The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acids derivatives and determination of their inhibition properties against human carbonic anhydrase I and II isoenzymes

Article abstract of DOI:10.3109/14756366.2015.1071808

The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acid (4) derivatives containing structural characteristics that can be used for the synthesis of several active molecules, is presented. Some of the butenoic acid derivatives (4a, 4c, 4e, 4i, 4j, 4k) are

Full text of DOI:10.3109/14756366.2015.1071808

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
The synthesis of (Z)-4-oxo-4-(arylamino)but-2-
enoic acids derivatives and determination of their
inhibition properties against human carbonic
anhydrase I and II isoenzymes
Koray Oktay, Leyla Polat Köse, Kıvılcım Şendil, Mehmet Serdar Gültekin,
İlhami Gülçin & Claudiu T. Supuran
To cite this article: Koray Oktay, Leyla Polat Köse, Kıvılcım Şendil, Mehmet Serdar Gültekin,
İlhami Gülçin & Claudiu T. Supuran (2015): The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic
acids derivatives and determination of their inhibition properties against human carbonic
anhydrase I and II isoenzymes, Journal of Enzyme Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1071808
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1071808
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RESEARCH ARTICLE
The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acids derivatives
and determination of their inhibition properties against human
carbonic anhydrase I and II isoenzymes
1
1
2
1
Koray Oktay , Leyla Polat Ko¨se , Kıvılcım S¸endil , Mehmet Serdar Gu¨ltekin , Ilhami Gu¨lc¸in1,3, and
_
Claudiu T. Supuran4,5
2
1Faculty of Science, Department of Chemistry, Ataturk University, Erzurum, Turkey, Faculty of Science and Arts, Department of Chemistry, Kafkas
3
4
University, Kars, Turkey, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, Dipartimento di Chimica Ugo
5
Schiff, Universita degli Studi di Firenze, Sesto Fiorentino (Firenze), Italy, and Neurofarba Department, Section of Pharmaceutical and Nutriceutical
`
`
Sciences, Universita degli Studi di Firenze, Sesto Fiorentino (Florence), Italy
Abstract
Keywords
The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acid (4) derivatives containing structural
characteristics that can be used for the synthesis of several active molecules, is presented. Some
of the butenoic acid derivatives (4a, 4c, 4e, 4i, 4j, 4k) are synthesized following literature
procedures and at the end of the reaction. In addition, structures of all synthesized derivatives
Butenoic acid, carbonic anhydrase, enzyme
inhibition, enzyme purification, isoenzymes
History
1
(4a–4m) were determined by H-NMR, 13C-NMR and IR spectroscopy. Carbonic anhydrase is a
Received 25 June 2015
Revised 7 July 2015
Accepted 8 July 2015
metalloenzyme involved in many crucial physiologic processes as it catalyzes a simple but
fundamental reaction, the reversible hydration of carbon dioxide to bicarbonate and protons.
Significant results were obtained by evaluating the enzyme inhibitory activities of these
derivatives against human carbonic anhydrase hCA I and II isoenzymes (hCA I and II). Butenoic
acid derivatives (4a–4m) strongly inhibited hCA I and II with Kis in the low nanomolar range of
1.85 0.58 to 5.04 1.46 nM against hCA I and in the range of 2.01 0.52 to 2.94 1.31 nM
against hCA II.
Published online 24 August 2015
Introduction
Literature offers several methods for synthesizing maleimide
structures; among these methods, the main strategy is to obtain
amino carboxylic acid (4) through the reaction of maleic
anhydride with primary amines. But-2-enoic acid (4) derivatives
are synthesized as the result of reaction of amine group with
lactone carbonyl through acid catalysis2,3. Opening of lactone ring
is a critical step that can affect the reaction time and the cost of
The name maleimide (1) was derived from maleic acid (3) and
imide functional group1. Maleimides are important structural
blocks with unsaturated structures in organic synthesis. In
polymer chemistry, such structure blocks are used as monomers.
When double bonds in maleimide molecules are saturated with
hydrogenation reaction, the succinimide structural blocks are
obtained (2).
maleimides4–6
.
