Synthesis and characterisation of two novel proton transfer compounds and their inhibition studies on carbonic anhydrase isoenzymes

Article abstract of DOI:10.3109/14756361003733639

Two novel proton transfer compounds were prepared between 2,4-dichloro-5-sulphamoylbenzoic acid (lasamide) (Hsba) and ethylenediamine (en), namely ethane-1,2-diaminium 2,4-dichloro-5-sulphamoylbenzoate (1), and also between Hsba and 2-amino-3-methylpyridine (2-amino-3-picoline) (amp), namely 2-amino-3-methylpyridinium 2,4-dichloro-5-sulphamoylbenzoate (2). All these were characterised by elemental, spectral (IR and UV-vis), thermal analyses, and single crystal X-ray diffraction studies. Compounds 1 and 2 crystallised in the P-1 and P21/c space groups, respectively. Intermolecular non-covalent interactions, such as ion pairing, hydrogen bonding, and πI€-πI€ stacking were observed for these ionic compounds. The free ligands Hsba, en and amp, the products 1 and 2, and acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on the human carbonic anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cells by affinity chromatography for their hydratase and esterase activities. The half maximal inhibitory concentration (IC50) values for products 1 and 2 with respect to hydratase activity are 0.15 and 0.32 μ M for hCA I and 0.06 and 0.15 μ M for hCA II, respectively. The IC50 values of the same inhibitors for esterase activity are 0.13 and 0.8 μ M for hCA I and 0.14 and 0.1 μ M for hCA II, respectively. In relation to esterase activities, the inhibition equilibrium constants (Ki) were also determined and found to be 0.137 and 0.99 μ M on hCA I and 0.157 and 0.075 μ M on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of the newly synthesised compounds 1 and 2 to the parent compounds Hsba and amp and also to AAZ indicated that 1 and 2 have an effective inhibitory activity on hCA I and II, and might be used as potential inhibitors.

Full text of DOI:10.3109/14756361003733639

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(1): 104–114
Copyright © 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756361003733639
RESEARCH ARTICLE
Synthesis and characterisation of two novel proton transfer
compounds and their inhibition studies on carbonic
anhydrase isoenzymes
Cengiz Yenikaya1, Musa Sarı2, Metin Bülbül1, Halil İlkimen1, Burcu Çınar1, and Orhan
Büyükgüngör3
1Department of Chemistry, Faculty of Arts and Sciences, Dumlupınar University, Kütahya, Turkey, 2Department of Physics
Education, Gazi University, Beşevler, Ankara, Turkey, and 3Department of Physics, Faculty of Arts and Sciences, Ondokuz
Mayıs University, Kurupelit, Samsun, Turkey
Abstract
Two novel proton transfer compounds were prepared between 2,4-dichloro-5-sulphamoylbenzoic acid (lasamide)
(Hsba) and ethylenediamine (en), namely ethane-1,2-diaminium 2,4-dichloro-5-sulphamoylbenzoate (1), and also
between Hsba and 2-amino-3-methylpyridine (2-amino-3-picoline) (amp), namely 2-amino-3-methylpyridinium
2,4-dichloro-5-sulphamoylbenzoate (2). All these were characterised by elemental, spectral (IR and UV-vis), thermal
analyses, and single crystal X-ray diffraction studies. Compounds 1 and 2 crystallised in the P-1 and P21/c space
groups, respectively. Intermolecular non-covalent interactions, such as ion pairing, hydrogen bonding, and π-π
stacking were observed for these ionic compounds. The free ligands Hsba, en and amp, the products 1 and 2, and
acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on the human
carbonic anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cells by affinity chromatography for
their hydratase and esterase activities. The half maximal inhibitory concentration (IC50) values for products 1 and 2
with respect to hydratase activity are 0.15 and 0.32 µM for hCA I and 0.06 and 0.15 µM for hCA II, respectively. The
IC50 values of the same inhibitors for esterase activity are 0.13 and 0.8 µM for hCA I and 0.14 and 0.1 µM for hCA II,
respectively. In relation to esterase activities, the inhibition equilibrium constants (Ki) were also determined and
found to be 0.137 and 0.99 µM on hCA I and 0.157 and 0.075 µM on hCA II for 1 and 2, respectively. The comparison
of the inhibition studies of the newly synthesised compounds 1 and 2 to the parent compounds Hsba and amp
and also to AAZ indicated that 1 and 2 have an effective inhibitory activity on hCA I and II, and might be used as
potential inhibitors.
