In vitro inhibition effect and structure-activity relationships of some saccharin derivatives on erythrocyte carbonic anhydrase i and II

Article abstract of DOI:10.3109/14756366.2012.757222

In this study, in vitro inhibitory effects of some saccharin derivatives on purified carbonic anhydrase I and II were investigated using CO2 as a substrate. The results showed that all compounds inhibited the hCA I and hCA II enzyme activities. Among the compounds, 6-(p-tolylthiourenyl) saccharin (6m) was found to be the most active one for hCA I activity (IC50 = 13.67 μM) and 6-(m-methoxyphenylurenyl) saccharin (6b) was found to be the most active one for hCA II activity (IC50 = 6.54 μM). Structure-activity relationships (SARs) study showed that, generally, thiourea derivatives (6l - v) inhibited more hCA I and hCA II than urea derivatives (6a-k). All compounds (excluding 6c and 6r) have higher inhibitory activity on hCA II than on hCA I.

Full text of DOI:10.3109/14756366.2012.757222

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(1): 118–123
2014 Informa UK Ltd. DOI: 10.3109/14756366.2012.757222
!
In vitro inhibition effect and structure–activity relationships of some
saccharin derivatives on erythrocyte carbonic anhydrase I and II
Fatih Sonmez1, Cigdem Bilen2, Sinem Sumersan2, Nahit Gencer2, Semra Isik2, Oktay Arslan2,
and Mustafa Kucukislamoglu3
2
1Department of Food Technology, Pamukova Vocational High School, Sakarya University, Sakarya, Turkey, Department of Chemistry,
3
Faculty of Art and Sciences, Balikesir University, Balikesir, Turkey, and Department of Chemistry, Faculty of Art and Sciences,
Sakarya University, Sakarya, Turkey
Abstract
Keywords
In this study, in vitro inhibitory effects of some saccharin derivatives on purified carbonic
anhydrase I and II were investigated using CO2 as a substrate. The results showed that all
compounds inhibited the hCA I and hCA II enzyme activities. Among the compounds,
6-(p-tolylthiourenyl) saccharin (6m) was found to be the most active one for hCA I activity
(IC50 ¼ 13.67 mM) and 6-(m-methoxyphenylurenyl) saccharin (6b) was found to be the most active
one for hCA II activity (IC50 ¼ 6.54 mM). Structure–activity relationships (SARs) study showed that,
generally, thiourea derivatives (6l–v) inhibited more hCA I and hCA II than urea derivatives (6a–k).
All compounds (excluding 6c and 6r) have higher inhibitory activity on hCA II than on hCA I.
Carbonic anhydrase, inhibition, saccharin,
thiourea, urea
History
Received 27 September 2012
Revised 6 December 2012
Accepted 6 December 2012
Published online 23 January 2013
Introduction
character of the molecule and suggests its potential to interact
with the zinc ion at the floor of the binding pocket of carbonic
Carbonic anhydrase (CA, EC 4.2.1.1) is a ubiquitous zinc enzyme.
Basically, there are several cytosolic forms (CA-I, CA-II, CA-III
and CA-VII), four membrane-bound forms (CA-IV, CA-IX, CA-XII
and CA-XIV), one mitochondrial form (CA-V), as well as a secreted
CA form (CA-VI)1,2. They all catalyze a very simple physiological
reaction, the interconversion between carbon dioxide and the
bicarbonate ion, and are thus involved in crucial physiological
processes connected with respiration and transport of CO2/
bicarbonate between metabolizing tissues and the lungs, pH and
CO2 homeostasis, electrolyte secretion in a variety of tissues/
organs, biosynthetic reactions (such as the gluconeogenesis,
lipogenesis and ureagenesis), bone resorption, calcification,
tumorigenicity and many other physiologic or pathologic pro-
cesses1–3. CAinhibitorshavenowbeenamainstayofhumanclinical
intervention for several decades, with at least 25 clinically used
drugs that are CA inhibitors4. Although there are many studies on
this enzyme, the CA enzyme family continues to capture the
attention of drug discovery scientists and clinicians as the knowl-
edge regarding the therapeutic implications associated with this
¨
anhydrases. Kohler et al. presented saccharin as zinc-binding
portion based on the X-ray structure7.
Sulfonamides are the best known inhibitors of CA enzymes
and are used for the treatment of glaucoma in medicinal
chemistry8. Acetazolamide (AAZ), dorzolamide (DZA) and
brinzolamide (BRZ) are sulfonamide derivatives and are used in
the treatment of glaucoma. However, these drugs have several
side effects such as numbness and tingling in the fingers and toes,
blurred vision, kidney stones, an increase in urination, upset
stomach, dry eye and headache or dizziness2,9
.
There are reports10,11 of many such aromatic sulfonamides or
bis-sulfonamide moieties incorporating urea or thiourea groups as
very potent inhibitors against three isozymes, human CA I, human
CA II and bovine isozyme CA. A small series of five ureido-
substituted benzenesulfonamide derivatives were recently investi-
gated as inhibitors of the cytosolic isoform hCA II by one of these
groups. It has been observed that their potency varied between 3.3
and 226 nM, and by means of X-ray crystallography a highly
variable orientation of the R-ureido moieties was evidenced when
the inhibitor was bound within the enzyme active site11.
enzyme class continues to grow4,5
.
Saccharin, 1,2-benzisothiazole-3-one-1,1-dioxide, is a well-
known heterocyclic compound and has been used as a sweetener
in the form of its sodium salt since 1885. Yet it is also a
heterocycle of pharmaceutical importance, being a key structural
element of certain CNS-active drugs6. Chemically, saccharin
consists of a sulfimide with a lactam and cyclic sulfonamide
moiety. The latter functionality is responsible for the acidic
In this study, we evaluated 6-(phenylurenyl/thiourenyl) sac-
charin derivatives (6a–v), synthesized in the previous work12,
effects on hCA I and hCA II purified from human erythrocytes.
Additionally, we presented SAR analyses.
Materials and methods
General
Sepharose 4B, L-tyrosine, sulfonamide, synthetic starting material,
reagents and solvents were purchased from Merck (Darmstadt,
Germany), Alfa Easer (Ward Hill, MA), Sigma-Aldrich
(Taufkirchen, Germany) and Fluka (Taufkirchen, Germany).
Address for correspondence: Nahit Gencer, Department of Chemistry,
Faculty of Art and Sciences, Balikesir University, Balikesir, 10145,
Turkey. Tel: þ90266 612 1278. Fax: þ90266 612 1215. E-mail: ngen-
cer@balikesir.edu.tr

