Secondary sulfonamides as effective lactoperoxidase inhibitors

Article abstract of DOI:10.3390/molecules22060793

Secondary sulfonamides (4a-8h) incorporating acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide, and trihydroxybenzamide and possessing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole groups were synthesized. Lactoperoxidase (LPO, E.C.1.11.1.7), as a natural antibacterial agent, is a peroxidase enzyme secreted from salivary, mammary, and other mucosal glands. In the present study, the in vitro inhibitory effects of some secondary sulfonamide derivatives (4a-8h) were examined against LPO. The obtained results reveal that secondary sulfonamide derivatives (4a-8h) are effective LPO inhibitors. The Ki values of secondary sulfonamide derivatives (4a-8h) were found in the range of 1.096 ± 10-3 to 1203.83 μM against LPO. However, the most effective inhibition was found for N-(sulfathiazole)-3,4,5-triacetoxy.

Full text of DOI:10.3390/molecules22060793

molecules
Article
Secondary Sulfonamides as Effective
Lactoperoxidase Inhibitors
Zeynep Köksal 1, Ramazan Kalin 2,3, Yasemin Camadan 4, Hande Usanmaz 5, Züleyha Almaz 6,
Ilhami Gülçin 2,*, Taner Gokcen 7,8, Ahmet Ceyhan Gören 7 and Hasan Ozdemir 2
˙
1
˙
˙
Department of Chemistry, Faculty of Sciences, Istanbul Medeniyet University, 34730 Istanbul, Turkey;
zeynep.koksal@medeniyet.edu.tr
2
Department of Chemistry, Faculty of Science, Ataturk University, 25240 Erzurum, Turkey;
rkalin@atauni.edu.tr (R.K.); hozdemir@atauni.edu.tr (H.O.)
Department of Basic Science, Faculty of Science, Erzurum Technical University, 25240 Erzurum, Turkey
Pharmacy Services Program, Vocational School of Health Services, Artvin Coruh University,
08000 Artvin, Turkey; yasem_can@hotmail.com
3
4
5
6
7
Department of Bioengineering, Faculty of Engineering and Architecture, Sinop University,
57000 Sinop, Turkey; husanmaz@sinop.edu.tr
Department of Molecular Biology and Genetics, Faculty of Sciences and Arts, Mus¸ Alparslan University,
49250 Mus¸, Turkey; z.turkoglu@alparslan.edu.tr
TUBITAK UME, Chemistry Group Laboratories, P.O. Box: 54, 41470 Gebze Kocaeli, Turkey;
taner.gokcen@tubitak.gov.tr (T.G.); ahmet.goren@acgpubs.org (A.C.G.)
Department of Organic Chemistry, Faculty of Science, Istanbul Technical University, 34469 Istanbul, Turkey
Correspondence: igulcin@atauni.edu.tr or igulcin@yahoo.com; Tel.: +90-442-231-4375; Fax: +90-442-231-4109
8
*
Academic Editor: Claudiu T. Supuran
Received: 28 February 2017; Accepted: 9 May 2017; Published: 24 May 2017
Abstract: Secondary sulfonamides (4a–8h) incorporating acetoxybenzamide, triacetoxybenzamide,
hydroxybenzamide, and trihydroxybenzamide and possessing thiazole, pyrimidine, pyridine,
isoxazole and thiadiazole groups were synthesized. Lactoperoxidase (LPO, E.C.1.11.1.7), as a natural
antibacterial agent, is a peroxidase enzyme secreted from salivary, mammary, and other mucosal
glands. In the present study, the in vitro inhibitory effects of some secondary sulfonamide derivatives
(
4a
derivatives (4a
4a 8h) were found in the range of 1.096
–
8h) were examined against LPO. The obtained results reveal that secondary sulfonamide
8h) are effective LPO inhibitors. The Ki values of secondary sulfonamide derivatives
10−3 to 1203.83
M against LPO. However, the most
–
(
–
×
µ
effective inhibition was found for N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a), with Ki values of
1.096 × 10−3 ± 0.471 × 10−3 µM as non-competitive inhibition.
Keywords: lactoperoxidase; secondary sulfonamide; enzyme purification; enzyme inhibition
1. Introduction
Sulfonamides constitute a privileged class among pharmacological agents by possessing
properties including carbonic anhydrase enzyme (CA) inhibition, as well as diuretic, hypoglycemic,
anticancer, antibacterial, antiviral, and metalloprotease inhibitory effects. Although many years passed
since their first discovery in the 1930s as a chemotherapeutic agent for antibacterial properties they
are receiving increasing attention due to their newly-discovered pharmacological properties [1–5].
