Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors: synthesis, molecular docking and anticonvulsant studies

Article abstract of DOI:10.3109/14756366.2015.1063624

Among many others, coumarin derivatives are known to show human carbonic anhydrase (hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-seizure properties. There are also several studies reporting compounds with an imidazole and/or benzimidazole moiety which exert these pharmacological properties. In this study, we prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and evaluated their hCA inhibitory activities and along with fourteen previously synthesized derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA isoforms I and II, some of them also proved protective against Maximal electroshock seizure (MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin derivatives have revealed that these compounds bind to the active pocket of the enzyme in a similar fashion to that previously described for coumarin derivatives.

Full text of DOI:10.3109/14756366.2015.1063624

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J Enzyme Inhib Med Chem, Early Online: 1–13
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1063624
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RESEARCH ARTICLE
Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors:
synthesis, molecular docking and anticonvulsant studies
Mert Olgun Karatas¸1, Harun Uslu2, Suat Sarı3, Mehmet Abdullah Alago¨z2, Arzu Karakurt2, Bu¨lent Alıcı1,
Cigdem Bilen4, Emre Yavuz4, Nahit Gencer4, and Oktay Arslan4
2
1Department of Chemistry, Faculty of Arts and Science and Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Inonu University,
3
4
Malatya, Turkey, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey, and Department of
Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir, Turkey
Abstract
Keywords
Among many others, coumarin derivatives are known to show human carbonic anhydrase
(hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account
for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-
seizure properties. There are also several studies reporting compounds with an imidazole
and/or benzimidazole moiety which exert these pharmacological properties. In this study, we
prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine
novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and
evaluated their hCA inhibitory activities and along with fourteen previously synthesized
derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA
isoforms I and II, some of them also proved protective against Maximal electroshock seizure
(MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin
derivatives have revealed that these compounds bind to the active pocket of the enzyme in a
similar fashion to that previously described for coumarin derivatives.
Anticonvulsant, benzoxazinone, carbonic
anhydrase, coumarin, epilepsy, inhibition
History
Received 24 February 2015
Revised 12 June 2015
Accepted 13 June 2015
Published online 24 July 2015
Introduction
molecule, the former polarizing the oxygen of the water molecule
to make it a stronger nucleophile for CO2. CAs fall into four major
classes, namely, cytosolic, mitochondrial, secreted and mem-
brane-bound CAs, which in turn involve several isoforms. Among
these, cytosolic and membrane-bound CAs are more widely
distributed and more important as drug targets than other classes,
especially isoform II is almost ubiquitous25.
Epilepsy is a common neurological disorder that manifests itself
in spontaneous, recurring attacks. Affecting more than 50 million
people worldwide, epilepsy usually continues for a lifetime and
requires permanent AED treatment, which is mainly symptomatic
for the exact reason of this brain pathology remains unclear.
Demand for new AEDs is high since the currently available ones
fail to control seizures for approximately 30% of the patients and
The CA inhibitors (CAIs) can be divided into four main
classes26: (i) sulfonamides (and their isosteres, such as sulfamates,
sulfamides and similar derivatives), (ii) phenols, (iii) polyamines,
such as spermine, spermidine and (iv) coumarins and thiocou-
marins. The primary sulfonamides (RSO2NH2) are classical CAIs.
They are in clinical use for more than 50 years as antiglaucoma
drugs. Furthermore, it has recently been reported that they have
potential anticonvulsant properties. Sulthiame, topiramate, zoni-
samide27–29 and lacosamide30 are clinically used antiepileptics
also showing potent inhibition of many hCA isozymes present in
the brain. Most CAs inhibitors exert a Zn (II) dependent inhibition
in that they either substitute Zn(II) or add to the coordination
network. Unlike the other three groups, coumarins and thiocou-
marins have an inhibition mechanism not dependent to Zn(II).
The natural product, coumarin 6-(1S-hydroxy-3-methylbutyl)-7-
methoxy-2H-chromen-2-one was shown to be hydrolyzed within
the CA active site forming 2-hydroxy-cinnamic acid derivative,
which possesses significant CA inhibitory properties. The latter,
in turn, bound to the enzyme’s active site in a completely
unprecedented manner, not interacting with the Zn(II) ion but
blocking the active site for substrate entrance31,32. Most recently,
several serious side effects are reported1–9
.
Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the physio-
logical hydration of CO2 to yield bicarbonate and a proton.
This reaction is involved in many physiological and pathological
processes, including respiration and transport of CO2 and
bicarbonate between metabolizing tissues and lungs; pH and
CO2 homeostasis; electrolyte secretion in various tissues
and organs; biosynthetic reactions such as gluconeogenesis,
lipogenesis and ureagenesis; bone resorption; calcification; and
tumorigenicity10–18. Many CA isoenzymes involved in these
processes are important therapeutic targets to treat a range of
disorders including edema, glaucoma, obesity, cancer, epilepsy
and osteoporosis19–24. The active site of most CAs contains a zinc
ion, hence the name metalloenzyme. Zn(II)forms three coordin-
ation bonds with histidine side chains and one with a water
Address for correspondence: Dr. Nahit Genc¸er, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University,
06100 Ankara, Turkey. E-mail: ngencer@balikesir.edu.tr

2
M. O. Karatas¸ et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
it was reported that some carboxylic acid derivatives inhibited derivatives, we performed molecular docking studies to obtain
hCA I, II, IX, XII with different inhibition mechanisms and this insights into binding mode of these ligands with key residues of
report makes carboxylic acids an interesting class of compound as the active site of hCA II.
inhibitors of metalloenzymes such as CAs33.
Some of the CA inhibitors are clinically used to treat epilepsy. Experimental
CA inhibition is a suggested mechanism among a few others for
All reactions for the preparation of imidazolium and benzimida-
seizure control. Therefore, the elevation of CO2 levels in the brain
zolium salts were carried out in standard Schlenk type
may play a crucial role in anti-seizure activity34. hCA isoforms II,
flasks. Chemicals were purchased from Sigma Aldrich
VII and XIV are expressed in the choroid plexus, glial cells and
(Istanbul, Turkey), Merck (Istanbul, Turkey), Alfa Aesar
oligodendrocytes and thought to play a role in signaling
(Istanbul, Turkey). 6-(Chloroacetyl)-2H-1,4-benzoxazine-3(4H)-
processes35,36. Targeting only carbonic anhydrase inhibition for
one, 1-methylimidazole, 1-butylimidazole and 1,2-dimethylimi-
the design of new anticonvulsant agents will not completely
1
dazole were supplied commercially, controlled by H-NMR and
resolve seizures because several other molecular factors are
used without further purification. Melting points were determined
involved in epilepsy etiology7.
by Electrothermal-9200 melting point apparatus. FT-IR spectra
Coumarins are a member of a class of compounds called
were recorded on ATR unit in the range of 400–4000 cmꢀ1 with
benzopyrones. Coumarin derivatives show various biological and
Perkin Elmer Spectrum 100 Spectrophotometer (Istanbul,
pharmacological activities37,38. In addition to numerous activities
Turkey). H-NMR and 13C-NMR spectra were recorded using a
of coumarin derivatives, they were reported to inhibit carbonic
Bruker AC300P FT spectrometer (Istanbul, Turkey) operating at
anhydrase activity31,32 and control seizures induced with ScMet39.
300.13 MHz (1H), 75,47 MHz (13C). Chemical shifts are given in
Benzimidazole consists of the fusion of benzene and imidazole.
ppm relative to TMS, coupling constants (J) in Hz. Elemental
Benzimidazole bearing bioactive compounds was reported as
analyses were performed by IBTAM (Inonu University Scientific
having antihypertensive, anti-inflammatory, antimicrobial, anti-
and Technological Research Central).
1
viral, antioxidant, antitumor, lipid modulator and anticoagulant
properties40. 1,4-Oxazinone derivatives have been used exten-
Synthesis and characterization of the compounds
sively for building bioactive compounds. Anticancer41, antiul-
cer42, antihypertensive43, anti-inflammatory44 and other Synthesis of 1-alkylbenzimidazoles and 1,10-benzimidazoles
biological activities of 1,4-oxazinones and benzoxazines were
1-Alkylbenzimidazole derivatives were synthesized by the pro-
reported45–48. Beside various biological activities of 1,4-oxazi-
cedure described by Ozdemir et al.49.
none and benzoxazine derivatives, anticonvulsant activities of
benzoxazinones were reported in the literature48.