Keywords: 2,4-dichloro-5-sulphamoylbenzoic acid, inhibition, 2-amino-3-methylpyridine, ethylenediamine, proton
transfer, glaucoma, crystal structure
Introduction
obtained, such as the antiglaucoma sulphonamides that
can 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. e CA is able to catalyse the reversible hydration of
carbon dioxide to bicarbonate and protons, a very simple
but critically important physiological reaction for organ-
isms [5–10]. is enzyme has 16 different isoenzymes
currently known. Several of these isoenzymes (hCA II
and hCA IV) are present in human eyes [11–14] causing
e sulphonamides constitute an important class of
drugs, with several types of pharmacological actions
including antibacterial, anti-glaucoma, diuretic, hypo-
glycemic, antithyroid, protease inhibitory and antican-
cer activities [1–4]. Diversification of the sulphonylamide
structure has led to the development of improved antibi-
otics, insulin-releasing hypoglycaemic drugs, and antihy-
pertensive drugs. With sulphonamides as lead structures,
different classes of pharmacological agents have been
Address for Correspondence: C. Yenikaya, Department of Chemistry, Dumlupinar University, 43100, Kütahya, Turkey. Tel: +902742652051.
Fax: +902742652056. E-mail: yenikaya@dumlupinar.edu.tr
(Received 14 December 2009; revised 22 February 2010; accepted 23 February 2010)
104

Synthesis and characterisation of two novel proton transfer compounds
105
glaucoma, which is a group of diseases characterised by
a gradual loss of the visual field due to an elevation in
intraocular pressure (IOP), and being the second leading
cause of blindness worldwide [14,15]. Since CA inhibi-
tors have been shown to reduce intraocular pressure
exclusively by lowering the aqueous humour flow and
these compounds have been used for the treatment of
glaucoma for years [16,17].
In recent years the different aspects of proton transfer
systems have been studied by chemists [18–22]. ere
have been several attempts at employing proton transfer
from carboxylic acids to amines [23–27] and at preparing
their complexes with different metal ions [28–33]. e
first proton transfer compound obtained between Hsba
(carboxylic acid including the sulphonylamide group)
and amp, with its metal complex have been prepared in
our laboratories [34].
In this study, we have prepared proton transfer com-
pounds between 2,4-dichloro-5-sulphamoylbenzoic acid
(lasamide) (Hsba) and ethylenediamine (en), namely eth-
ane-1,2-diaminium 2,4-dichloro-5-sulphamoylbenzoate
(1), and between Hsba and 2-amino-3-methylpyridine
(2-amino-3-picoline) (amp), namely 2-amino-3-meth-
ylpyridinium2,4-dichloro-5-sulphamoylbenzoate(2).ey
have been characterised by elemental, spectral (IR and
UV-vis) and thermal analyses, as well as structural studies
by single X-ray diffractions. Furthermore, we have investi-
gated the potential use of these compounds as new inhibi-
tors of hCA isoenzymes in the treatment of glaucoma.
H, N and S were performed on an Elementar Vario III
EL (Hanau, Germany). FT-IR spectra were recorded in
the 4000–400cm−1 region with aBruker Optics, Vertex
70 FT-IR spectrometer (Ettlingen, Germany) using ATR
techniques. ermal analyses were performed on the SII
Exstar 6000 TG/DTA 6300 model (Shimadzu Co, Japan)
using a platinum crucible with 10mg samples. TG/DTA
measurements were taken in static air, within a 20–700°C
temperature range. e UV–Vis spectra were obtained for
aqueous solution and DMSO solution of the compounds
1 and 2 (10−3 M) and the free ligands, amp and Hsba (10−3
M), with a SHIMADZU UV-2550 spectrometer (Shimadzu
Co, Japan) in the range of 900–200nm.