Due to the belief that sulfonamides must be be primary sulfonamides to exhibit those pharmacological
properties, secondary sulfonamides are more investigated from the synthetic view and have found
less pharmacological applications. However, recent investigations shown that secondary sulfonamides
have a great potential not only for their selective inhibition characteristics on CA isozymes [6–8]
Molecules 2017, 22, 793; doi:10.3390/molecules22060793
www.mdpi.com/journal/molecules

Molecules 2017, 22, 793
2 of 9
but also for their good inhibition properties over cancer-related CA isoenzymes [
9], for anticandidal
properties [10] and also for their glutamate carboxypeptidase II inhibition properties [11].
It was also reported that sulfonamides had a number of interesting functionalities. Some example
can be given for secondary and tertiary and sulfonylureas represent particularly important structural
motifs in several classes of drugs. Sulfonamides possessed some biological activities as antibiotics
(sulfhamethoxazole), PDE5 inhibitors for the treatment of erectile dysfunction (sildenafil), protease
inhibitor for treatment of HIV (darunavir), sulfonylureas for treatment of diabetes mellitus
(glibenclamide), hepatitis C anti-viral RNA polymerase inhibitors, and non-steroidal anti-inflammatory
COX-2 inhibitors (oxicam class) [12].
Because secondary sulfonamides have been reported to be effective agents, in this study, new designs
of secondary sulfonamide drugs containing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole
moieties were utilized to obtain acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide and
trihydroxybenzamide sulfonamide derivatives to achieve the synergistic effects of sulfonamides and
polyphenols in one molecule. Since acetyl protection of hydroxyl groups is a well-known application in
pharmacology to enhance oral bioavailability and to increase therapeutic concentration of the drug in
the bloodstream, both acetylated and non-acetylated forms of compounds were examined together.
Milk contains a variety of constituents that protect the neonate and the milk itself from a host
of deleterious microorganisms. One of the constituents is the lactoperoxidase (LPO) system [13
–15].
This system is a naturally-occurring antimicrobial system [16 18] which is inherently available in raw
–
milk and human body fluids such as saliva. There are three primary components in the LPO system:
haeme-containing LPO, hydrogen peroxide (H2O2), and thiocyanate (SCN−). H2O2 is produced by
a number of microorganisms such as lactobacilli, Lactococcus and streptococci through enzymatic
oxidation of some biomolecules including ascorbic acid and glucose [19
released from mucosal glands and can be found in secretions like saliva, milk or tears. The potential of
LPO to inhibit bacterial growth in milk has been recognized [22 23]. LPO catalyses the H2O2-dependent
–21]. Additionally, LPO is
,
−
−
oxidation of thiocyanate (SCN ) to hypothiocyanite (OSCN ). The latter ion is a potent antimicrobial
agent against gram-negative and gram-positive bacteria, fungi, and viruses [16 24]. This reaction makes
the LPO system potentially useful in improving food safety [16 25]. LPO has crucial applications
,
,
in various fields. For example, LPO protects the intestinal tract system of newborn infants against
pathogenic microorganisms by catalysing halides and pseudohalides. LPO is one of the important
proteins in bovine whey, and it has been known to play a key role in protection of the lactating
mammary gland and the intestinal tract of newborn infants against pathogenic microorganisms [26–28].
The aim of the present study was to assess the inhibition effects of a new class of secondary
sulfonamides against LPO enzyme, one of the prominent enzymes generally found in several sources,
such as bovine milk, saliva, and tears.
2. Materials and Methods
2.1. Chemicals and Materials
Fresh bovine milk was obtained from the local dairy. L-Tyrosine, Amberlite CG-50-NH4+ resin,
CNBr-activated-Sepharose 4B, protein assay reagent sulfanilamide, and chemicals for electrophoresis
were purchased from Sigma-Aldrich Co. (Sigma-Aldrich Chemie GmbH Export Department
Eschenstrasse 5, 82024 Taufkirchen, Germany).
2.2. General Procedure for Sulfonamide Derivatives
Secondary sulfonamides were synthesized using naturally available 4-hydroxy and
3,4,5-trihydroxy benzoic acids (gallic acid) in various plants and fruits, which were reacted with acetic
anhydride to obtain 4-acetoxy and 3,4,5-triacetoxy benzoic acids for the protection of hydroxyl groups.
Then, they were converted to their corresponding chloride derivatives by treating with thionyl chloride
to produce benzoyl chlorides, which underwent reactions with secondary sulfonamides having

Molecules 2017, 22, 793
3 of 9
thiazole, pyrimidine, pyridine, isoxazole and thiadiazole groups to obtain secondary sulfonamide
derivatives of acetoxybenzamides and triacetoxybenzamides as one part of target compounds.
Deacetylation under acidic conditions gave the other part of our targeted sulfonamide derivatives [28].
The reaction scheme is given in Figure 1.
Figure 1. The synthesis route of targeted secondary sulfonamide derivatives (1a–8h).
