Zn1–N1
N2–Zn1–N2i
O1i–Zn1–N2i
O1–Zn1–N2i
78.26(11)
164.88(9)
87.08(11)
78.26(11)
28.3(2)
O3–S1–N3
O4–S1–N3
145.38(11)
117.13(11)
O1–Zn1–O1i
Symmetry codes: (i) x, ꢀy+1/2, z.
0
0
of compound 2 contains singlet peaks for H1–H1 and H2–H2 on
aromatic ring of sba at 8.22 and 7.76 ppm with the intensity of
two H atoms, respectively. However, in compound 1, these peaks
are seen at 7.91 ppm (H1) and 7.62 ppm (H2) with the intensity
Hydrogen bond between the uncoordinated water molecule and
N–H group of amp plays an important role in stabilizing the crystal

934
C. Yenikaya et al. / Bioorg. Med. Chem. 18 (2010) 930–938
0
of one H atom, which clearly indicate the formation of the proton
transfer compound with 1:1 ratio of amp and Hsba. The broad sin-
glet peaks at 4.3 ppm (H9, with one H intensity) and 7.74 ppm (H3–
H3 , with four H intensity) are assigned to –NH and –SO2NH2
groups in compound 2. This result also indicates that one hydrogen
atom was lost from NH2 group of amp to give NHꢀ in order to bal-
Table 3
1H NMR chemical shifts (ppm) with coupling constants and assignments for compounds 1 and 2
5
H
H6
4H
2'
NH93
H
Cl
-OOC
Cl
7
H
8
8
H2C
N
H2C
Cl
N
N
OCO
4
H
11
1
H
H2
Zn
9 H
10
H2O
OH2
N
3'
1H'
H1
SO2NH2
OCO
H23NO2S
H7
Cl
5H
Cl
6H
2H
SO2NH23
Cl
H8
4.19 (2H, s)
H8
H9
H6
H7
4.16 (2H, s)
H5
7.39 (1H, t) [3JH5–H4,6 = 6.15 Hz]
7.43 (1H, d) [3JH4–H5 = 7.82 Hz]
7.62 (1H, s)
4.30 (1H, s) (broad)
7.51 (1H, t) [3JH6–H5,7 = 6.36 Hz]
7.54 (1H, d) [3JH7–H6 = 8.07 Hz]
7.74 (4H, s) (broad)
7.76 (2H, s)
H4
H2
H6
7.83 (1H, t) [3JH6–H5,7 = 7.72 Hz]
7.91 (1H, s)
H3, H3
H2, H2
H5
0
0
H1
H7
8.57 (1H, d) [3JH7–H6 = 4.65 Hz]
Not observed
8.0 (1H, t) [3JH5–H4,6 = 7.61 Hz]
8.22 (2H, s)
H9, H3
H1, H1
0
H4
8.61 (1H, d) [3JH4–H5 = 6.36 Hz]
Not observed
H
10, H11
Figure 5. 1H NMR spectrum of compound 1.

C. Yenikaya et al. / Bioorg. Med. Chem. 18 (2010) 930–938
935
Figure 6. 1H NMR spectrum of compound 2.
TGA
DTA
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
20.0
500.0
450.0
400.0
350.0
300.0
250.0
200.0
150.0
100.0
50.0
0.0
-20.0
-40.0
-60.0
-80.0
-100.0
DTG
0.0
-50.0
100.0
200.0
300.0
400.0
500.0
Temp Cel
Figure 7. The TG-DTG and DTA curves of 1.
ance the charge in complex compound (2). The hydrogen atoms, on
–NH3 (H9), –SO2NH2 (H3) in compound 1, and on both water mol-
ecules (H10 and H11) in compound 2, have not been observed in the
1H NMR spectra.