Synthesis of proton transfer compounds 1 and 2
Solutions of amines (0.3 g, 5 mmol en for compound 1
and 0.54 g, 5 mmol amp for compound 2) in 10 mL dis-
tilled water were added to the solutions of Hsba (1.35 g,
5 mmol) in 10 ml water separately. e mixtures were
refluxed for 3 h, and then were cooled to room tem-
perature. e reaction mixtures were kept at room tem-
perature for 2 weeks to give colourless needle crystals for
both studies (1.95 g, 65 % yield for 1, 1.51 g, 80.1% yield
for 2). e single crystals suitable for X-ray analyses were
obtained by slow evaporation of water solutions for both
compounds (Figure 1).
Anal. Calcd. for 1 (C8H9Cl2N2O4S): C, 32.01; H, 3.02;
N, 9.33; S, 10.68. Found: C, 32.03; H, 3; N, 9.32; S, 10.7;
for 2 (C13H13Cl2N3O4S): C, 41.28; H, 3.46; N, 11.11; S, 8.48.
Found: C, 41.29; H, 3.45; N, 11.10; S, 8.46.
Materials and methods
Crystal structure determinations of 1 (C8H9Cl2N2O4S)
and 2 (C13H13Cl2N3O4S)
e crystal and instrumental parameters used in
the unit-cell determination and data collection are
All chemicals and reagents were of analytical grade and
used as received from a commercial source (Sigma-
Aldrich, Munich, Germany). Elemental analyses for C,
A
−
+
NH
OOC
Cl
Cl
COOH
3
NH
2
+
NH
+
3
NH
2
H NO S
Cl
Cl
2
2
SO NH
2
2
2
B
−OOC
Cl
COOH
NH2
NH2
Cl
H3C
CH3
+
NH
N
+
H2NO2S
Cl
Cl
SO2NH2
Figure 1. Syntheses of compounds; (A) for 1, and (B) for 2.
© 2011 Informa UK, Ltd.

106
C. Yenikaya et al.
summarised in Table 1 for compounds 1 and 2.
Measurements of 1 and 2 were made on a Stoe IPDS
II CCD X-ray diffractometer (STOE & Cie GmbH,
Darmstadt, Germany) employing plane graphite mono-
chromatised with Mo Kα radiation (λ= 0.71073 Å),
using the ω - 2θ scan mode. e unit cell parameters
were refined from the setting angles of the 25 centred
reflections in the range of 1.69 ≤ θ ≤ 27.54 for 1 and 1.46
≤ θ ≤ 26.19 for 2. e structures were solved by the direct
methods using SHELXS-97 and refined by full-matrix
least-squares techniques on F2 with SHELXL-97 [35]. e
empirical absorption corrections were applied by the
integration method via X-RED software [36]. All non-hy-
drogen atoms were refined with the anisotropic displace-
ment parameters and the hydrogen atoms were included
in their idealised positions and refined isotropically,
except for the N1 (in compound 1) and N3 (in compound
2). e hydrogen atoms of N1 for compound 1 and of N1
and N3 for compound 2 were located from the difference
maps and refined with the isotropic thermal parameters.
e crystal data and structure refinement details for com-
pounds 1 and 2 are given in Table 1. e ORTEP drawings
[37] of the molecules with a 40% probability displace-
ment thermal ellipsoids and atom-labelling schemes are
shown in Figure 2 for 1 and Figure 3 for 2.