2.3. Biochemical Assays
LPO activities were determined by the procedure of Shindler and Bardsley [29] with
slight modification [30,31]. This method is based on the oxidation of chromogenic substrate
2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by hydrogen peroxide (H2O2), which
results in a product which had absorbance at 412 nm [32].
The affinity matrix was synthesised by coupling sulfanilamide as the ligand and L-tyrosine as
the spacer arm to CNBr-activated-Sepharose 4B, following the previously published procedure [22
]
with a slight modification [33,34]. The protein flow in the column eluates was spectrophotometrically
determined at 280 nm [35]. All purification steps were performed at 4 ◦C. The protein quantity was
determined at 595 nm according to the Bradford method [36]. Bovine serum albumin was used as the
standard protein [37–39]. For determination of LPO purity, sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) was used according to the procedure of Laemmli [40]. In this
application, the imaging method was performed in 10% and 3% acrylamide for the running and
the stacking gel, respectively, with 0.1% SDS [41–43].
The effects of secondary sulfonamide derivatives (4a
by affinity chromatography technique were previously determined [22]. In our study, LPO activity was
measured in the presence of different concentrations of secondary sulfanilamide derivatives (4a 8h).
–8h) on LPO purified from fresh bovine milk
–

Molecules 2017, 22, 793
4 of 9
A control sample without secondary sulfonamide was taken as 100%. An activity (%)-[Secondary
sulfonamide] plot was drawn. For the determination of the inhibition constant (Ki) values, three
different secondary sulfonamide derivative (4a–8h) concentrations were used. Additionally, ABTS
was used as a substrate at five different concentrations. Lineweaver–Burk plots (1/V-1/[ABTS])
were obtained for secondary sulfonamide; the Ki and the inhibition type were calculated from these
plots [44]. The data obtained were analysed by t-test and the results are given as Means ± SD.
3. Results and Discussion
Enzymes are biological macromolecules that accelerate or catalyse chemical reactions in biological
systems [45
by specific enzyme inhibitions [47
first systemically used antibiotics and paved the way for the antibiotic revolution in medicine. They
were also largely investigated by means of physiological, kinetic, and pharmacological studies [49 51].
,46]. At low concentrations, some chemicals and drugs alter normal enzyme activities
,48]. On the other hand, sulfonamide derivative drugs were the
–
In addition, these molecules are the most important and largely used zinc binding function for the
design of CA isoenzymes inhibitors. It was highlighted that the sulfonamide groups are an ideal ligand
for some enzyme active site [52,53].
Although sulfonamide derivatives are used in therapies, there has been no reported LPO activity
for these synthesized compounds (4a–8h). As show in Table 1, LPO was separately purified from
bovine milk by Sepharose 4B-L-tyrosine-sulfonamide affinity chromatography technique. The LPO
was purified 407.0-fold with a specific activity of 24.45 EU/mg and overall yield of 75.6% (Table 1).
The purification of LPO after Sepharose 4B-L-tyrosine-sulfonamide-affinity chromatography was
controlled by SDS-PAGE and a single band was observed for LPO (Figure 2). For secondary
sulfonamides (4a–8h), the inhibitor concentrations causing up to 50% inhibition (IC50 values) were
determined from the regression analysis graphs. IC50 values obtained for LPO are shown in Table 2.
From in vitro studies, it was understood that LPO was effectively inhibited by these secondary
sulfonamide derivatives (4a
used for designing new antithyroid agents [45]. To understand the nature of LPO inhibition, kinetic
experiments with different concentrations of secondary sulfonamides (4a 8h), varying the respective
concentrations of the substrates ABTS for each concentration of the sulfonamide derivatives (4a 8h
were performed. The Lineweaver–Burk plots were obtained by plotting 1/v versus 1/[ABTS], which
showed parallel lines for different concentrations of secondary sulfonamide derivatives (4a 8h).
The lines do not intersect at a common point as shown in Table 2, indicating that secondary sulfonamide
derivatives (4a 8h) inhibition effects against LPO are commonly non-competitive with respect to the
ABTS substrates. In this assay, it was suggested that the secondary sulfonamides (4a 8h) do not
directly react with LPO. However, any variation in the concentration of ABTS does not affect the
reactivity of secondary sulfonamides (4a 8h). This is also reflected in the non-competitive nature of
the inhibition by the compounds with respect to ABTS. The inhibition data of secondary sulfonamides
4a 8h) reported here are summarized in Table 2, and the following comments can be drawn from
these data. Additionally, LPO inhibition by sulfonamide derivatives (4a 8h) is dependent on the
–8h). The inhibition of LPO-catalysed iodination has sometimes been
–
–
)
–
–
–
–
(
–
–
positioning of the inhibitor in the active site; i.e., the distance between the atoms in the secondary
sulfonamide derivatives (4a–8h) and active site amino acids.
