Synthesis of 3-chloro-1-substituted aryl pyrrolidine-2,5-dione derivatives: discovery of potent human carbonic anhydrase inhibitors

Article abstract of DOI:10.1007/s00044-017-1865-2

Carbonic anhydrase isoenzymes are important metalloenzymes that are involved in many physiologic processes, in which they catalyze the reversible hydration of carbon dioxide (CO₂) to bicarbonate (HCO₃ ^–) and protons (H

Full text of DOI:10.1007/s00044-017-1865-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1865-2
ORIGINAL RESEARCH
Synthesis of 3-chloro-1-substituted aryl pyrrolidine-2,5-dione
derivatives: discovery of potent human carbonic anhydrase
inhibitors
Koray Oktay1 Leyla Polat Kose1 Kıvılcım Şendil2 Mehmet Serdar Gültekin1
●
●
●
●
1,3
İlhami Gülçın
Received: 18 February 2016 / Accepted: 4 March 2017
© Springer Science+Business Media New York 2017
Abstract Carbonic anhydrase isoenzymes are important
metalloenzymes that are involved in many physiologic
processes, in which they catalyze the reversible hydration of
The 3-chloro-1-aryl pyrrolidine-2,5-diones strongly inhib-
ited the activity of human carbonic anhydrase I and II, with
Ki values in the low nanomolar range of 23.27–36.83 nmol/
L against human carbonic anhydrase I and 10.64–31.86
nmol/L against human carbonic anhydrase II.
–
carbon dioxide (CO2) to bicarbonate (HCO3 ) and protons
(H+) via a metal hydroxide nucleophilic mechanism.
Because of their known biological activities and potential as
carbonic anhydrase inhibitors, the present study focused on
developing of a convenient route to synthesize 3-chloro-1-
aryl pyrrolidine-2,5-diones (2b). This synthetic route started
with (Z)-4-oxo-4-(arylamino)but-2-enoic acid (3b) and
sulfurous dichloride (SOCl2) and resulted in a ring closing
reaction that produced a series of 3-chloro-N-aryl maleimide
derivatives (20–29) in good yields. This is the first report of
the syntheses of 3-chloro-1-aryl pyyrolidine-2,5-diones 20–
23 and 27–29 by ring-closing reactions of (Z)-4-oxo-4-
(arylamino)but-2-enoic acid. The structures of all of the
products were determined by 1H-NMR, 13C-NMR and
infrared spectroscopy. Their biological activities were
studied against human carbonic anhydrase I, and II.
●
●
Keywords Aryl pyrrolidine Carbonic anhydrase
●
●
Isoenzymes Enzyme inhibition Enzyme purification
Introduction
Carbonic anhydrases (CAs) are a superfamily of metal-
loenzymes that catalyze the interconversion between carbon
–
dioxide (CO2) and bicarbonate (HCO3 ) via a metal
hydroxide nucleophilic mechanism. The interconversion
–
between CO2 and HCO3 supports many biochemical and
physiological processes associated with pH control, fluid
secretion and ion transport (Boztaş et al. 2015). CAs are
ubiquitous in almost all living organisms (Supuran 2013;
Scozzafava et al. 2015a; Carta et al. 2015; Akıncıoğlu et al.
2015). In humans, CAs are present in many tissues,
including the gastrointestinal tract, nervous system, repro-
ductive tract, lungs, kidneys, skin, and eyes (Supuran 2008;
Yıldırım et al. 2015). Six distinct CA families, the α-, β-, γ-,
δ-, ζ- and η-CAs, have been described to date, and these
families present an interesting example of convergent evo-
lution at the molecular level (Scozzafava et al. 2015b;
Arabaci et al. 2015). Sixteen different CA isoenzymes that
have been found in various organisms (Ceruso et al. 2014),
and these isoenzymes differ in their subcellular localization,
catalytic activity, and susceptibility to different classes of
inhibitors. Some of them are cytosolic (CA I, CA II, CA III,
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1865-2) contains supplementary material,
which is available to authorized users.