Synthesis of 4-chloromethyl-6,8-dimethylcoumarin and 6,8-
In this article, with the prospect of potent CA inhibitory and
dimethylcoumarin bearing imidazolium and benzimidazolium
anticonvulsant activities, we prepared fifteen novel coumarin and
salts (1a–i)
nine novel benzoxazinone-bearing imidazolium and benzimida-
zolium chloride derivatives (Schemes 1–4) and evaluated their 4-Chloromethyl-6,8-dimethylcoumarin (1) was synthesized
inhibitory activities against purified hCA I and II isoforms. according to the procedure described by Frasinyuk50
Together with fourteen derivatives available from our previous (Scheme 1). Later, imidazolium and benzimidazolium salts
studies (Table 1), we investigated their inhibitor activities against (1a–i) were synthesized. About 10 mmol 1-alkylbenzimidazole
seizures induced in mice by maximal electroshock (MES) and or 1-alkylimidazole was dissolved in 5 mL DMF and 10 mmol
subcutaneous metrazole (ScMet). For selected coumarin compound 1 was added to this solution and the resulting mixture
OH
O
Compound
R
R
2
1
O
O
Cl
-CH
-H
1a
1b
1c
3
3
R
1
-CH
-CH
+
3
N
R
-(CH ) CH
2 3
2
-H
3
O
O
N
−
Cl
+
N
H SO , 24h.
Compound
R
3
2
4
-CH
3
N
1d
1e
1f
R
2
R
-CH -CH=CH
2
1
3
O
O
1a-c
DMF, 80°C, 24h.
-(CH ) CH
2 3
3
O
O
R
3
CH
1g
2
N
O
O
Cl
1
N
−
Cl
1h
O
CH
CH
2
R
3
N
N
2
1i
1d-i
Scheme 1. Synthesis of 6,8-dimethyl coumarin bearing imidazolium and benzimidazolium salts (1a–i).

DOI: 10.3109/14756366.2015.1063624
Coumarin or benzoxazinone-based inhibitors
3
OH
O
Cl
+
HO
O
O
Compound
2g
R
HO
O
O
HClO , 3h.
4
R
CH
2
2
HO
O
O
N
−
DMF, 90°C, 48h.
Cl
+
R
N
N
N
CH
2h
Cl
2
2g,h
Scheme 2. Synthesis of 7-hydroxy coumarin bearing benzimidazolium salts (2g, h).
OH
Compound
OH
O
O
R
R
2
1
HO
O
O
-CH
-CH
-H
HO
HO
Cl
3a
3b
3c
3
R
1
-CH
3
3
+
N
R
-(CH ) CH
2 3
-H
2
3
O
−
Cl
+
N
N
HClO , 3h.
4
N
R
OH
2
R
1
3a-c
HO
O
O
HO
DMF, 90°C, 48h.
HO
O
O
N
Cl
3
−
Cl
N
N
N
3j
Scheme 3. Synthesis of 7,8-dihydroxy coumarin bearing imidazolium and benzimidazolium salts (3a–c, j).
was heated for 24 h at 80 ꢁC. Then, the mixture was cooled to 1-Methyl-2-methyl-3-(4-methylene-6,8-dimethyl-2H-chromen-2-
ambient temperature and 20 mL diethyl ether was added and the one)imidazolium chloride (1b). White solid, yield; 92%, m.p.:
resulting precipitate was collected by filtration. The crude product 288–289 ꢁC; Anal Calcd for C17H19ClO2N2; C: 64.05, H: 6.01, N:
was washed with hexane (2 ꢂ 10 mL) and diethyl ether (10 mL) 8.79, found: C: 64.23, H: 6.23, N: 8.62; IR (cmꢀ1): 1714 (–C¼O);
then dried under reduced pressure.