936
C. Yenikaya et al. / Bioorg. Med. Chem. 18 (2010) 930–938
The room temperature 1H NMR spectra, 500 MHz, for both com-
Table 4
Optical properties of compounds 1 and 2, and the free ligands amp and Hsba in water
and DMSO
pounds ( Figs. 5 and 6) clearly indicate the formation of proton
transfer compound and complex compound, and confirm that solid
state structure of 2 is retained in solution.48
kmax (e)
Amp
Hsba
(1)
(2)
3.4. Thermal analyses of 1 (C13H13Cl2N3O4S) and 2
(C20H18Cl4N4O10S2Zn)
Water
DMSO
209(25520)
236(12340)
209(26150)
209(23460)
236(10530)
262(27370)
261(44340)
280(10200)
257(30100)
264(41020)
280(15750)
257(35120)
285(11230)
256(24450)
283(16800)
Figures 7 and 8 show the TG-DTG and DTA curves of compounds
1 and 2, respectively. The endothermic first stage (DTGmax
=
182 °C), between 30 and 188 °C, corresponds to the loss of the
NH3 from amine group of 1 (found 4.50, calcd 4.50). The second
stage, an endothermic peak (DTGmax = 241, 254 and 261 °C) be-
tween 188 and 263 °C, corresponds to the loss of C6H6N from
amine group of 1 (found 24.5, calcd 24.4). Sulfonamide (SO2NH2)
and carboxylate (COO) groups of Hsba are decomposed in the third
stage between 236 and 450 °C with DTGmax at 273, 276, 280 and
314 °C (found 32.8, calcd 32.8). The final stage was the decomposi-
tion of the residue of sba, C6H2Cl2, between 450 and 560 °C with
DTGmax at 496 °C (found 38.2, calcd 38.3).
3.5. UV–vis spectrum, magnetic susceptibility and conductivity
The electronic spectra of compounds 1 and 2, and the free li-
gands amp and Hsba were recorded in solution at a 1 ꢂ 10ꢀ3 M
concentration at room temperature (Table 4). Characteristic
p–p*
transitions are in the spectra of 1 (209 and 280 nm) and of 2
(209 and 236 nm) in water solution with the same profiles as the
free ligands Hsba (209 and 236 nm) and amp (262 nm). Two sets
For the compound 2, the first stage, an endothermic peak
(DTGmax = 173 and 175 °C) between 30 and 184 °C, corresponds
to the loss of the 2 mol of crystal water molecules (found 4.2, calcd
4.8). The endothermic second stage, (DTGmax = 187 °C) between
184 and 266 °C, is consistent to the loss of the C2H3N from amine
group of 2 (found 5.2, calcd 5.5). The residue of amine group
(C4H3N) and 2 moles sulfonamide (SO2NH2) are decomposed endo-
thermically in the third stage between 266 and 371 °C with DTGmax
at 302, 319, 334 and 345 °C (found 30.0, calcd 30.1). The fourth
stage, an endothermic peak (DTGmax = 372 and 443 °C) between
371 and 485 °C, agrees to the loss of the 4 moles of chlorine of
sba unit in compound 2. In the fifth stage, decomposition of the
residue of sba is observed between 485 and 600 °C with DTGmax
at 568 and 595 °C (found 32.0, calcd 31.8). The final decomposition
product was ZnO identified by IR spectroscopy (found 9.7%, calcd
10.8).
of characteristic
except amp in DMSO solutions, which were located around 257
and 283 nm. The more energetic * transition bands in water
around 209 nm for all compounds, except amp, were red shifted
to around 257 nm in DMSO solution spectra. The other
absorption bands at 283 nm for compound 1 in water were also ob-
served in the same region (280 nm) in the spectra recorded in
DMSO. The intensities of the absorption bands for all compounds
in DMSO are, in general, higher than in water.
All the data which we obtained from the electronic spectra of
the free ligands do not show any marked differences from those
of either proton transfer compound or Zn(II) complex in both
solutions.
p–p* transitions were observed for all compounds,
p–p
p–p*
Zn(II) complex does not show any magnetic properties on mea-
surements at room temperature due to d10 electronic structure of
TGA
100.0
100.0
80.0
60.0
40.0
20.0
0.0
600.0
400.0
200.0
0.0
80.0
60.0
40.0
20.0
DTG
DTA
-200.0
-400.0
-600.0
-20.0
-40.0
-60.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
Temp Cel
Figure 8. The TG-DTG and DTA curves of 2.