* Mehmet Serdar Gültekin
gultekin@atauni.edu.tr
* İlhami Gülçın
igulcin@atauni.edu.tr
1
Faculty of Science, Department of Chemistry, Ataturk University,
Erzurum 25240, Turkey
2
Faculty of Science and Arts, Department of Chemistry, Kafkas
University, Kars 36100, Turkey
3
College of Science, Department of Zoology, King Saud
University, Riyadh, Saudi Arabia

Med Chem Res
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)
(Supuran and Scozzafava 2002; Göçer et al. 2015). It was
recently report that the CA XV isoform is not expressed in
humans or primates, although it is found in rodents and
other higher vertebrates. There are only three known a
catalytic forms called the CA-related proteins VIII, X, and
XI (CARP VIII, X, and XI) (Akbaba et al. 2014; Göksu
et al. 2014). Malfunctioning or altered expression of CA
inhibitors (CAIs) could promote pathological situations.
CAIs have been used as therapeutics in clinical appli-
cations for decades (Carta et al. 2015). CAIs include
anticancer, antiglaucoma, and anti-osteoporosis agents as
well as diuretics, anti-obesity and anti-infective drugs. CAIs
have also been used to manage Alzheimer’s disease and a
variety of neurological disorders. Recent reports have
described many new CAIs and their potential applications
(Güney et al. 2014). Over the past 15 years, much effort has
been devoted to the development of CAIs for specific
applications, and excellent progress has been made (Neri
and Supuran 2011; Pacchiano et al. 2011; Topal and Gülçin
2014). One such inhibitor is acetazolamide (AZA), which is
used clinically as a CAI. AZA is broad-spectrum inhibitor
that is approved for treating of a range of conditions,
including glaucoma, epilepsy, and altitude sickness (Yıl-
dırım et al. 2015). The development of isoenzyme-specific
inhibitors will be beneficial for obtaining novel classes of
drugs that have fewer undesirable side effects than broad
spectrum inhibitors do (Çoban et al. 2007; Boztaş et al.
2015).
Macromolecules containing an imide group have high
thermochemical stability and biological activity and are
promising scaffolds for application as CAIs (Colquhoun
et al. 2003). However, imide groups are difficult to syn-
thesize in high yields, and the methods for their synthesis
are typically expensive. Consequently, the application of
imide groups in synthetic, pharmaceutical and polymer
chemistry has been limited (Colquhoun et al. 2003). Mal-
eimide (1a), which was named because of its structural
similarities to maleic acid (3a) and the imide functional
group (Boros et al. 2007), and its derivatives have
5-membered rings. These compounds are known to be more
reactive in many reactions, such as Diels–Alder reactions
and Michael additions, than are compounds with 6- or
7-membered rings (Faturacı and Coskun 2012). Because of
its unsaturated structure, maleimide is an important building
block in organic synthesis, including polymer and phar-
maceutical chemistry. Saturation of the double bond in
maleimide by hydrogenation reactions yields succinimide
(2a) as a building block. Maleimides are biological sub-
strates because they can be inserted into the structures of
some proteins (Faturacı and Coskun 2012; Kumar et al.
2013). They are frequently used as photon initiators for
polymerization of free radicals in polymer chemistry,
(Faturacı and Coskun 2012; Khattab et al. 2012; Li et al.
2012; Saedi 2013) and used as monomers in the synthesis of
poly-maleimides and their copolymers. Phthalimides, which
are derivatives of maleimides, are commonly used for the
protection of amino acids and in pharmaceutical applica-
tions. Many studies have investigated the use of maleimides
and their derivatives, because they have a number of useful
functions, including their use as antimicrobial agents (Isobe
et al. 2001; Khattab et al. 2012) and for bonding natural
rubbers (El-Gaby et al. 2002; Isobe et al. 2001). Addition-
ally, they have many uses in aerospace, where they are used
for the resin encapsulation of indocyanine dyes and as
structural adhesives in composites (El-Gaby et al. 2002;
Kaur et al. 2013). In a recent study (Ol’shevskaya et al.
2012), 3-nitryl- and 3-acetoxy aryl maleimides were suc-
cessfully synthesized and found to have good antifungal
activities.
Several methods have been detailed in the literature for
the synthesis of maleimide derivatives (1b), with the main
strategy involving the production of amino carboxylic acid
(3b) from maleic anhydride and primary amines (Oktay
et al. 2016). This route results in maleimide (1a) synthesis
through an attack of the amine group on the acid carbonyl
under acid catalysis and produces water as a byproduct
(Faturacı and Coskun 2012). N-Aryl maleimides have been
synthesized in two stages from maleic anhydride and aniline
derivatives (Faturacı and Coskun 2012; Saedi 2013).