1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 7.45–7.82 (m, 4H, Ar-H,
NCH ¼ CHN), 5.89 (s, 2H, coumarin–CH2–N–), 5.78 (s, 1H,
–C¼C–H), 3.84 (s, 3H, –N–CH3), 2.60 (s, 3H, –NC(CH3)N–),
2.40 (s, 3H, Ar-CH3), 2.37 (s, 3H, Ar-CH3); 13C-NMR (DMSO-
d6, 75 MHz) ꢀ/ppm:160.0, 150.1, 149.8, 146.4, 135.1, 133.7,
125.7, 123.7, 122.5, 122.1, 116.8, 111.7, 48.0, 35.6, 20.9, 15.6,
9.8.
1-Methyl-3-(4-methylene-6,8-dimethyl-2H-chromen-2-one)imida-
zolium chloride (1a). White solid, yield; 89%, m.p.: 271–
273 ꢁC; Anal Calcd for C16H17ClO2N2; C: 63.06, H: 5.62, N:
9.19, found: C: 62.88, H: 5.88, N: 9.30; IR (cmꢀ1): 1707 (–C¼O),
1
1604 (–C¼C–); H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 9.41(s,
1H, –NCHN–), 7.42–7.91(4H, Ar-H, –NCH ¼ CHN–), 6.06 (s,
1H, –C¼C–H), 5.86 (s, 2H, coumarin–CH2–N–), 3.91 (s, 3H,
–N–CH3), 2.35 (s, 3H, Ar-CH3), 2.34 (s, 3H, Ar-CH3); 13C-NMR
(DMSO-d6, 75 MHz) ꢀ/ppm: 160.0, 150.0, 149.9, 138.7, 135.2,
134.0, 125.9, 124.7, 123.4, 122.3, 116.8, 113.7, 48.8, 36.6, 20.8,
15.6.
1-Butyl-3-(4-methylene-6,8-dimethyl-2H-chromen-2-one)imidazo-
lium chloride (1c). White solid, yield; 87%, m.p.: 262–264 ꢁC;
Anal Calcd for C19H23ClO2N2; C: 65.79, H: 6.68, N: 8.08, found:
C: 65.65, H: 6.99, N: 7.98; IR (cmꢀ1): 3114 (–C¼C–H), 1716
(–C¼O); 1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 9.60 (s, 1H,

4
M. O. Karatas¸ et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
R
R'
Compound
4a
4b
4c
-CH
-CH
-H
-CH
-H
3
R
3
3
O
H
N
-(CH ) CH
2 3
3
R
N
R'
N
N
Cl
O
R'
R''
-CH
Compound
4d
N
3
O
O
H
N
4a-c
4e
4f
-CH CH=CH
2
2
Cl
O
DMF,80°C,8h.
-(CH ) CH
2 3
3
O
R''
R''
O
H
4g
4h
4i
CH
4
N
N
2
N
Cl
O
O
N
N
O
CH
O
2
O
4d-i
CH
2
Scheme 4. Synthesis of benzoxazinone bearing imidazolium and benzimidazolium salts (4a–i).
NCHN), 7.96 (s, 2H, NCH¼CHN), 7.53 (s, 1H, Ar-H), 7.41 1-Butyl-3-(4-methylene-6,8-dimethyl-2H-chromen-2-one)benzimi-
(s, 1H, Ar-H) 6.13 (s, 1H, C¼C–H), 5.86 (s, 2H, coumarin–CH2– dazolium chloride (1f). White solid, yield; 85%, m.p.: 253–
N–), 4.24 (t, 2H, J ¼ 7.1 Hz, NCH2CH2CH2CH3), 2.36 (s, 3H, Ar- 255 ꢁC; Anal Calcd for C23H25ClO2N2; C: 69.60, H: 6.35, N:
CH3), 2.35 (s, 3H, Ar-CH3), 1.82 (five, 2H, J ¼ 7.2 Hz, NCH2CH2 7.06, found: C: 69.41, H: 6.59, N: 7.18; IR (cmꢀ1): 1722 (–C¼O);
CH2CH3), 1.26 (six, 2H, J ¼ 7.5 Hz, NCH2CH2CH2CH3), 0.91 1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 9.95 (s, 1H, NCHN),
(t, 3H, J ¼ 7.4 Hz, NCH2CH2CH2CH3) 13C-NMR (DMSO-d6, 7.16–8.19 (m, 6H, Ar-H), 6.10 (s, 2H, coumarin–CH2–N–), 5.89
75 MHz) ꢀ/ppm: 159.9, 150.0, 149.5, 137.7, 135.2, 133.9, (s, 1H, –C¼C–H), 4.84 (t, 2H, J ¼ 7.3 Hz, –NCH2CH2CH2CH3),
125.9, 123.6, 123.5, 122.3, 116.8, 114.5, 49.4, 48.9, 31.6, 19.3, 2.36 (s, 3H, Ar-CH3), 2.33 (s, 3H, Ar-CH3), 1.89 (five, 2H,
15.6, 13.4.