Table 1. Purification of lactoperoxidase (LPO) from bovine milk by Sepharose 4B-L-tyrosine-
sulfonamide affinity chromatography.
Total
Volume
(mL)
Enzyme
Activity
(EU/mL)
Total Enzyme
Activity
Total
Protein
(mg)
Specific
Activity
(EU/mg)
Protein
(mg/mL)
Yield
(%)
Purification
Fold
Purification Steps
(EU/mL·min)
Homogenate
Sepharose
4B-L-tyrosine-sulfonamide
60.0
10.0
1.0
4.5
60.0
15.0
900.0
1.84
0.06
100
1.00
45.0
0.184
24.45
75.6
407.0

Molecules 2017, 22, 793
5 of 9
Figure 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) band of LPO.
Columns a, b and c are purified LPO from bovine milk by affinity column chromatography. Column d
is standard proteins (Line 1: 250 kDa, Line 2: 150 kDa, Line 3: 100 kDa, Line 4: 70 kDa, Line 5: 50 kDa,
Line 6: 40 kDa, Line 7: 30 kDa, Line 8: 20 kDa).
Table 2. The inhibition types, IC50 and Ki values of some secondary sulfonamide derivatives (4a–8h)
on LPO purified from bovine milk.
No.
Compound Names
IC50 (µM)
Ki (µM)
Inhibition Type
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
Sulfathiazole
Sulfadiazine
Sulfamethazine
Sulfapyridine
Sulfisoxazole
Sulfamethizole
Sulfamerazine
Sulfanilamide
231.00
92.24
346.50
227.00
221.70
115.50
18.73
8.48
99.00
0.015
0.010
10.04
3.03
38.43 ± 6.06
90.66 ± 2.52
198.00 ± 24.46
65.00 ± 3.61
182.66 ± 34.07
21.18 ± 5.66
20.52 ± 2.14
35.70 ± 4.88
56.06 ± 23.56
0.026 ± 0.004
0.008 ± 0.003
49.15 ± 19.99
0.934 ± 0.357
0.324 ± 0.115
13.39 ± 6.19
2.29 ± 1.02
Non-competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Competitive
Competitive
Competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Non-competitive
Non-competitive
Noncompetitive
Non-competitive
Competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Competitive
Competitive
N-(sulfathiazole)-p-acetoxybenzamide
N-(sulfadiazine)-p-acetoxybenzamide
N-(sulfamethazine)-p-acetoxybenzamide
N-(sulfapyridine)-p-acetoxybenzamide
N-(sulfisoxazole)-p-acetoxybenzamide
N-(sulfamethizole)-p-acetoxybenzamide
N-(sulfamerazine)-p-acetoxybenzamide
N-(sulfanilamide)-p-acetoxybenzamide
N-(sulfathiazole)-3,4,5-triacetoxybenzamide
N-(sulfadiazine)-3,4,5-triacetoxybenzamide
N-(sulfamethazine)-3,4,5-triacetoxybenzamide
N-(sulfapyridine)-3,4,5-triacetoxybenzamide
N-(sulfisoxazole)-3,4,5-triacetoxybenzamide
N-(sulfamethizole)-3,4,5-triacetoxybenzamide
N-(sulfamerazine)-3,4,5-triacetoxybenzamide
N-(sulfanilamide)-3,4,5-triacetoxybenzamide
N-(sulfathiazole)-p-hydroxybenzamide
N-(sulfadiazine)-p-hydroxybenzamide
N-(sulfamethazine)-p-hydroxybenzamide
N-(sulfapyridine)-p-hydroxybenzamide
N-(sulfisoxazole)-p-hydroxybenzamide
N-(sulfamethizole)-p-hydroxybenzamide
N-(sulfamerazine)-p-hydroxybenzamide
N-(sulfanilamide)-p-hydroxybenzamide
N-(sulfathiazole)-3,4,5-trihydroxybenzamide
N-(sulfadiazine)-3,4,5-trihydroxybenzamide
N-(sulfamethazine)-3,4,5-trihydroxybenzamide
N-(sulfapyridine)-3,4,5-trihydroxybenzamide
N-(sulfisoxazole)-3,4,5-trihydroxybenzamide
N-(sulfamethizole)-3,4,5-trihydroxybenzamide
N-(sulfamerazine)-3,4,5-trihydroxybenzamide
N-(sulfanilamide)-3,4,5-trihydroxybenzamide
0.238
3.94
3.053
−3
−3
−3
0.656 × 10
13.86
138.60
1.56
1.096 × 10 ± 0.471 × 10
2.90 ± 1.13
152.03 ± 48.69
1.39 ± 0.15
2.03
6.66
1.28
3.05
1.07 ± 0.02
8.55 ± 1.45
1.49 ± 0.28
2.29 ± 0.98
15.06
0.040
0.426
2.96
10.83
0.799
1.759
0.285
0.014
0.715
2.53
10.19
1.99
173.25
1.484
1.782
6.85 ± 1.81
0.044 ± 0.022
0.484 ± 0.146
1.41 ± 0.13
15.08 ± 3.90
0.387 ± 0.150
3.939 ± 1.098
0.217 ± 0.099
0.025 ± 0.010
1.292 ± 0.465
2.17 ± 0.88
7g
7h
8a
8b
8c
8d
8e
8f
5.85 ± 1.28
1.44 ± 0.52
Competitive
292.78 ± 61.96
3.87 ± 0.43
Non-competitive
Non-competitive
Non-competitive
8g