DOI: 10.3109/14756366.2012.757222
Inhibitory effects of saccharin derivatives 119
General procedure for 6-(phenylurenyl/thiourenyl)
saccharin derivatives
Biological evaluation of saccharin derivatives for hCA I
and hCA II inhibitory activities
Phenylisocyanate or phenylisothiocyanate derivatives (1 mmol) For evaluating the hCA I and hCA II inhibitory effects, all
were added to a solution of 6-aminosaccharin (1 mmol) and compounds were subjected to hCA I and hCA II inhibition assay
triethyl amine (1 mL) in dry DMF. The mixture was stirred at with CO2 as a substrate. The result showed that all compounds
room temperature for 12 h and then poured into cold 1 M HCl. (6a–v) inhibited the hCA I and hCA II enzyme activity.
The precipitate was filtered and washed with cold water. The The IC50 values and inhibition constants of 6a–v analogues
crude products were recrystallized from ethanol over 99% purity. against hCA I and hCA II are summarized in Table 1 and the IC50
The synthetic procedures are depicted in Scheme 1.
graphs are given in Figure 1. The IC50 figures are presented as
supporting information.
Preparation of hemolysate and purification from blood
red cells
We have determined the IC50 values of 13.57–74.90 mM for
the inhibition of hCA I and 6.54–49.00 mM for the inhibition of hCA
II. Among all compounds, 6m (IC50 ¼ 13.67 mM) was found to be
the most active one for hCA I inhibitory activity and 6b
(IC50 ¼ 6.54 mM) showed the highest hCA II inhibitory activity.
6d (IC50 ¼ 30.81 mM) was found to be the most active one for hCA I
inhibitory activity and 6b (IC50 ¼ 6.54 mM) showed the highest
hCA II inhibitory activity for the urea derivatives. Among the
thiourea derivatives, 6m (IC50 ¼ 13.67 mM) showed the highest
hCA I inhibitory activity and 6q (IC50 ¼ 8.10 mM) showed the
highest hCA II inhibitory activity.
Preparation of hemolysate and purification from blood red cells
made by the literature13 was presented in supporting information.
CA enzyme assay
CA activity measured by the Maren method14 was presented in
supporting information.
In vitro inhibition studies
It was reported7 that saccharin most likely coordinates in a
deprotonated state through its nitrogen atom to the catalytically
active zinc ion. Additionally, ureido-substituted benzenesulfona-
mide moieties were evidenced when the inhibitor was bound within
the enzyme active site10,11. We believe that the synthesized
saccharin urea/thiourea derivatives inhibited hCA I and II in the
same way.
For the inhibition studies of saccharin, different concentrations of
these compounds were added to the enzyme. Activity percentage
values of CA for different concentrations of each saccharin were
determined by regression analysis using Microsoft Office 2000
Excel. CA enzyme activity without a saccharin solution was
accepted as 100% activity.
Results and discussion
Structure–activity relationships
Chemistry
Generally, we have seen that all compounds (excluding 6c and 6r)
have a higher inhibitory activity on hCA II than hCA I in the
SARs study. When same substituents bonded to phenyl ring, most
of the thiourea derivatives (6l–v) exhibited higher hCA I and hCA
The synthetic procedures are depicted in Scheme 1.
6-(Phenylurenyl/thiourenyl) saccharin compounds (6a–v) were
synthesized from 4-nitrotoluene (1) in five steps by known
procedures12.
Scheme 1. Synthesis of 6-(phenylurenyl/thiourenyl) saccharin (6a–v) derivatives.