Dehydration is an important step in maleimide synthesis
and can affect both the length and cost of the reaction
(Khattab et al. 2012). N-Aryl maleimides with an acetate or
chloride group at the 3-position can be synthesized by ring-
closing reactions of 3b using acetic anhydride (Ac2O) and
sulfurous dichloride (SOCl2) (Haval et al. 2006; El-Gaby
et al. 2002; Faturacı and Coskun 2012). In the acetic
anhydride reactions, ring opening of maleic anhydride
occurs first, and this is followed by conversion to an amino
carboxylic acid derivative (3b) (Colquhoun et al. 2003;
Oktay et al. 2016). In the final stage, the N-aryl maleimide is
formed in high yield and can then be used for the synthesis
of 3-chloro aryl maleimides. However, the formation of
excess acetic acid occurs in the final stage of the reaction,
which is undesirable for chromatographic studies.
In the present study, SOCl2 was used for dehydration
instead of Ac2O to avoid production of excess acetic acid
and to reduce the need for the chromatographic purification
of the product. Ring closing products (2b, 3-chloro-1-aryl
pyrrolidine-2,5-diones 20–29) were produced in high yields
(Scheme 1). However, the 3-chloro-1-aryl pyrrolidine-2,5-
diones 30, 31, and 32 were not obtained from the corre-
sponding butenoic acid derivatives (17, 18, and 19). The
synthesized molecules were investigated for their inhibition

Med Chem Res
Scheme 1 The synthestic route
for 3-chloro-1-aryl pyrrolidine-
2,5-diones
properties against of human CA (hCA) I, and II isoenzymes,
which have attracted much interest as the slow cytosolic
isoform (hCA I) and dominant physiological isoform (hCA
II). The inhibition properties of the prepared compounds
were compared to that of AZA.
Experimental procedure for synthesis of 3-chloro-1-
substituted arylpyrrolidine-2,5-dione derivatives
General procedure 1.0 eq. (Z)-4-oxo-4-(arylamino) but-2-
enoic acid molecules (7–19) was solved with 5 ml SOCl2
and mixture refluxed and the reaction monitored by thin
layer chromatograph (TLC). All of the reaction time rea-
lized between 5 and 8 h in reflux condition. End of the
reaction, the reaction mixture added to 20 mL saturated
aqueous NaHCO3, and mixture stirred for 10 min. The
organic layer was separated and then washed with water
(100 mL), dried with MgSO4, and concentrated to give the
corresponding 3-chloro-1-substituted arylpyrrolidine-2,5-
dione derivatives.
Materials and method
Experimental
Known
(3-chloro-1-(4-nitrophenyl)pyrrolidine-2,5-dione
(24), 3-chloro-1-(3-nitrophenyl) pyrrolidine-2,5-dione (25),
3-chloro-1-(2-nitrophenyl) pyrrolidine-2,5-dione (26)) and
unknown (3-chloro-1-(4-ethylphenyl)pyrrolidine-2,5-dione
(20), 3-chloro-1-mesityl pyrrolidine-2,5-dione (21), 3-
chloro-1-(naphthalen-1-yl) pyrrolidine-2,5-dione (22), 3-
chloro-1-(4-(trifluoromethyl)quinolin-5-yl)pyrrolidine-2,5-
dione (23) and 4-(3-chloro-2,5-dioxopyrrolidin-1-yl)ben-
zoic acid (27), 3-(3-chloro-2,5-dioxopyrrolidin-1-yl)benzoic
acid (28), 4-(3-chloro-2,5-dioxopyrrolidin-1-yl)benzonitrile
(29)) maleimide derivatives were synthesized using estab-
lished methods as detailed in the electrospray ionization
(ESI). Compounds with amino carboxylic acid (3b) struc-
tures were synthesized by reaction of maleic anhydride with
the relevant aromatic amine as described in the literature
(Colquhoun et al. 2003). Compounds with a (Z)-4-oxo-4-
(aryl)but-2-enoic acid (4) skeleton were obtained via ring-
opening reactions at room temperature (General synthetic
procedure). These compounds were then treated with SOCl2
to yield the corresponding 3-chloro-aryl maleimide deriva-
tives (20–29) via ring-closing reactions. This is the first
report of the synthesis of compounds 20–23 and 27–29, and
their structures determined by NMR spectroscopy (see ESI).