8h
3.104 ± 0.578

Molecules 2017, 22, 793
6 of 9
All synthesized secondary sulfonamides derivatives (4a–8h) exhibited effective inhibitory
activity against LPO, one of the prominent enzymes in milk, with a Ki values in the range
of 1.096 0.471 616.78 M (Table 2). Additionally, N-(sulfathiazole)-
×
10−3 10−3–1203.83
3,4,5-triacetoxybenzamide (6a) demonstrated the most powerful LPO inhibition effect with
±
×
±
µ
low nanomolar Ki value (1.096 nM). On the other hand, among the synthesized secondary
sulfonamides (4a–8h) N-(sulfadiazine)-p-acetoxy-benzamide (5b, 260 nM), N-(sulfamethazine)-p-
acetoxybenzamide (5c, 80 nM), N-(sulfisoxazole) p-acetoxybenzamide (5e, 934 nM), N-(sulfamethizole)-
p-acetoxybenzamide (5f, 324 nM), N-(sulfanilamide)-p-hydroxybenzamide (7h, 217 nM) and
N-(sulfathiazole)-3,4,5-trihydroxybenzamide (8a, 25 nM), showed effective inhibition profiles against
bovine milk LPO. Additionally, in general, the acetylated sulfonamides are better inhibitors than
the non-acetylated sulfonamides, although the acetylated acids are poorer inhibitors than the
non-acetylated acids.
4. Conclusions
Secondary sulfonamides incorporating acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide
and trihydroxybenzamide–all possessing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole
groups–were synthesized, and biological activities were evaluated. Milk is a vital liquid secreted from
the mammary glands of females of all mammal species. So, the inhibition of the LPO is very important
in terms of the LPO system, which catalyses the oxidation of several different reactions by H2O2 of
a large range of substrates, such as the oxidation of endogenous thiocyanate (SCN−) to the antibacterial
hypothiocyanite (OSCN−). Secondary sulfonamides (4a
–8h) have shown micromolar to nanomolar
inhibition against LPO that is vital activity for the innate immune system because of removing bacteria
from milk and mucosal secretions. If LPO activity is reduced, this means that the immune system is
weakened. This is particularly undesirable, since this affects the immune system of infants that used
sulfonamides as a drug.
Author Contributions: Y.C. and H.U. purified lactoperoxidase enzyme from bovine milk by Sepharose
4B-L-tyrosine-sulfonamide affinity chromatography. Z.K., R.K. and Z.A. determined the inhibition effects of
sulfonamide derivatives on LPO enzyme. T.G. and A.C.G. synthesised secondary sulfonamide derivatives. I.G.
and H.O. participated in the study design and coordination, and drafted the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Supuran, C.T.; Innocenti, A.; Mastrolorenzo, A.; Scozzafava, A. Antiviral sulfonamide derivatives. Mini Rev.
Med. Chem. 2004, 4, 189–200. [CrossRef] [PubMed]
˙
Göçer, H.; Akıncıog˘lu, A.; Öztas¸kın, N.; Göksu, S.; Gülçin, I. Synthesis, antioxidant and antiacetylcholinesterase
activities of sulfonamide derivatives of dopamine related compounds. Arch. Pharm. 2013, 346, 783–792.
˙
Akbaba, Y.; Akıncıog˘lu, A.; Göçer, H.; Göksu, S.; Gülçin, I.; Supuran, C.T. Carbonic anhydrase inhibitory
properties of novel sulfonamide derivatives of aminoindanes and aminotetralins. J. Enzyme Inhib. Med. Chem.
2014, 29, 35–42. [CrossRef] [PubMed]
˙
4.
5.
6.
Akıncıog˘lu, A.; Topal, M.; Gülçin, I.; Göksu, S. Novel sulfamides and sulfonamides incorporating tetralin
scaffold as carbonic anhydrase and acetylcholine esterase inhibitors. Arch. Pharm. 2014, 347, 68–76.
Gokcen, T.; Al, M.; Topal, M.; Gülçin, I.; Ozturk, T.; Goren, A.C. Synthesis of some natural sulphonamide
derivatives as carbonic anhydrase inhibitors. Org. Commun. 2017, 10, 15–23.