120 F. Sonmez et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 118–123
Table 1. Inhibitory effects of saccharin derivatives on hCA I and hCA II.
hCA I IC50
(mM)
hCA II IC50
(mM)
hCA I IC50
(mM)
hCA II IC50
(mM)
Compound
X
R
Compound
X
R
5
–
–
H
54.90
63.06
41.65
34.55
30.81
38.99
63.03
37.37
37.47
54.24
44.71
42.64
49.00
21.13
6.54
6l
6m
6n
6o
6p
6q
6r
6s
S
S
S
S
S
S
S
S
S
S
S
H
4-CH3
4-OCH3
2-F
46.74
13.67
43.31
74.90
46.70
55.87
20.21
33.41
59.38
26.10
47.73
40.24
11.14
30.32
30.23
14.55
8.10
30.70
17.19
12.48
11.04
43.49
6a
6b
6c
6d
6e
6f
6g
6h
6i
O
O
O
O
O
O
O
O
O
O
O
3-OCH3
4-OCH3
4-CH3
3-Cl
37.66
24.65
17.79
23.67
12.67
32.74
38.87
41.61
17.14
3-F
4-F
3-I
3-Cl
4-Cl
3,4-di-Cl
3-NO2
4-NO2
2-F
6t
6u
6v
2,4-di-Cl
3,5-di-Cl
4-NO2
6j
6k
4-F
120
120
100
80
100
80
60
40
20
0
hCA I
hCA II
hCA I
hCA II
60
40
20
0
0
0
0
0
10
10
10
20
[5]
30
40
10
20
30
[6a] µM
40
µ
M
120
100
80
120
100
80
hCA I
hCA I
hCA II
hCA II
60
60
40
40
20
20
0
0
0
10
20
30
40
20
30
[6b] µM
40
-20
[6c] µM
120
100
80
60
40
20
0
120
100
80
hCA I
hCA II
hCA I
hCA II
60
40
20
0
0
20
30
40
10
20
30
40
[6d] µM
[6e] µM
Figure 1. IC50 graphics of saccharin derivatives (5, 6a–v) on hCA I and hCA II.

DOI: 10.3109/14756366.2012.757222
Inhibitory effects of saccharin derivatives 121
120
120
100
80
60
40
20
0
100
80
60
40
20
0
hCA I
hCA II
hCA I
hCA II
0
10
20
30
40
0
10
20
30
40
[6g] µM
[6f] µM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
hCA I
hCA II
hCA I
hCA II
0
10
20
30
40
0
10
20
30
40
[6h] µM
[6i] µM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
hCA I
hCA I
hCA II
hCA II
0
10
20
30
40
0
10
20
30
40
[6j]
µM
[6k] µM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
hCA I
hCA II
hCA I
hCA II
0
10
20
30
40
0
10
20
30
40
[6l]
µM
[6m] µM
Figure 1. Continued.
II inhibitory activity than urea derivatives (6a–k). Additionally,
the following results were obtained.
(a) For urea derivatives:
bonded to meta position of phenyl ring (6b) had the
highest hCA II inhibitory activity (IC50 ¼ 6.54 mM).
(ii) Electron-donating groups (methoxy, methyl) bonded
to para position of the phenyl ring (6c and 6d)
inhibited hCA I activity more than halogens and
electron-withdrawing groups bonded. On the other
hand, halogen groups bonded to the para position of
(i) Although electron-withdrawing groups (nitro and
halogens) bonded to meta position of the phenyl
ring (6e, 6g and 6h) increased the inhibitory activity
on hCA I, electron-donating groups (methoxy)