Melting points recorded for all new compounds and
reported in the ESI. The methods for preparation of the
known compounds have been reported elsewhere
(see Table 1) (Mohammed and Mustapha 2010; Molla and
Ghosh 2012). The yields of all the prepared compounds
shown in Table 1.
The known molecules in literature
3-Chloro-1-(4-nitrophenyl)pyrrolidine-2,5-dione (24) (Gaina
and Gaina 2005), 3-chloro-1-(3-nitrophenyl)pyrrolidine-
2,5-dione (25) (Kumar et al. 1986), 3-chloro-1-(2-
nitrophenyl) pyrrolidine-2,5-dione (26) (Hiran et al. 2007).
Spectroscopic data for the unknown molecules (20–23,
27–29) in literature
3-Chloro-1-(4-ethylphenyl) pyrrolidine-2,5-dione (20)
Compound 20 recrystallized by n-hexane-CH2Cl2 solvent
mixture and it obtained as light yellowish solid. Melting
point: 158 °C, IR (cm−1, KBr): 2964, 1698, 1507, 1406,
1323, 1268, 1180, 1017, 972, 850, 640. 1H-NMR
(400 MHz, CDCl3, ppm): 1.25 (t, J = 7.7 Hz, 3H), 2.70
(q, J = 7.7 Hz, 2H), 3.10 (dd, J = 4.0, 18.7), 1H), 3.46 (dd,
J = 8.8, 18.7 Hz, 1H), 4.76 (d, J = 4.0), 7.20 (m, 2H), 7.26
(m, 2H). 13C-NMR (100 MHz, CDCl3, ppm): 17.6 (CH3, C-
8,), 17.9 (CH3, C-8′), 21.3 (CH3, C-11), 39.9 (CH2, C-4),
49.4 (CHCl, C-3), 129.7 (CH, C-9), 129.8 (CH, C-9′), 135.3
(C, C-7), 135.8 (C, C-7′), 140.1 (C, C-10), 142.8 (C, C-6),
172.1 (2C=O, C-2, -5).
3-Chloro-1-mesitylpyrrolidine-2,5-dione (21) Compound
21 recrystallized by n-hexane-ethyl acetate solvent mixture

Med Chem Res
Table 1 The synthesis of 3-chloro-substituted aryl maleimide (5) skeleton structures
O
COOH
SOCl2
N-Ar-X
Yields %
CONHAr-X
Cl
O
(Z)-4-(Arylamino)-
3-Chloro-1-
4-oxobut-2-enoic acid
(aryl)pyrrolidine-2,5-dione
X: Et, Me, CN, NO2, COOH, CF3, SO2NH2
Entry
1
(Z)-4-oxo-4-(arylamino)
Yields (%)
92
3-Chloro-1-aryl
but-2-enoic acidsa
pyrrolidine-2,5-dionesa
71a
20
21
2
90
812
3
4
5
6
89
96
83
85
913
22
23
101a
2415
1114

Med Chem Res
Table 1 continued
Entry
(Z)-4-oxo-4-(arylamino)
but-2-enoic acidsa
Yields (%)
3-Chloro-1-aryl
pyrrolidine-2,5-dionesa
2517
1216
7
8
87
76
2616
1316
141a
27
28
9
92
85
151a
10
1618
29
11
12
–
–
No reaction
1719
No reaction
No reaction
181a
13
–
191a
References number of known molecules in literature
a

Med Chem Res
and it obtained as yellow solid. Melting point: 125 °C, IR
(cm−1, KBr): 2996, 2859, 1713, 1607, 1484, 1372, 1289,
1199, 1031, 949, 857. H-NMR (400 MHz, CDCl3, ppm):
2.04 (s, 3H), 2.11 (s, 3H), 2.30 (s, 3H), 3.10 (dm, J = 18.8
Hz, 1H), 3.49 (dd, J = 18.8, 8.5 Hz, 1H), 4.81 (m, 1H), 6.98
(m, 2H). 13C-NMR (100 MHz, CDCl3, ppm): 17.6 (CH3, C-
8,), 17.9 (CH3, C-8′), 21.3 (CH3, C-11), 39.9 (CH2, C-4),
49.4, (CHCl, C-3), 129.7 (CH, C-9), 129.8 (CH, C-9′),
135.3 (CH, C-7), 135.8 (CH, C-7′), 140.1 (C, C-10), 142.8
(C, C-6), 172.1 (2C=O, C-2, -5).