J ¼ 7.3 Hz, NCH2CH2CH2CH3), 1.28 (six, 2H, J ¼ 7.3 Hz,
NCH2CH2CH2CH3), 0.88 (t, 3H,
J ¼ 7.3 Hz,
NCH2CH2CH2CH3); 13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm:
160.1, 149.9, 149.1, 143.5, 135.3, 134.1, 131.8, 131.6, 127.6,
127.5, 126.0, 122.3, 116.7, 114.5, 114.1, 113.0, 47.4, 47.0, 30.7,
20.8, 19.5, 15.5, 13.8.
1-Methyl-3-(4-methylene-6,8-dimethyl-2H-chromen-2-one)benzi-
midazolium chloride (1d). White solid, yield; 88%, m.p.:
164–166 ꢁC; Anal Calcd for C20H19ClO2N2; C: 67.70, H: 5.40,
N: 7.89, found: C: 67.55, H: 5.68, N: 7.80; IR (cmꢀ1): 1714
(–C¼O); 1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 10.0 (s, 1H,
NCHN), 7.46–8.14 (m, 6H, Ar-H), 6.23 (s, 2H, coum–CH2–N–), 1-Benzyl-3-(4-methylene-6,8-dimethyl-2H-chromen-2-one)benzi-
5.94 (s, 1H, –C¼C–H), 4.16 (s, 3H, N–CH3), 2.42 (s, 3H, Ar- midazolium chloride (1 g). White solid, yield; 77%, m.p.: 219–
CH3), 2.38 (s, 3H, Ar-CH3); 13C-NMR (DMSO-d6, 75 MHz) 221 ꢁC; Anal Calcd for C26H23ClO2N2; C: 72.47, H: 5.38, N:
ꢀ/ppm: 160.0, 149.9, 149.5, 144.3, 135.2, 133.8, 132.6, 132.1, 6.50, found: C: 72.34, H: 5.58, N: 6.40; IR (cmꢀ1): 1721 (–C¼O);
131.5, 127.4, 127.0, 125.8, 122.6, 116.8, 114.4, 114.0, 112.6, 1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 10.24 (s, 1H, NCHN),
46.8, 34.1, 20.9, 15.6.
7.36–8.13 (m, 11H, Ar-H), 6.23 (s, 2H, –CH2–N–), 6.06 (s, 1H,
–C¼C–H), 5.85 (s, 2H, –PhCH2–N–), 2.40 (s, 3H, Ar-CH3), 2.38
(s, 3H, Ar-CH3); 13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 159.9,
150.0, 149.1, 144.1, 135.2, 134.2, 133.8, 131.9, 131.6, 129.5,
129.4, 129.3, 127.6, 127.5, 125.1, 122.5, 116.9, 114.7, 114.5,
113.3, 50.7, 47.2, 20.9, 15.6.