Ivanova, J.; Carta, F.; Vullo, D.; Leitans, J.; Kazaks, A.; Tars, K.; Žalubovskis, R.; Supuran, C.T. N-Substituted
and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-associated carbonic
anhydrases IX and XII. Bioorg. Med. Chem. 2017. [CrossRef] [PubMed]
7.
Carta, F.; Birkmann, A.; Pfaff, T.; Buschmann, H.; Schwab, W.; Zimmermann, H.; Maresca, A.; Supuran, C.T.
Lead development of thiazoylsulfonamides with carbonic anhydrase inhibitory action. J. Med. Chem. 2017
60, 3154–3164. [CrossRef] [PubMed]
,

Molecules 2017, 22, 793
7 of 9
8.
9.
Petrou, A.; Geronikaki, A.; Terzi, E.; Guler, O.O.; Tiziano, T.; Supuran, C.T. Inhibition of carbonic anhydrase
isoforms I, II, IX, and XII with secondary sulphonamides incorporating benzothiazole scaffolds. J. Enzyme
Inhib. Med. Chem. 2016, 31, 1306–1311. [CrossRef] [PubMed]
Moeker, J.; Peat, T.S.; Bornaghi, L.F.; Vullo, D.; Supuran, C.T.; Poulsen, S.A. Cyclic secondary sulfonamides:
Unusually good inhibitors of cancer-related carbonic anhydrase enzymes. J. Med. Chem. 2014, 57, 3522–3531.
[CrossRef] [PubMed]
10. Qandil, A.M.; Hassan, M.A.; Al-Shar’i, N.A. Synthesis and anticandidal activity of azole-containing
sulfonamides. Arch. Pharm. 2008, 341, 99–112. [CrossRef] [PubMed]
11. Blank, B.R.; Alayog˘lu, P.; Engen, W.; Choi, J.K.; Berkman, C.E.; Anderson, M.O. N-Substituted glutamyl
sulphonamides as inhibitors of glutamate carboxypeptidase II (GCP2). Chem. Biol. Drug. Des. 2011, 77,
241–247. [CrossRef] [PubMed]
12. Burhop, A.; Weck, R.; Atzrodt, J.; Derdau, V. Hydrogen-isotope exchange (HIE) reactions of secondary and
tertiary sulfonamides and sulfonylureas with iridium (I) catalysts. Eur. J. Org. Chem. 2017, 11, 1418–1424.
[CrossRef]
13. Ueda, T.; Sakamaki, K.; Kuroki, T.; Yano, I.; Nagata, S. Molecular cloning and characterization of the
chromosomal gene for human lactoperoxidase. Eur. J. Biochem. 1976, 243, 32–41. [CrossRef]
˙
14. S¸is¸eciog˘lu, M.; Çankaya, M.; Gülçin, I.; Özdemir, M. The Inhibitory effect of propofol on lactoperoxidase.
Protein Pept. Lett. 2009, 16, 46–49.
15. S¸is¸eciog˘lu, M.; Çankaya, M.; Gülçin, I.; Özdemir, M. Interactions of melatonin and serotonin to
˙
lactoperoxidase enzyme. J. Enzyme Inhib. Med. Chem. 2010, 25, 779–783.
16. Elliot, R.M.; McLay, J.C.; Kennedy, M.J.; Simmonds, R.S. Inhibition of foodborne bacteria by the
lactoperoxidase system in a beef cube system. Int. J. Food Microbiol. 2004, 91, 73–81. [CrossRef]
˙
17. S¸is¸eciog˘lu, M.; Kireçci, E.; Çankaya, M.; Özdemir, H.; Gülçin, I.; Atasever, A. The prohibitive effect of
lactoperoxidase system (LPS) on some pathogen fungi and bacteria. Afr. J. Pharm. Pharmacol. 2010, 4,
671–677.
˙
18. S¸is¸eciog˘lu, M.; Gülçin, I.; Çankaya, M.; Atasever, A.; Özdemir, H. The effects of norepinephrine on
lactoperoxidase enzyme (LPO). Sci. Res. Essays 2010, 5, 1351–1356.
19. Jooyandeh, H.; Aberoumand, A.; Nasehi, B. Application of lactoperoxidase system in fish and food products:
A review. Am. Eurasian J. Agric. Environ. Sci. 2011, 10, 89–96.
˙
20. S¸is¸eciog˘lu, M.; Uguz, M.T.; Çankaya, M.; Özdemir, H.; Gülçin, I. Effects of ceftazidime pentahydrate,
prednisolone, amikacin sulfate, ceftriaxone sodium and teicoplanin on bovine milk lactoperoxidase activity.
Int. J. Pharmacol. 2011, 7, 79–83.