C. Yenikaya et al. / Bioorg. Med. Chem. 18 (2010) 930–938
937
Table 5
IC50 values of hydratase and esterase activities on hCA I and hCA II isoenzymes and Ki values
Inhibitor
Hydratase IC50
(
l
M)
Esterase IC50
(
l
M)
Ki values (
l
M)
hCA II
3.8
No inhibition
22
hCA I
3.6
hCA II
hCA I
6.1
hCA II
hCA I
5.2
AAZ
Amp
Hsba
1
2.8
4.5
No inhibition
0.43
No inhibition
0.38
0.30
No inhibition
3.2
No inhibition
18.0
0.29
No inhibition
7.7
0.26
0.13
0.32
0.045
0.25
0.058
0.22
0.24
2
0.15
0.23
zinc(II) ion. The conductivity data in DMSO and in water are the 4.1
liminary biological evaluation suggests that derivatives of such
compounds should be subject to further inhibition in vivo tests.
Proton transfer compound and coordination compound of zinc(II)
ion, which are prepared from the same ligands, have shown almost
similar inhibition result on hCA-II isoenyzme. These results also
suggest that further studies on such compounds are worthwhile
to obtain correlation of inhibitory effects on hCA-II.
and 11.5
X
ꢀ1 cm2 molꢀ1, respectively, indicating that the complex
2 is non-ionic.59 This result agrees that one mole of sba anion and
one mole of amp anion in asymmetric unit to form neutral coordi-
nation compound.
3.6. In vitro inhibition studies
Inhibition effects of parents compounds (amp and Hsba), newly
synthesized proton transfer compound (1) and Zn(II) complex (2)
and AAZ as the control compound on hCA I and hCA II enzymes
were studied by hydratase and esterase activity methods and then
Ki values were determined for each compound (Table 5).
Acknowledgments
The authors acknowledge the support provided by Dumlupınar
University Research Fund (Grant No. 2007/2). In addition, the
authors would like to thank the Faculty of Arts and Sciences at
Ondokuz Mayıs University, Turkey, for use of the Stoe IPDS-2 dif-
fractometer purchased under Grant F.279 of the University Re-
search Fund.
According to in vitro studies, the IC50 values of hydratase activ-
ities of newly synthesized compounds 1 and 2 (0.26 and 0.13
for hCA I and 0.30 and 0.15 M for hCA II, respectively) are lower
than IC50 values of Hsba (0.43 and 0.38 M for hCA I and II) and
of AAZ (3.6 and 2.8 M for hCA I and II). The IC50 values obtained
from esterase activities of all compounds studied have been ob-
served in similar trend, which are 0.32 and 0.045 M for hCA I
and 0.29 and 0.23 M for hCA II for 1 and 2, respectively. Hsba
and AAZ have esterase activities as 3.2 and 6.1 for hCA I and 18.0
and 4.5 M for hCA II, respectively. Other parent compound,
lM
l
l
l
Supplementary data
l
Crystallographic data (excluding structure factors) for the struc-
tures in this paper have been deposited with the Cambridge Crys-
tallographic Data Centre as supplementary publication No. CCDC
745613 for 2. Copies of the data can be obtained, free of charge,
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK,
(fax: +44 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk). Sup-
plementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2009.11.031.
l
l
amp, does not present any hydratase and esterase activities.
In relation to esterase activities, the inhibition equilibrium con-
stants (Ki) were also determined. Novel compounds 1 and 2 have
significant Ki values which are, respectively 0.25 and 0.058
for hCA I and 0.22 and 0.24 M for hCA II compared to Hsba (7.7
and 22.0 M for hCA I and II) and AAZ (5.2 and 3.8 M for hCA I
and II). Coordination compound (2) has shown remarkable inhibi-
tion on hCA I (Ki: 0.058 M), showing higher inhibition than 1,
lM
l
l
l
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