Purification of isoenzymes hCA I and II from human
erythrocytes
In order to purify the hCA I and II isoenzymes, human
blood was centrifuged at 1500 rpm for 20 min, and
after the removal of the plasma, the erythrocytes were
washed with an isotonic solution (0.9% NaCl). After that,
the erythrocytes were lysed with 1.5 volume of ice-cold
water. elysatewascentrifugedat20000 rpmfor30 min
to remove any cell membranes and non-lysed cells. e
pH of the supernatant was adjusted to 8.7 with tris and
was then loaded onto an affinity column containing
Sepharose-4B-L-tyrosine-p-aminobenzene sulphon-
amide as the binding group. After extensive washing
with 25 mM tris–HCl/22mM Na2SO4 (pH 8.7), the hCA
I and II isoenzymes were eluted with 1 M NaCl/25 mM
Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4
(pH 5.6) [38,39]. e amount of purified protein was
estimated by the Bradford method [40] and SDS–PAGE
was carried out to determine whether the elute con-
tained the enzyme (Figure 4) [41].
Hydratase activity assay
e carbonic anhydrase activity was assayed by following
the hydration of CO2 according to the method described
by Wilbur and Anderson [42]. e CO2-hydratase activity
Table 1. Crystal data and structure refinement details for compounds 1 and 2.
Compound 1
Compound 2
C13 H13 Cl2 N3 O4 S
378.23
Chemical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system, space group
Unit cell dimensions: (Å, °)
a
C8 H9 Cl2 N2 O4 S
300.14
293(2)
293(2)
0.71073
0.71073
Triclinic, P-1
Monoclinic, P 21/c
6.2835(5)
8.4174(5)
27.8110(16)
7.2793(4)
b
7.5041(6)
c
12.371(9)
83.195(6)
α
79.421(6)
113.104(4)
β
88.442(6)
γ
Volume (Å3)
569.35(8)
1567.38(16)
Z
2
4
Calculated density (Mg m−3)
Absorbtion coefficient (mm−1)
F(000)
1.751
1.603
0.757
0.570
306
776
Crystal size (mm)
eta range for data collection(°)
Reflections collected
Independent reflections
Number of reflections used
Number of parameters
Max and min transmission
Refinement method
Limiting indices
Final R indices [I≥2σ(I)]
0.460×0.387×0.28
0.23×0.183×0.1
1.69 to 27.54
1.46 to 26.19
10653
7034
2624
2408
3140
2357
171
218
0.809 and 0.713
Full-matrix least–squares on F2
−8≤h≤8, −9≤k≤9,−16≤l≤16
0.945 and 0.882
Full-matrix least–squares on F2
−10≤h≤9, −34≤k≤34,−9≤l≤8
R1 =0.0433,
wR2 =0.1192
R1= 0.0577,
wR2 =0.1684
Goodness of fit on F2
1.049
1.067
Largest diff peak and hole
0.729 and −0.625 (e.Å−3)
0.511 and −0.547 (e.Å−3)
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and characterisation of two novel proton transfer compounds
107
C12
N1
O1
C3
S1
C2
C4
C5
O2
C1
C6
C7
C11
O4
N2
O3
C8
C8i
N2i
Figure 2. e molecular structure and atomic labeling scheme of 1. Displacement ellipsoids are drawn at the 40% probability level.
synthesised compounds 1 and 2, with acetazolamide
C12
(AAZ) as the control compound.
Esterase activity assay
O2
C3
C2
C1
N3
e carbonic anhydrase activity was assayed by following
the change in absorbance at 348 nm of the 4-nitropheny-
lacetate (NPA) to the 4-nitrophenylate ion over a period
of 3 min at 25°C using a spectrophotometer (CHEBIOS
UV–VIS, Rome, Italy) according to the method described
in the literature [43,44]. e enzymatic reaction, in a total
volume of 3 mL, contained 1.4 mL of 0.05 M tris–SO4 buf-
fer (pH 7.4), 1 mL of 3 mM 4-nitrophenylacetate, 0.5 mL
H2O and 0.1 mL enzyme solution. A reference measure-
ment was obtained by preparing the same cuvette with-
out enzyme solution. e IC50 values have been obtained
as in vitro for the free ligands en, amp and Hsba, and the
synthesised compounds 1 and 2, with AAZ as the control
compound.