Several derivatives involving the imide group have consider-
ably high biological activities; however, the synthesis of imide
groups in high yields and the development of low-cost synthesis
methods for these groups are restricted to applications in synthetic
and polymer chemistry. Cyclic structure involving the imide
group occupy an important place in synthetic organic and drug
chemistry. Especially, phthalimides are commonly used for the
protecting amino acids in addition to their remarkable use in the
field of medicine. Maleimides have easily usable characteristics in
Michael Addition and Diels–Alder reactions in synthetic organic
chemistry since 5-membered ring maleimides are more reactive
O
O
O
O
OH
OH
OH
NH
NH
H
N
Ar
O
O
O
O
1
2
3
4
Address for correspondence: Mehmet Serdar Gu¨ltekin, Faculty of
Science, Department of Chemistry, Ataturk University, 25240 Erzurum,
Turkey. Tel: +90-442 231 4428. Fax: +90 442 231 4109. E-mail:
gultekin@atauni.edu.tr
Ilhami Gu¨lc¸in, Faculty of Science, Department of Chemistry, Ataturk
University, 25240 Erzurum, Turkey. Tel: +90-442 231 4375. Fax: +90
442 231 4109. E-mail: igulcin@atauni.edu.tr
compared to those with 6-membered and 7-memeberd rings2,3
As maleimides can fit in the chemical structure of some proteins,
they make up an important category of biological substrates3,7
.
.
_
They can frequently be used as photon-initiator for the polymer-
ization of free radicals in polymer chemistry3–8, as well as

2
K. Oktay et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
monomer in the synthesis of polymaleimides and their copoly- antiglaucoma and anti-osteoporosis agents. They are used as
mers. They are among the most commonly mentioned chemical diuretics, anti-obesity and anti-infective drugs. These CAIs have
structures in the literature with their recently found functions, also been used for managing Alzheimer’s disease and a variety of
such as the use of their derivatives in the form of pharmaceutical neurological disorders. Many types of CAI derivatives have been
mid-products because of their antibacterial property5,9 and their reported recently, together with their potential applications39,48
.
characteristics of bonding with natural rubbers9,10. In addition, These chemical groups are used for the clinical treatment of some
they are used vastly in the space industry for resin encapsulation conditions for decades47,49–52
of IC-dyes and as structural adhesives for fiber-empowered In this contest, many efforts have been made for the
.
composites9,11. In recent times, some research groups12,13 development of specific CAIs, and some remarkable results
achieved a successful synthesis of 3-nitryl- and 3-acetoxy aryl have been achieved in the past 15 years53–59. In this study, we
maleimides. In the mentioned synthesis, antifungal activities of investigated the inhibitory effects of butenoic acid derivatives
derivatives were investigated and were found to have biological (4a–4m) on hCA I and II.
activities also.
N-aryl maleimide molecules were obtained from aryl aromatic
Experimental
compounds and maleic anhydrate using the methods mentioned in
literature. During the reaction, N-aryl maleimides associated with
dehydration produced high yields (Scheme 1).
The known and unknown derivatives, having maleimide skeleton
structure, were synthesized using the methods commonly known
in the literature, and biological activities of such derivatives were
investigated. Derivatives with amino carboxylic acid (4) skeleton
structure were synthesized through the reaction of maleic
anhydride with the related aromatic amine molecule. Due to
their structural features, activities of the synthesized derivatives
were investigated using enzymatic reactions. This work is the first
to report the syntheses of 4a, 4c, 4e, 4i, 4j, 4k derivatives, and
their structures were determinate by NMR spectroscopy
(Supplementary material). Melting points for all these new
derivatives are listed in Supplementary material, whereas that of
Enzyme activities of the synthesized derivatives were
investigated, as well as significant results obtained by these
syntheses were introduced in the literature. Carbonic anhydrases
(CA, E.C.4.2.1.1) are ubiquitous metalloenzymes that catalyze a
simple reaction, the interconversion between carbon dioxide
(CO2) and bicarbonate (HCO3) generate H+ ions in the hydration
reaction; thus, being one of the main players of pH regulation in
many tissues, organs and organisms14–18
.
CO2 þ H2O ! HCOꢀ3 þ Hþ
other derivatives are provided in the literature (Table 1)60–68
.