1-Allyl-3-(4-methylene-6,8-dimethyl-2H-chromen-2-one)benzimi-
dazolium chloride (1e). White solid, yield; 78%, m.p.: 243–
246 ꢁC; Anal Calcd for C22H21ClO2N2; C: 69.38, H: 5.56, N:
7.34, found: C: 69.22, H: 5.79, N: 7.29; IR (cmꢀ1): 1706 (–C¼O);
1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 10.06 (s, 1H, –NCHN–),
7.47–8.14 (m, 6H, Ar-H), 6.17 (s, 2H, coumarin–CH2–N–), 6.16 1-(3,4,5-Trimethoxybenzyl)-3-(4-methylene-6,8-dimethyl-2H-
(ddt, 1H, –CH2–CH ¼ CH0H00, JH–CH2 ¼ 5.9 Hz, JH–H ¼ 10.3 Hz, chromen-2-one)benzimidazolium chloride (1h). White solid,
0
JH–H ¼ 16.3 Hz), 5.99 (s, 1H, –C¼C–H), 5.55–5.44 (dd, 2H, yield; 90%, m.p.: 158–160 ꢁC; Anal Calcd for C29H29ClO5N2;
00
–CH2–CH¼CH0H00, JH –H ¼ 1.1 Hz, JH–H ¼ 15.99 Hz), 5.25 (d, C: 66.86, H: 5.61, N: 5.38, found: C: 66.67, H: 5.79, N: 5.28; IR
2H, –CH2–CH¼CH0H00, JCH2–H ¼ 5.9 Hz), 2.42 (s, 3H, Ar-CH3), (cmꢀ1): 1719 (–C¼O), 1650 (–C¼C–); 1H-NMR (DMSO-d6,
2.38 (s, 3H, Ar-CH3); 13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 300 MHz) ꢀ/ppm; 10.13 (s, 1H, NCHN), 7.01–8.23 (m, 8H, Ar-
159.9, 149.9, 149.3, 144.1, 135.2, 133.8, 131.8, 131.4, 127.5, H), 6.21 (s, 2H, coumarin–CH2–N–), 5.97 (s, 1H, –C¼C–H), 5.70
127.3, 127.2, 125.9, 122.5, 121.7, 116.9, 114.7, 114.3, 112.9, (s, 2H, –N–CH2Ph), 3.78 (s, 6H, Ar-OCH3), 3.74 (s, 3H, Ar-
0
00
00
49.7, 47.1, 20.9, 15.6.
OCH3), 2.39 (s, 3H, Ar-CH3), 2.37 (s, 3H, Ar-CH3); 13C-NMR

DOI: 10.3109/14756366.2015.1063624
Coumarin or benzoxazinone-based inhibitors
5
Table 1. Structures of 2a–j and 3d–i (these compounds were available from previous studies).
R
2
HO
O
O
HO
O
O
−
Cl
−
Cl
R
3
N
N
+
N
N
R
1
2c-f, 3d-i
2a,b
HO
O
O
O
O
OH
−
−
Cl
Cl
(CH )n
2
N
N
N
N
2i,j
Compounds
R1
–CH3
R2
R3
n
2a
2b
2c
2d
2e
2f
–
–
–H
–H
–H
–H
–
–
–
–
–
–
–
–
–
4
5
–
–
–
–
–
–
–(CH2)3CH3
–
–
–
–
–
–
–
–
–
–
–
–
–CH3
– (CH2)3CH3
Benzyl
3,4,5-Trimethoxybenzyl
2i
2j
–
–
–
3d
3e
3f
3g
3h
3i
–OH
–OH
–OH
–OH
–OH
–OH
–CH3
–CH3–CH ¼ CH2
–(CH2)3CH3
Benzyl
3-Methylbenzyl
3,4,5-Trimethoxybenzyl
(DMSO-d6, 75 MHz) ꢀ/ppm: 163.8, 159.9, 153.7, 149.9, 133.8, 133.3, 133.2, 132.0, 131.8, 131.6, 129.3, 128.4, 128.2,
149.4, 143.8, 138.1, 135.2, 133.8, 131.9, 131.7, 129.3, 127.6, 127.6, 127.5, 127.3, 127.2, 126.3, 125.9, 122.5, 116.9, 114.7,
127.4, 122.5, 116.8, 114.7, 112.9, 107.1, 60.5, 56.5, 36.3, 114.5, 114.4, 113.2, 51.0, 47.2, 20.9, 15.6.