S1
O1
C4
C5
C11
C6
C7
O3
O4
C10
C11
C12
C9
C13
N2
Determination of Ki values
e method for determination of the Ki values is
described elsewhere [45–49]. In the first part of this
study, the IC50 values have been obtained as in vitro. e
IC50 of the inhibitors (free ligands en, amp and Hsba, and
the synthesised compounds 1 and 2, with AAZ as the
control compound) were assayed by the hydrolysis of
p-nitrophenylacetate on the esterase activities of the CA
isoenzymes in the presence of various inhibitor concen-
trations. e absorbance was determined at 348 nm after
3 min [45]. Regression analysis graphs were drawn by
plotting inhibitor concentrations versus enzyme activity
using Microsoft Excel [50].
N1
C8
Figure 3. e molecular structure and atomic labeling scheme of 2.
Displacement ellipsoids are drawn at the 40% probability level.
as an enzyme unit (EU) was calculated by using the equa-
tion ((t0-tc)/tc) where t0 and tc are the times for pH
change of the nonenzymatic and the enzymatic reactions,
respectively. e concentration of inhibitor producing a
50% inhibition of CA activity (IC50) values were obtained
in vitro for the free ligands en, amp and Hsba, and the
In the second part of the study, the enzyme activity
was measured in the presence of five different substrate
© 2011 Informa UK, Ltd.

108
C. Yenikaya et al.
concentrations. At each of these inhibitor concentra-
tions (30%, 50%, and 70%) the data were linearised using
the Lineweaver-Burk plot in order to obtain the Ki values
as shown in Figure 5.
the S1 and C7 atoms deviate from the mean plane of
the sba anion with −0.3316(8) Å and −0.0923(10) Å
for compound 1 and −0.3095(14) Å and −0.0238(17) Å for
compound 2. e plane of amp is out of the plane of sba
by 51.04(4)° in compound 2.
e non-covalent interactions between neighbour-
ing molecules, such as ion pairing, hydrogen bonding,
and π-π stacking, were observed in these ionic com-
pounds as they have been considered as an effective
factor on the stability of biological and chemical sys-
tems [51,54–57].
Results and discussion
Descriptions of the structures of 1 (C8H9Cl2N2O4S) and 2
(C13H13Cl2N3O4S)
e molecular structures of compounds 1 and 2 with
theiratomic numbering schemes are shown in Figures
2 and 3, respectively, whereas the selected bond lengths
and angles are presented in Table 2. Compounds 1 and 2
crystallised in the P-1 and P 21/c space groups, respec-
tively. e asymmetric unit of 1 was found to contain
one sba anion and half of the ethylenediammonium
cation. All bond lengths and angles for both compounds
are consistent with those found in related compounds
[51–53]. In the symmetric units of both compounds,
FTIR Measurements
e FTIR spectra of all the compounds studied are given
in Figure 6. In the high frequency region, weak bands
between 3098 and 3072 and between 3005 and 2849 cm−1
are attributed to the stretching vibrations υ(C–H)aromatic
and υ(C–H)aliphatic, respectively.
e NH2 vibrations at 3357 and 3281 cm−1 in en were
shifted to lower frequencies in compound 1 as 2794,
2676, 2547 cm−1 due to the proton transfer to the amine
group [58]. e absorption bands at 3317 and 3175 cm−1
of the NH2 group of amp were slightly shifted from those
found for compound 2 (3327 and 3162 cm−1) due to the
weak intermolecular interactions. e relatively weak
and broad band at 2500 cm−1 can be attributed to the
ν(N-H)pyridinium vibration of the compound 2 due to proton
transfer to the pyridine nitrogen [59].
e NH2 vibrations of the sulphonamides in free Hsba
(3425, 3278 cm−1), for compounds 1 (3326, 3156 cm−1) and
2 (3473, 3328 cm−1) were observed with a similar pattern.