In humans, CAs are present in a large variety of tissues, such as
the gastrointestinal tract, the nervous system, the reproductive
tract, lungs, kidneys, skin and eyes19–24. This regulatory reaction
supports many biochemical and physiological processes asso-
ciated with pH control, fluid secretion and ion transport.
First, for the synthesis of (Z)-4-oxo-4-(aryl)but-2-enoic acid
skeleton structure, the derivatives 5–17 were treated with maleic
anhydrate at room temperature. High-yield derivatives, having
(Z)-4-oxo-4-(aryl)but-2-enoic acid (4) skeleton structures, were
obtained via ring-opening reaction at room temperature. Finally,
these derivatives were reacted with aromatic amine compounds
for the synthesis of (Z)-4-oxo-4-(aryl)but-2-enoic acid (4). The
yields of all the derivatives are showed in Table 1.
Six distinct genetic CA families, the a-, b-, g-, d-, f- and
Z-CAs, are known to date, constituting an interesting example of
convergent evolution at the molecular level25–31. Also, up to now,
16 different x-CA isoenzymes have been described in various
organisms32–35. These enzymes differ in their subcellular local-
ization, catalytic activity and susceptibility to different classes of
inhibitors. Some of them are cytosolic (CA I, CA II, CA III, CA
VII and CA XIII), others are membrane bound (CA IV, CA IX,
CA XII and CA XIV), two are mitochondrial (CA VA and CA
VB) and one is secreted in saliva (CA VI)36–41. The disregulated
activity of some of these isoenzymes leads to a large spectrum of
diseases, including retinal or cerebral edema (in which hCA I is
involved); epilepsy, glaucoma, edema, high altitude sickness
(hCA II seems to be the main, but not the only isoenzyme
involved in these conditions); oxidative stress (hCA III); retinitis
pigmentosa (hCA IV); obesity (hCA VA/VB); carcinogenesis
(hCA VI); epilepsy (hCA VII); tumorigenesis (hCA IX and XII;
but hCA XII is also implicated in glaucoma); sterility (hCA XIII)
and various retinopathies (in which hCA XIV is the main isoform
Affinity chromatography is the science of separation of
biomolecules based on their specific interactions, such as that
between enzyme and substrate to solid phase-coupled ligands.
Due to its ability to enrich selective targets, affinity chromatog-
raphy has remained a mainstay technique in separation chemis-
try69–72. In this study, both hCA I and II isoenzymes were purified
by Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatog-
raphy73–76. The affinity chromatography consists Sepharose-4B-
L-tyrosine-sulfanilamide that acts as an affinity matrix for
selective retention of CA isoenzymes77–79. The column material
was prepared according to a previous method80,81. Thus,
homogenate solution acidity was adjusted and supernatant was
transferred to the previously prepared column82. The protein flow
in the column eluates was spectrophotometrically determined at
280 nm. Sodium dodecyl sulphate-polyacrylamide gel electro-
phoresis (SDS-PAGE) was applied for the detection of both the
isoenzymes’ purity83–86. This technique is widely used in
biochemistry, genetics, forensics, molecular biology and biotech-
nology for the separation of biological macromolecules including
proteins, according to their electrophoretic mobility87–89. After
visualizing the SDS-PAGE process, a single band was observed
for hCA I and II isoenzymes. This protein-imaging method was
previously described90–93. In this application, the imaging method
was performed using 10 and 3% acrylamide for the running and
the stacking gel, respectively, with 0.1% SDS94.
involved)33,42–47
.
Inhibitors of carbonic anhydrase enzymes (CAIs) have a large
number of applications in therapy, including anticancer,
O
O
OH
NH -Ar
2
O
NH-Ar
hCA I and II isoenzyme activities were determined according
to the method of Verpoorte et al.95 The protein quantity was
spectrophotometrically measured at 595 nm according to the
Bradford method96. Bovine serum albumin was used as the
standard protein90. For determining the inhibition effect of each
O
4
O
Scheme 1. General synthesis (Z)-4-oxo-4-(arylamino)but-2-enoic acids
derivatives from maleic acid in catalytic acidic media.

DOI: 10.3109/14756366.2015.1071808
Inhibitory effects of butenoic acid derivatives on hCA I and II
3
Table 1. The synthesis of (Z)-4-oxo-4-(aryl) but-2-enoic acid (4) and
3-chloro-aryl maleimide (5) skeleton structures.