˙
21. S¸is¸eciog˘lu, M.; Gülçin, I.; Çankaya, M.; Özdemir, H. The inhibitory effects of L-Adrenaline on lactoperoxidase
enzyme (LPO) purified from buffalo milk. Int. J. Food Propert. 2012, 15, 1182–1189.
22. Munsch-Alatossava, P.; Quintyn, R.; De Man, I.; Alatossava, T.; Gauchi, J.P. Efficiency of N2 gas flushing
compared to the lactoperoxidase system at controlling bacterial growth in bovine raw milk stored at mild
temperatures. Front. Microbiol. 2016, 7, 839. [CrossRef] [PubMed]
˙
˙
23. Atasever, A.; Özdemir, H.; Gülçin, I.; Küfreviog˘lu, Ö.I. One-step purification of lactoperoxidase from bovine
milk by affinity chromatography. Food Chem. 2013, 136, 864–870. [CrossRef] [PubMed]
˙
24. Köksal, Z.; Usanmaz, H.; Özdemir, H.; Gülçin, I.; Güney, M. Inhibition effects of some phenolic and dimeric
phenolic compounds on bovine lactoperoxidase (LPO) enzyme. Int. J. Acad. Res. 2014, 6, 27–32.
25. Golmohamadi, A.; Morra, M.J.; Popova, I.; Nindo, C.I. Optimizing the use of Sinapis alba seed meal extracts
−
as a source of thiocyanate (SCN ) for the lactoperoxidase system. Lebensm. Wiss. Technol. 2016, 72, 416–422.
[CrossRef]
˙
26. Ozturk Sarikaya, S.B.; Sisecioglu, M.; Cankaya, M.; Gülçin, I.; Ozdemir, H. Inhibition profile of a series of
phenolic acids on bovine lactoperoxidase enzyme. J. Enzyme Inhib. Med. Chem. 2015, 30, 479–483. [CrossRef]
[PubMed]
˙
27. Koksal, Z.; Kalın, R.; Gülçin, I.; Özdemir, H.; Atasever, A. The impact of some avermectins on lactoperoxidase
from bovine milk. Int. J. Food Propert. 2016, 19, 1207–1216. [CrossRef]
˙
28. Gokcen, T.; Gülçin, I.; Ozturk, T.; Goren, A.C. A Class of Sulfonamides as carbonic anhydrase inhibitor.
J. Enzyme Inhib. Med. Chem. 2016, 31, 180–188. [CrossRef] [PubMed]
29. Shindler, J.S.; Bardsley, W. Steady-state kinetics of lactoperoxidase with ABST as chromogens. Biochem.
Biophys. Res. Commun. 1975, 67, 1307–1312. [CrossRef]

Molecules 2017, 22, 793
8 of 9
30. Jacob, B.M.; Anthony, K.E.; Sreekumar, B.; Haridas, M. Thiocyanate mediated antifungal and antibacterial
property of goat milk lactoperoxidase. Life Sci. 2000, 66, 2433–2439. [CrossRef]
˙
31. Gülçin, I.; Scozzafava, A.; Supuran, C.T.; Akıncıog˘lu, H.; Koksal, Z.; Turkan, F.; Alwasel, S. The effect of
caffeic acid phenethyl ester (CAPE) metabolic enzymes including acetylcholinesterase, butyrylcholinesterase,
glutathione s-transferase, lactoperoxidase and carbonic anhydrase ısoenzymes I, II, IX and XII. J. Enzyme
Inhib. Med. Chem. 2016, 31, 1095–1101.
˙
32. Gülçin, I.; Scozzafava, A.; Supuran, C.T.; Koksal, Z.; Turkan, F.; Çetinkaya, S.; Bingöl, Z.; Huyut, Z.;
Alwasel, S.H. Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase,
lactoperoxidase, acetylcholinesterase, butyrylcholinesterase, and carbonic anhydrase isoenzymes. J. Enzyme
Inhib. Med. Chem. 2016, 31, 1698–1702.
33. Cuatrecasa, P. Protein purification by affinity chromatography. J. Biol. Chem. 1970, 245, 3059–3065.
34. Arslan, O.; Nalbantoglu, B.; Demir, N.; Ozdemir, H.; Kufrevioglu, O.I. A new method for the purification of
carbonic anhydrase isozymes by affinity chromatography. Turk. J. Med. Sci. 1996, 26, 163–166.
˙
35. Boztas¸, M.; Çetinkaya, Y.; Topal, M.; Gülçin, I.; Menzek, A.; S¸ahin, E.; Tanc, M.; Supuran, C.T. Synthesis and
carbonic anhydrase isoenzymes I, II, IX, and XII inhibitory effects of dimethoxy-bromophenol derivatives
incorporating cyclopropane moieties. J. Med. Chem. 2015, 58, 640–650.
36. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–251. [CrossRef]
˙
37. Yıldırım, A.; Atmaca, U.; Keskin, A.; Topal, M.; Çelik, M.; Gülçin, I.; Supuran, C.T. N-Acylsulfonamides
strongly inhibit human carbonic anhydrase isoenzymes I and II. Bioorg. Med. Chem. 2015, 23, 2598–2605.
38. Aydin, B.; Gülcin, I.; Alwasel, S.H. Purification and characterization of polyphenol oxidase from Hems¸in
apple (Malus communis L.). Int. J. Food Propert. 2015, 18, 2735–2745. [CrossRef]
˙
39. Arabaci, B.; Gülçin, I.; Alwasel, S. Capsaicin: A potent inhibitor of carbonic anhydrase isoenzymes. Molecules
2014, 19, 10103–10114. [CrossRef] [PubMed]
40. Laemmli, D.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature
1975, 227, 680–685. [CrossRef]
˙
41. Göksu, S.; Naderi, A.; Akbaba, Y.; Kalın, P.; Akıncıog˘lu, A.; Gulcin, I.; Durdag˘i, S.; Salmas, R.E. Carbonic
anhydrase inhibitory properties of novel benzylsulfamides using molecular modeling and experimental
studies. Bioorg. Chem. 2014, 56, 75–82.
42. Güney, M.; Cos¸kun, A.; Topal, F.; Das¸tan, A.; Gülçin, I.; Supuran, C.T. Oxidation of
˙
cyanobenzocycloheptatrienes: Synthesis, photooxygenation reaction and carbonic anhydrase isoenzymes
inhibition properties of some new benzotropone derivatives. Bioorg. Med. Chem. 2014, 22, 3537–3543.
˙
43. Topal, M.; Gülçin, I. Rosmarinic acid: A potent carbonic anhydrase isoenzymes inhibitor. Turk. J. Chem. 2014
,
38, 894–902. [CrossRef]
44. Lineweaver, H.; Burk, D. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 1934, 56,
658–666. [CrossRef]
˙
45. Aksu, K.; Nar, M.; Tanç, M.; Vullo, D.; Gülçin, I.; Göksu, S.; Tümer, F.; Supuran, C.T. The synthesis of
sulfamide analogues of dopamine related compounds and their carbonic anhydrase inhibitory properties.
Bioorg. Med. Chem. 2013, 21, 2925–2931. [CrossRef] [PubMed]
˙
46. Gülçin, I.; Beydemir, S. Phenolic compounds as antioxidants: Carbonic anhydrase isoenzymes inhibitors.
Mini Rev. Med. Chem. 2013, 13, 408–430.
47. Akıncıog˘lu, A.; Akbaba, Y.; Göçer, H.; Göksu, S.; Gülçin, I.; Supuran, C.T. Novel sulfamides as potential
˙
carbonic anhydrase isoenzymes inhibitors. Bioorg. Med. Chem. 2013, 21, 1379–1385.
˙
48. Öztürk, Sarı; kaya, S.B.; Topal, F.; S¸entürk, M.; Gülçin, I.; Supuran, C.T. In vitro inhibition of
α-carbonic
anhydrase isozymes by some phenolic compounds. Bioorg. Med. Chem. Lett. 2011, 21, 4259–4262.
49. Temperini, C.; Scozzafava, A.; Supuran, C.T. Carbonic anhydrase inhibitors. X-ray crystal studies of the
carbonic anhydrase II-trithiocarbonate adduct-An inhibitor mimicking the sulfonamide and urea binding to
the enzyme. Bioorg. Med. Chem. Lett. 2010, 20, 474–478. [CrossRef] [PubMed]
50. Thiry, A.; Dogné, J.M.; Supuran, C.T.; Masereel, B. Anticonvulsant sulfonamides/sulfamates/sulfamides
with carbonic anhydrase inhibitory activity: Drug design and mechanism of action. Curr. Pharm. Des. 2008
14, 661–671. [PubMed]
,

Molecules 2017, 22, 793
9 of 9
51. Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C.T.; De Simone, G. Multiple binding modes of inhibitors
to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem. Rev. 2012, 112,
4421–4468. [CrossRef] [PubMed]
˙
52. Topal, F.; Gülçin, I.; Dastan, A; Guney, M. Novel eugenol derivatives: Potent acetylcholinesterase and
carbonic anhydrase inhibitors. Int. J. Biol. Macromol. 2017, 94, 845–851. [CrossRef] [PubMed]
53. Kirthana, M.V.; Khan, F.N.; Sivakumar, P.M.; Doble, M.; Manivel, P.; Prabakaran, K.; Krishnakumar, V.
Antithyroid agents and QSAR studies: Inhibition of lactoperoxidase-catalyzed iodination reaction by
isochromene-1-thiones. Med. Chem. Res. 2013, 22, 4810–4817. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).