e absorption bands for compound 1 has shifted almost
100 cm−1 to the lower frequencies while compound 2 has
shifted almost 50 cm−1 to the upper frequencies due to
molecularinteractioninthesolidstate.estrongabsorp-
tion bands for the SO2 groups in Hsba for compounds 1
and 2 were observed at the region of 1400–1170cm−1 with
similar profiles and almost similar vibrations [60].
e relatively weak and broad band at 2900 cm−1 can
be attributed to the COOH group of Hsba. e strong
a
b
c
d
Figure 4. SDS-PAGE analyses of CA isoenzymes: (a) Standard hCA I,
(b) isolated hCA I, (c) Standard hCA II and (d) isolated hCA II.
0.15
0.12
0.09
0.06
0.03
0
-0.5
0
0.5
1
1.5
-0.03
1/[S]×103(M−1
)
Figure 5. Ki graph obtained from in vitro studies for compound 1 on hCA II.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and characterisation of two novel proton transfer compounds
109
Table 2. Selected bond distances (Å) and angles (°) for compounds 1 and 2.
Compound 1
Cl2- C3
Compound 2
1.428 (3)
S1-O1
1.4268 (15)
1.438 (16)
1.5995 (18)
1.7767 (19)
1.731 (2)
1.724 (2)
1.485 (3)
1.262 (2)
1.238 (2)
1.506 (4)
105.16 (9)
107.49 (10)
110.1(2)
S1- O1
S1- O2
S1- N3
S1- C4
N2- C8
Cl1- C1
Cl2- C3
1.5 (6)
S1-O2
N2- C8
1.434 (3)
1.581 (3)
1.780 (3)
1.736 (4)
1.719 (3)
S1-N1
O3- C7
S1-C4
O4- C7
C8- C8i
Cl1-C1
O1-S1- O2
O1-S1- N1
O2-S1- N1
O1-S1- C4
118.58 (10)
108.37 (10)
107 (10)
O2- S1- C4
N1- S1- C4
N2- C8- C8i
O1- S1- O2
O1- S1- N3
O2- S1- N3
O1- S1- C4
119.59 (17)
107.7 (18)
109.77 (17)
105.92 (15)
O2- S1- C4
N3- S1- C4
O4- C7- O3
N1- C8- N2
106.99 (16)
106.02 (18)
125 (3)
109.72 (9)
124.4 (4)
Symmetry code: (i) –x-1, -y, -z
266°C, corresponds to the loss of the CH5N from half
of the ethylenediammonium group in the asymmetric
unit of 1 (found 10.5, calcd 10.33). One mole of sul-
phonamide (SO2NH2) and one mole of the carboxylate
(COO) groups of Hsba are decomposed in the second
stage between 266 and 362°C with a DTGmax at 307 and
318°C (found 41.4, calcd 41.34). e residue of sba,
C6H2Cl2, was decomposed between 362 and 590°C with
DTGmax at 548 and 580°C (found 48.1, calcd 48.3).
For compound 2, the first stage, an endothermic
peak (DTGmax = 222°C) between 30 and 232°C, cor-
responded to the loss of the CH3N2 from the amine
group of 2 (found 11.4, calcd 11.4). e second stage,
an endothermic peak (DTGmax = 282°C) between 232
and 282°C, corresponded to the loss of C5H6 from the
amine group of 2 (found 17.5, calcd 17.5). e sul-
phonamide (SO2NH2) and carboxylate (COO) groups
of Hsba were decomposed in the third stage between
282 and 360°C with a DTGmax at 325 and 364°C (found
32.7, calcd 32.7). e final stage was the decompo-
sition of the residue of sba, C6H2Cl2, between 360
and 575°C with a DTGmax at 559°C (found 38.4, calcd
38.5).
en
1
Hsba
2
amp
4000
3500
3000
2500
Wavenumber cm
2000
1500
1000
500
−1
Figure 6. FTIR spectra of en, Hsba, amp, compounds 1 and 2 (KBr).