(continued )

4
K. Oktay et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Table 2. Human carbonic anhydrase isoenzymes I and II inhibition profile of some butenoic acid derivatives (4a–4m).
IC50 (nM)
Ki (nM)
Bromophenols
hCA I
r2
hCA II
r2
hCA I
hCA II
4a
4b
4c
4d
4e
42.77
35.54
30.43
38.82
28.72
32.25
30.43
32.88
31.87
32.59
37.42
40.91
41.07
6.07
0.9933
0.9902
0.9975
0.9924
0.9979
0.9948
0.9989
0.9986
0.9958
0.9990
0.9958
0.9933
0.9975
0.9154
34.49
26.04
23.45
29.64
23.04
32.71
27.66
32.63
30.19
27.48
29.61
27.79
28.95
5.50
0.959
41.18 12.17
32.47 7.72
33.00 4.69
37.96 5.51
29.05 9.23
26.32 6.49
22.15 7.76
33.86 10.30
29.19 6.94
32.23 9.73
35.18 11.50
41.79 13.19
31.95 3.45
6.76 2.55
29.05 5.21
21.35 3.04
24.03 7.52
21.74 1.64
21.34 6.26
23.52 3.67
22.34 4.65
28.31 4.93
24.92 4.24
20.91 3.14
23.21 5.22
23.03 4.57
28.38 5.18
5.85 2.56
0.9956
0.9931
0.9857
0.9973
0.9945
0.9921
0.9863
0.9823
0.9932
0.9889
0.9934
0.9855
0.9636
4f
4g
4h
4i
4j
4k
4l
4m
AZA*
*AZA (Acetazolamide) was used as a standard inhibitor for both hCA enzymes.
butenoic acid derivative, an activity (%)-[butenoic acid deriva- a nitro group (–NO2). It is well known that these groups are
tives] graph was drawn. To determine Ki values, three different biologically very reactive and the molecules in these groups
butenoic acid derivative concentrations were tested. Also, differ- demonstrated effective CA isoenzyme inhibition properties. On
ent substrate concentrations were used and Lineweaver–Burk the other hand, acetazolamide (AZA) is considered a broad-
curves were drawn97, as previously described52.
specificity CA inhibitor due to its widespread inhibition of CAs,
showing Ki value of 6.76 2.55 nM against hCA I isoenzyme.
The inhibition effects of all butenoic acid derivatives (4a–4m) are
close to acetazolamide. AZA is considered a good CA inhibitor
and approved for the treatment of a range of conditions, including
glaucoma, epilepsy and altitude sickness5.
Results and discussion
Carbonic anhydrase enzyme inhibitors (CAIs) have a large
spectrum of applications in therapy, including blood pressure-
lowering, anticancer, vasodilator effects, antiglaucoma and anti-
osteoporosis agents. They are used as diuretics, anti-obesity and
anti-infective drugs. It has been shown that the CA inhibition in
smooth muscle cells results in a rise in pH, leading to KCa
channel activation and vasorelaxation98,99. Also, these CAIs have
been used for managing Alzheimer’s disease and a variety of
neurological disorders. Many types of CAIs and new derivatives
have been reported recently, together with their potential appli-
cations. In our study, physiologically relevant hCA I and II
isomers are studied. A dozen of butenoic acid derivatives (4a–
4m) were examined for their hCA I and II isoenzymes inhibition
properties. All butenoic acid derivatives (4a–4m) have shown
efficient inhibition against both isoforms. The chemical structures
of butenoic acid derivatives (4a–4m) are given in Table 1. Also,
CA I and II inhibiting effects of a dozen of butenoic acid
derivatives (4a–4m) are summarized in Table 2. It is well known
that developing isoenzyme-specific CAIs is highly beneficial in
obtaining novel classes of drugs devoid of various undesired side
effects.