3-(3-Chloro-2,5-dioxopyrrolidin-1-yl)benzoic acid (28)
The molecule 28 obtained as white color solid and it
recrystallized by n-hexane-ethylacetate solvent mixture.
Melting point: 177 °C. IR (cm−1, KBr): 2957, 1717, 1605,
1
1
1590, 1489, 1379, 1277, 1170, 1083, 951, 859, 751. H-
NMR (400 MHz, CDCl3, ppm: 3.19 (dd, J = 3.0, 19.5 Hz,
1H), 3.69 (dd, J = 7.8, 19.5 Hz, 1H), 5.02 (m, 1H), 7.76 (m,
3H), 8.25 (m, 1H). 13C-NMR (100 MHz, CDCl3, ppm):
39.1 (CH2, C-4), 52.5 (CHCl, C-3), 118.9 (CH, C-7), 125.1
(CH, C-9), 129.3 (C, C-8), 130.1 (CH, C-10), 132.7 (CH,
C-11), 134.2 (C, C-6), 161.1 (C=O, C-2), 166.3 (COOH,
C-12), 176.5 (C=O, C-5).
3-Chloro-1-(naphthalen-1-yl) pyrrolidine-2,5-dione (22)
The molecule 22 obtained light yellow solid and recrys-
tallized by n-hexane-ethylacetate solvent mixture. Melting
point: 157oC. IR (cm−1, KBr): 3020, 1868, 1706, 1634,
4-(3-Chloro-2,5-dioxopyrrolidin-1-yl)benzonitrile
(29)
Compound 29 obtained as white color solid and it recrys-
tallized by n-hexane-ethylacetate solvent mixture. Melting
point: 174 °C. IR (cm−1, KBr): 3288, 2225, 1706, 1625,
1
1524, 1496, 1347, 1228, 1012, 970, 892, 788. H-NMR
(400 MHz, CDCl3, ppm): 8.01–7.91 (m, 2H), 7.61–7.50 (m,
4H), 7.45–7.29 (m, 1H), 4.91 (dd, J = 8.5, 3.6 Hz, 1H), 3.61
(dd, J = 19.0, 8.5 Hz, 1H), 3.23 (dd, J = 19.0, 3.6 Hz, 1H).
13C-NMR (100 MHz, CDCl3, ppm): 39.9 (CH2, C-4), 49.3
(–CHCl–, C-3), 121.4 (CH, C-7), 125.0 (CH, C-9), 125.3
(CH, C-14), 126.4 (C, C-15), 126.9 (CH, C-13), 127.7 (CH,
C-12), 128.8 (CH, C-8), 130.6 (CH, C-11), 134.4 (C, C-10),
134.5 (C, C-6), 172.4 (2C=O, C-2, -5).
1
1590, 1404, 1320, 1259, 1161, 971, 898, 836, 752. H-
NMR (400 MHz, CDCl3, ppm: 3.11 (dd, J = 4.0, 20.1 Hz,
1H), 3.52 (dd, J = 8.7, 20.1 Hz, 1H), 4.82 (dd, J = 4.0, 8.7
Hz, 1H), 7.56 (m, 2H), 7.77 (m, 2H). 13C-NMR (100 MHz,
CDCl3, ppm): 39.3 (CH2, C-4), 48.7 (CHCl, C-3), 112.8 (C,
C-9), 117.8 (CN, C-10), 126.7 (2CH, C-7, -7′), 133.1 (2CH,
C-8, -8′), 135.1 (C, C-6), 171.2 (C=O, C-2), 171.3 (C=O,
C-5).
3-Chloro-1-(4-(trifluoromethyl)quinolin-5-yl)pyrrolidine-
2,5-dione (23) Compound 23 obtained as yellowish nee-
dles solid and it recrystallized by n-hexane-methylene
Biochemical studies
chloride solvent mixture. Melting point: 192 °C. IR (cm−1
,
In this study, the activities of the synthesized compounds
were tested against two physiologically relevant CAs, hCA
I, and II. Both hCA isoenzymes were purified by affinity
chromatography with a Sepharose-4B-L-tyrosine-sulfanila-
mide matrix for selective retention of CAs (Aksu et al.