UV–Vis Spectra
C=O vibration at 1684 cm−1 of Hsba is shifted to 1634 and
1670 cm−1 for compounds 1 and 2, respectively, indicat-
ing the difference between the carboxylic acid group
of the free Hsba and proton transfer compounds 1 and
2 [61]. e ν(C-O) vibration of Hsba also shifted from
1169 cm−1 to 1073 and 1074 cm−1 in 1 and 2 [62,63].
e strong absorption bands at the region of 1400–
1580cm−1 are attributed to the ν(C=N) and ν(C=C) vibra-
tions in free ligands, amp and Hsba, and compounds 1
and 2 [64].
e electronic spectra of compounds 1 and 2, and the
free ligands, amp and Hsba were recorded in solution at
a 1 × 10−3 M concentration at room temperature (Figure
9 and Table 3). e characteristic π- π* transitions are
in the spectra of 1 (207 nm) and of 2 (209 and 291 nm)
in a water solution with the same profiles as the free
ligands, Hsba (209 nm), amp (226 and 291 nm). Two sets
of characteristic π- π* transitions were observed for all
compounds in the DMSO solutions, which were located
around 255 and 290 nm. e more energetic π-π* tran-
sition bands in water around 210 nm for all compounds
were red shifted to around 255 nm in the DMSO solution
spectra. e other π- π* absorption bands at 291 nm for
both amp and compound 2 in water were also observed
in almost the same region (296 and 294 nm respectively)
in the spectra recorded in the DMSO. All the data which
we obtained from the electronic spectra of the free ligands
did not show any marked differences from those of the
proton transfer compounds for both solutions
e ring wagging vibrations of the pyridine groups are
also observed at 768 and 701cm−1 for amp and 743 and
683 cm−1 for compound 2.
Thermal Analyses of 1 (C8H9Cl2N2O4S) and 2
(C13H13Cl2N3O4S)
Figures 7 and 8 show the TG-DTG and DTA curves of
compounds 1 and 2, respectively. e endothermic
first stage (DTGmax = 240 and 260°C), between 25 and
© 2011 Informa UK, Ltd.

110
C. Yenikaya et al.
TGA
100.0
80.0
60.0
40.0
20.0
0.0
100.0
1.500
1.000
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
0.500
DTG
DTA
0.000
−20.0
−40.0
−60.0
−0.500
−1.000
100.0
200.0
300.0
400.0
Temp Cel
500.0
600.0
700.0
Figure 7. e TG-DTG and DTA curves of 1.
TGA
100.0
300.0
1.500
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
250.0
200.0
150.0
100.0
50.0
1.000
0.500
DTG
DTA
0.000
0.0
−0.500
−1.000
−50.0
−100.0
100.0
200.0
300.0
400.0
Temp Cel
500.0
600.0
700.0
Figure 8. e TG-DTG and DTA curves of 2.
compound on the hCA I and hCA II enzymes were
studied by both hydratase and esterase activity meth-
ods and then the Ki values were determined for each
compound (Table 4).
In vitro inhibition studies
e inhibition effects of the parent compounds (amp
and Hsba), newly synthesised proton transfer com-
pounds (1) and (2) together with AAZ as the control
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and characterisation of two novel proton transfer compounds
111
A
3.000
Hsba
3.000
2.000
1.000
0.000
Hsba
1
2.000
2
amp
1.000
en
0.000
200.00
300.00
400.00
500.00
200.00
300.00
400.00
500.00
nm.
nm.
B
4.000
3.000
2.000
1.000
0.000
4.000
3.000
2.000
1.000
0.000
2
Hsba
Hsba
amp
1
en
300.00
200.00
300.00
400.00
500.00
200.00
400.00
500.00
nm.
nm.
Figure 9. UV-Vis spectra of en, Hsba, amp, compounds 1 and 2; (A) in water, (B) in DMSO.
e other parent compounds, amp and en, did not present
any hydratase and esterase activities.