CA II is involved in several diseases, including glaucoma,
epilepsy, altitude sickness and edema. Compared to the physio-
logically dominant isoform hCA II, all butenoic acid derivatives
(4a–4m) showed Ki values ranging from 20.91 3.14 to
29.05 5.21 nM (Table 2). The butenoic acid derivative 4j,
derived from 3-aminobenzoic acid, had two carbonyl groups
(–C¼O), an amine group (–NH2), a hydroxyl group (–OH) and an
acetate group (–COO), being the best hCA II inhibitor (Ki:
20.91 3.14 nM). The molecules with acetate, azide, cyanate,
sulfide and cyanide groups bind the active site of CA100. In
another study, the binding of acetate ions to bovine CA was
investigated by NMR and inhibition measurements. It was found
that two acetate ions were found to interact with the protein, but
only one is linked with the inhibition of the esterase activity of the
bovine CA101. However, all butenoic acid derivatives (4a–4m)
demonstrated similar hCA II inhibition properties. Also, the
results obtained from this study showed that butenoic acid
derivatives (4a–4m) had generally higher affinity toward hCA II
than that of hCA I isoform. Also, AZA, which may interact with
the distinct hydrophobic and hydrophilic halves of the CA II
active site, showed Ki value of 5.85 2.56 nM.
Cytosolic isoenzyme hCA I is found in many tissues; however,
it was demonstrated that this isoenzyme is involved in retinal and
cerebral edema, and its inhibition may be a valuable tool for
fighting these conditions. Also, it was reported that the Ki value of
a dozen of butenoic acid derivatives (4a–4m) was less than 50 nM
Conclusion
(Kis550 nM). The results obtained from this study clearly Although (Z)-4-oxo-4-(aryl)but-2-enoic acid molecules have very
indicate that a dozen of butenoic acid derivatives (4a–4m) had simple skeletons, they are very active biological compounds, so
potent inhibition profile against slow hCA I, and cytosolic their monomer property is used in polymer chemistry. Therefore,
dominant rapid isozymes hCA II with low nanomolar range the synthesis of these molecules is an important issue for
(Kis530 nM). These compounds derived from butenoic acid (4a– researches. (Z)-4-Oxo-4-(aryl)but-2-enoic acid (4) derivatives
4m) bind to hCA I in the low nanomolar range and were found as used in this study influence the activity of CA enzymes due to
stronger inhibitors of this isoform, with Kis ranging between the presence of different functional groups (–Et, –Me, –COOH,
22.15 7.76 and 41.79 13.19 nM. However, the most powerful –SO2NH2, –CN, –NO2, and –NH2) in their aromatic scaffold.
inhibition effect was found in butenoic acid derivative 4g derived These findings signify that substituted but-2-enoic acid deriva-
from 3-nitroaniline (11), with Ki value of 22.15 7.76 nM. tives may be used as leads for generating potent CAIs. In this
Butenoic acid derivative 4g possess two carbonyl groups study, a dozen of butenoic acid derivatives (4a–4m) were
(–C¼O), an amine group (–NH2), a hydroxyl group (–OH) and evaluated against cytosolic human carbonic anhydrase isoenzyme

DOI: 10.3109/14756366.2015.1071808
Inhibitory effects of butenoic acid derivatives on hCA I and II
5
(Oncorhynchus mykiss) erythrocytes in vitro and in vivo. Turk J Vet
Anim Sci 2005;29:841–5.
21. Supuran CT. Carbonic anhydrases: novel therapeutic applications for
I and II. All the butenoic acid derivatives (4a–4m) have shown
low nanomolar inhibition against cytosolic CA I and II. Both
isoenzymes were potently inhibited by butenoic acid derivatives
(4a–4m) with Kis in the range of 22.15 7.76–41.79 13.19 nM
against hCA I, and 20.91 3.14–29.05 5.21 nM against hCA II.
inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81.
¨
_
22. Innocenti A, Oztu¨rk Sarıkaya SB, Gu¨lc¸in I, Supuran CT. Carbonic
anhydrase inhibitors. Inhibition of mammalian isoforms I-XIV with
a series of natural product polyphenols and phenolic acids. Bioorg
Med Chem 2010;18:2159–64.
Declaration of interest
_
23. Innocenti A, Gu¨lc¸in I, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors. Antioxidant polyphenol natural products
effectively inhibit mammalian isoforms I-XV. Bioorg Med Chem
Lett 2010;20:5050–3.
The authors report there is no conflict of interests.
_
24. Gu¨lc¸in I, Beydemir S. Phenolic compounds as antioxidants:
Carbonic anhydrase isoenzymes inhibitors. Mini Rev Med Chem
2013;13:408–30.
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