122 F. Sonmez et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 118–123
120
120
100
80
60
40
20
0
hCA I
100
80
hCA II
hCA I
hCA II
60
40
20
0
0
10
20
30
40
0
10
20
30
40
[6n]
µM
[6o] µM
120
100
80
60
40
20
0
120
100
80
hCA I
hCA I
hCA II
hCA II
60
40
20
0
0
0
10
20
30
40
10
20
[6p] µM
30
40
[6q]µM
120
100
80
60
40
20
0
120
100
80
hCA I
hCA II
hCA I
hCA II
60
40
20
0
0
0
10
20
30
40
10
20
30
40
[6r]
µM
[6s] µM
120
100
80
120
100
80
60
40
20
0
hCA I
hCA II
hCA I
hCA II
60
40
20
0
0
10
20
30
40
0
10
20
30
40
[6t]
µM
[6u] µM
Figure 1. Continued.
the phenyl ring (6f, 6g and 6k) increased the
inhibitory activity on hCA II.
(b) For thiourea derivatives:
para (6q, IC50 ¼ 8.10 mM) positions led to major
enhancement of hCA II inhibitory activity.
(iii) In same moving for hCA
I
activity, 6p
(i) Electron-donating groups bonded to the para position
of the phenyl ring (6m and 6n) increased the hCA I
inhibitory activity.
(ii) Moving-F group on the phenyl ring from ortho (6o,
IC50 ¼ 30.23 mM) to meta (6p, IC50 ¼ 14.55 mM) and
(IC50 ¼ 46.70 mM) was more inhibited than 6o
(IC50 ¼ 74.90 mM) and 6q (IC50 ¼ 55.87 mM).
(iv) Halogen series on the meta position of the phenyl
ring showed a linear relationship for higher hCA I
inhibitory activity with increasing size and

DOI: 10.3109/14756366.2012.757222
Inhibitory effects of saccharin derivatives 123
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120
100
80
60
40
20
0
hCA I
hCA II
¨
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0
10
20
[6v]
30
40
µM
Figure 1. Continued.
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benzenesulfonamides potently inhibit carbonic anhydrase IX and
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polarizability (for size and polarizability, I4Cl4F,
for hCA inhibitory activity, 6r
(IC50 ¼ 20.21 mM)46s (IC50 ¼ 33.41 mM)46p
I
(IC50 ¼ 46.70 mM). Interestingly, this series showed
an inverse relationship for hCA II inhibitory activity
with increasing size and polarizability (for hCA II
inhibitory activity, 6r (IC50 ¼ 30.70 mM)56s
(IC50 ¼ 17.19 mM)56p (IC50 ¼ 14.55 mM).
14. Maren TH. A simplified micromethod for the determination of
carbonic anhydrase and its inhibitors.
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18. Kiranoglu S, Sinan S, Gencer N, et al. In vivo effects of oral
contraceptives on paraoxonase, catalase and carbonic anhydrase
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19. Sinan S, Kockar F, Gencer N, et al. Amphenicol and macrolide
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prophenoloxidase from Galleria mellonella L. Artif Cell Blood Sub
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In conclusion, we evaluated 6-(phenylurenyl/thiourenyl) saccharin
derivatives (6a–v) effects on hCA I and hCA II purified from
human erythrocytes and SARs were examined. All compounds
inhibited both hCA I and hCA II enzyme activities. Most of the
compounds had higher hCA II inhibitory activity than hCA I
activity. Most of the thiourea derivatives (6l–v) exhibited
higher hCA I and hCA II inhibitory activities than urea
derivatives (6a–k). The present study revealed that activity
could also be influenced by the type and position of the
substituent on the phenyl ring. Among all compounds, 6m
showed the highest hCA I inhibitory activity and 6b showed the
highest hCA II inhibitory activity.
In summary, enzyme inhibition is the most important issue for
drug design and biochemical applications15–27. Therefore, our
results suggested that saccharin derivatives are likely to be
adopted as candidates to treat glaucoma and may be taken for
further evaluation in in vivo studies.
Declaration of interest
The authors report no conflicts of interest.
This work was supported by Sakarya University Scientific Research
Project (Project No. 2011–50-02-020).
23. Arslan M, Genc¸er N, Arslan O, Guler OO. In vitro efficacy of some
cattle drugs on bovine serum paraoxonase 1 (PON1) activity.
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