2013; Göçer et al. 2013; Çetinkaya et al. 2014; Akıncıoğlu
et al. 2014), which was prepared according to an established
method (Akıncıoğlu et al. 2013; Gülçin and Beydemir
2013). For purification, the acidity of the homogenate
solution was adjusted with solid Tris, and the supernatant
was transferred to the prepared column (Atasaver et al.
2013; Nar et al. 2013). Proteins in the eluates were detected
spectrophotometrically at 280 nm (Gülçin et al. 2005;
Aydin et al. 2015). Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (0.1% sodium dodecyl sulfate) was used
to determine the purities of the CAs using an established
method with 10 and 3% acrylamide for the running and
stacking gels, respectively (Şişecioğlu et al. 2010; Şentürk
et al. 2011). A single band was observed for each CA
(Şişecioğlu et al. 2012; Köksal et al. 2012). The quantity of
each protein was measured spectrophotometrically at 595
nm during the purification according to the Bradford
method (1976).
KBr): 3055, 1605, 1589, 1479, 1339, 1133, 1099, 964, 814,
777, 681. H-NMR (400 MHz, CDCl3, ppm: 7.79 (dd, J =
1
8.7, 1.8 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.38 (dd, J = 8.7,
2.0 Hz, 1H), 6.80 (s, 1H), 4.78 (dd, J = 8.8, 4.1Hz, 1H),
3.49 (dd, J = 19.0, 8.8 Hz, 1H), 3.10 (dd, J = 19.0, 4.1 Hz,
1H. 13C-NMR (100 MHz, CDCl3, ppm): 39.4 (CH2, C-4),
48.7 (CHCl, C-3), 100.0 (CH, C-7), 113.7 (CH, C-13),
115.3 (CH, C-9), 117.0 (C, C-15), 117.1 (C, C-14), 122.4
(CF3, C-16), 126.2 (CH, C-8), 135.1 (C, C-6), 154.4 (C, C-
10), 158.0 (CH, C-12), 171.0 (C=O, C-2), 171.1 (C=O, C-
5).
4-(3-Chloro-2,5-dioxopyrrolidin-1-yl)benzoic acid (27)
Compound 27 obtained as white color solid and it recrys-
tallized by n-hexane-methylene chloride solvent mixture.
Melting point: 195 °C. IR (cm−1, KBr): 3030, 1706, 1515,
1
1429, 1395, 1282, 1174, 1020, 953, 859, 751. H-NMR
(400 MHz, CDCl3, ppm: 8.28–8.21 (m, 2H), 7.61–7.48 (m,
2H), 4.83 (dd, J = 8.8, 4.0, Hz, 1H), 3.54 (dd, J = 18.9, 8.8
Hz, 1H), 3.15 (dt, J = 18.9, 4.0 Hz, 1H). 13C-NMR (100
MHz, CDCl3, ppm): 37.4 (CH2, C-4), 46.7 (CHCl, C-3),
124.1 (CH, C-7), 124.3 (CH, C-7′), 129.2 (C, C-9), 130.2
(CH, C-8, -8′), 165.5 (C=O, C-2), 169.2 (COOH, C-10),
169.2 (C=O, C-5).
The activities of the CAs were determined according to
the method of Verpoorte et al. (1967), as described

Med Chem Res
previously (Şentürk et al. 2008). Bovine serum albumin was
used as the standard protein (Çoban et al. 2008). To
determine the inhibitor constant (Ki) of each bromophenol
derivative, three concentrations of each 3-chloro-1-
substituted aryl pyrrolidine-2,5-dione were tested, and the
activities (%) were plotted against the concentrations of the
inhibitors in Lineweaver–Burk plots (1934) as previously
described (Köksal and Gülçin 2008).
1-substituted aryl pyrrolidine-2,5-diones, 23, which has two
carbonyl groups (–CO) and one chlorine group (–Cl), was
the best hCA I inhibitor (Ki 23.27 0.68 nmol/L). It is well
known that compounds containing carbonyl (–CO) and
chlorine (–Cl) groups are effective CAIs.
Cytosolic hCA II is involved in many different diseases,
including edema, epilepsy, altitude sickness and glaucoma.