Table 3. Optical properties of compounds 1 and 2, and the free
ligands amp and Hsba, in water and DMSO.
λmax (nm) (ε (M−1cm−1))
In relation to esterase activities, the inhibition equi-
librium constants (Ki) were also determined. e novel
compounds 1 and 2 had significant Ki values which are,
respectively 0.137 and 0.157 µM for hCA I and 0.99 and
0.075 µM for hCA II compared to Hsba (7.7 and 232 µM for
hCA I and II) and AAZ (5.2 and 3.8 µM for hCA I and II).
e parent compound Hsba showed inhibitory effects
on hCA I and II as expected since it is a sulphonamide
compound [1–4]. Nevertheless, the other parent com-
pounds, amp and en, did not show any inhibitory effects
on these isoenzymes due to a lack of -SO2NH2 groups
in their structures. e novel compounds 1 and 2 bear
sulphonamide functional groups like Hsba and AAZ,
and bear additionally carboxylate and protonated nitro-
gen functional groups unlike the parent compounds
(Figure 1) and AAZ. ese novel compounds (1 and 2)
had higher inhibitory effects than the parent compounds
and AAZ, which might be due to the interactions between
1
Hsba
2
amp
Water 207 (22320) 209 (25520) 209 (19586) 226 (103000)
291 (12426) 291 (67800)
DMSO 256 (25810) 257 (35120) 258 (25780) 256 (15490)
285 (12830) 285 (11230) 294 (35870) 296 (34190)
According to the in vitro studies, the IC50 values for the
hydratase activities of the newly synthesised compounds
1 and 2 (0.15 and 0.32 µM for hCA I and 0.06 and 0.15 µM
for hCA II, respectively) were lower than the IC50 values for
Hsba (0.45 and 0.4 µM for hCA I and II) and of AAZ (3.6 and
2.8 µM for hCA I and II). e IC50 values obtained from the
esterase activities of all the compounds studied have been
observed in a similar trend, and are 0.13 and 0.8 µM for
hCA I and 0.14 and 0.1 µM for hCA II for 1 and 2, respec-
tively. e Hsba and AAZ have esterase activities as 3.2 and
6.1 for hCA I and 18 and 4.5 µM for hCA II, respectively.
© 2011 Informa UK, Ltd.

112
C. Yenikaya et al.
Table 4. IC50 and Ki values of hCA-I and hCA-II isoenzyme hydratase and esterase activities after inhibition experiments.
Hydratase IC50 (μM ) Esterase IC50 (μM ) Ki values (μM )
hCA-I
Inhibitor
AAZ
hCA-I
hCA-II
2.8
hCA-I
hCA-II
4.5
hCA-II
3.8
3.6
6.1
5.2
en
No Inhibition
No Inhibition
No Inhibition
No Inhibition
No Inhibition
No
Inhibition
amp
No Inhibition
No Inhibition
No Inhibition
No Inhibition
No Inhibition
No
Inhibition
Hsba
0.45
0.15
0.32
0.4
3.2
0.13
0.8
18
0.14
0.1
7.7
0.137
0.99
23
1
2
0.06
0.15
0.157
0.075
the functional groups (sulphonamide, carboxylate and
protonated nitrogen) in their structures and the zinc ion
and histidine residue in the active site of CA [65].
As the novel proton transfer compounds (1 and 2)
have a higher potential inhibitory effects than the parent
inhibitors (Hsba, amp and en) and also than AAZ as in
our previous study [34], they could be seen as candidates
for the treatment of glaucoma.
use of the Stoe IPDS-2 diffractometer purchased under
grant F.279 of the University Research Fund.
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e authors acknowledge the support provided by
DumlupınarUniversityResearchFund(grantNo.2007/2).
In addition, the authors would like to thank the Faculty of
Arts and Sciences, Ondokuz Mayıs University, Turkey, for
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and characterisation of two novel proton transfer compounds
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