In this study, all of the 3-chloro-1-substituted aryl pyrroli-
dine-2,5-diones (20–29) inhibited hCA II activity with a Ki
value in the range of 10.64 4.01 to 31.86 9.20 nmol/L
(Table 2). The 3-chloro-1-substituted aryl pyrrolidine-2,5-
dione 22, which has two carbonyl groups (–CO) and a
chlorine group (–Cl), was the best hCA II inhibitor (Ki =
10.64 4.01 nmol/L). Compared to AZA, which interacts
with the distinct hydrophobic and hydrophilic regions of the
hCA II active site with a Ki of 31.93 14.77 nmol/L, all of
the 3-chloro-1-substituted aryl pyrrolidine-2,5-diones (20–
29) exhibied greater inhibition of hCA II.
It is interesting that the compounds prepared in this study
are structurally similar to 1,3-dioxooctahydro-4,7-metha-
noisoindol-2-yl)-N-(quinolin-8-yl)benzamide, which was
recently reported as an inhibitor of the oncogenic canonical
Wnt response (Lanier et al. 2012).
The compounds prepared in this study contained carbo-
nyl groups as well as nitrogen and oxygen atoms, which all
have important roles in biological systems. Additionally,
these compounds are soluble in polar solvents such as
ethanol and methanol. Therefore, these compounds will be
easy to prepare and use in clinical applications.
Results and discussion
In this study, 3-chloro-1-aryl pyrrolidine-2,5-diones were
prepared from maleic anhydride and aromatic amines. The
structures of these 3-chloro-1-substituted aryl pyrrolidine-
2,5-diones (20–29) are given in Table 1. These compounds
were evaluated for their inhibition properties against the
physiologically relevant isoenzymes hCA I, and II. The
results for the inhibition tests with hCA I, and II are shown
in Table 2. Additionally, it was reported that if the Ki value
of a 3-chloro-1-substituted aryl pyrrolidine-2,5-dione (20–
29) was less than 10 nmol/L (Ki < 40 nmol/L). The 3-
chloro-1-substituted aryl pyrrolidine-2,5-diones (20–29)
prepared in this study effectively inhibited hCA I, and II
with Ki in the low nanomolar range (<40 nmol/L).
The hCA I isoenzyme is found in many tissues and
occurs in high concentrations in the blood and gastro-
intestinal tract. It is involved in retinal and cerebral edema,
and the inhibition of hCA I may be a valuable tool for
fighting these conditions. For hCA I, the Ki values were
between 23.27 0.68 and 36.83 7.18 nmol/L. In com-
parison, the Ki for the broad-spectrum CA inhibitor AZA
was 34.70 4.09 nmol/L against hCA I. The only 3-chloro-
1-substituted aryl pyrrolidine-2,5-dione with an inhibition
effect lower than that of AZA was 27. Among the 3-chloro-
Conclusion
In this work, 3-chloro-aryl maleimide derivatives 20–29
were prepared from the corresponding (Z)-4-oxo-4-
Table 2 The inhibition effects
Compounds
C50 (nM)
hCA I
KI (nM)
of a series of 3-chloro-1-
substituted aryl pyrrolidine-2,5-
dione derivatives (20–29)
against both human carbonic
anhydrase isoenzymes (hCA I,
and II)
r2
hCA II
r2
hCA I
hCA II
20
21
22
23
24
25
26
27
28
29
AZAa
a
35.22
33.56
31.89
25.70
34.31
34.98
33.62
36.83
35.46
41.51
40.46
0.9982
0.9971
0.9992
0.9906
0.9913
0.9932
0.9944
0.9941
0.9928
0.9979
0.9996
26.56
26.02
24.30
21.02
32.35
30.43
30.56
25.63
25.33
28.87
24.16
0.9749
0.9829
0.9951
0.9903
0.9971
0.9955
0.9967
0.9980
0.9970
0.9901
0.9833
30.63 4.95
31.85 3.97
31.50 6.77
23.27 0.68
31.29 3.19
34.06 5.08
29.48 3.41
36.83 7.18
33.19 7.41
34.63 5.86
34.70 4.09
25.19 5.01
20.07 2.25
10.64 4.01
24.41 9.39
31.86 9.20
26.87 4.88
33.97 6.04
19.69 3.09
17.87 3.70
23.34 1.52
31.93 14.77
Acetazolamide (AZA) was used as a standard inhibitor for both CAs

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Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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