31.2,20.9, 15.6.
Synthesis of 4-chloromethyl-7-hydroxycoumarin and 7-hydroxy-
coumarin bearing imidazolium and benzimidazolium salts (2g,
2h)
1-(Naphthalen-2-ylmethyl)-3-(4-methylene-6,8-dimethyl-2H-chro-
men-2-one)benzimidazolium chloride (1i). White solid, yield;
85%, m.p.: 283–286 ꢁC; Anal Calcd for C30H25ClO2N2; C: 74.91, 4-Chloromethyl-7-hydroxycoumarin (2) and compounds 2a–2f, 2i
H: 5.24, N: 5.82, found: C: 74.75, H: 5.49, N: 5.78; IR (cmꢀ1): and 2j were previously synthesized by our group51 (Scheme 2).
1717 (–C¼O); 1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 10.12 Following the synthesis of compound 2, benzimidazolium salts
(s, 1H, NCHN), 7.46–8.18 (m, 13H, Ar-H), 6.23 (s, 2H, (2g, 2h) were synthesized. About 10 mmol 1-alkylbenzimidazole
coumarin–CH2–N–), 6.07 (s, 1H, –C¼C–H), 6.01 (s, 2H, –N– was dissolved in 5 mL DMF and 10 mmol compound 2 was added
CH2Ph), 2.39 (s, 3H, Ar-CH3), 2.38 (s, 3H, Ar-CH3); 13C-NMR to this solution and the resulting mixture was heated for 48 h at
(DMSO-d6, 75 MHz) ꢀ/ppm: 159.9, 150.0, 149.2, 144.2, 135.2, 90 ꢁC. Later, the mixture was cooled to room temperature.

6
M. O. Karatas¸ et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
A 20 mL diethyl ether was added and the resulting precipitates J ¼ 1.9 Hz, NCH ¼ CHN), 7.14 (d, 1H, J ¼ 8.2 Hz, Ar-H), 6.91
were collected by filtration. The crude product was washed with (d, 1H, J ¼ 8.2 Hz, Ar-H), 5.69 (s, 2H, coumarin–CH2–N–), 5.51
hexane (2 ꢂ 10 mL) and diethyl ether (10 mL) then dried under (s, 1H, –C¼C–H), 2.88 (s, 3H, –N–CH3), 2.72 (s, 3H,
reduced pressure.
–NC(CH3)N–); 13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 163.1,
160.5, 150.4, 150.3, 146.2, 143.6, 123.5, 122.1, 115.2, 112.9,
110.3, 107.8, 47.9, 36.4, 9.7.
1-(2,3,4,5,6-Pentamethylbenzyl)-3-(4-methylene-7-hydroxy-2H-
chromen-2-one)benzimidazolium chloride (2g). White solid,
yield; 52%, m.p.: 203–206 ꢁC; Anal Calcd for C29H29ClO3N2; 1-Butyl-3-(4-methylene-7,8-dihydroxy-2H-chromen-2-one)imida-
C: 71.23, H: 5.98, N: 5.73, found: C: 71.37, H: 6.30, N: 5.85; IR zolium chloride (3c). Yellow solid, yield; 43%, m.p.: 234–
(cmꢀ1): 3260 (Ar-OH), 1710 (–C¼O), 1610 (–C¼C–); H-NMR 236 ꢁC; Anal Calcd for C17H19ClO4N2; C: 58.21, H: 5.46, N:
1
(DMSO-d6, 300 MHz) ꢀ/ppm; 11.02 (s, 1H, Ar-OH), 9.23 (s, 1H, 7.99, found: C: 58.05, H: 5.79, N: 7.78; IR (cmꢀ1): 3280, 1715
–NCHN–), 6.87–8.15 (m, 7H, Ar-H), 6.05 (s, 2H, coumarin–CH2– (–C¼O), 1616 (–C¼C–); 1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm;
N–), 5.78 (s, 2H, –N–CH2Ph), 5.58 (s, 1H, –C¼C–H), 2.25 (s, 3H, 9.46 (s, 1H, –NCHN–), 7.93 (s, 2H, NCH ¼ CHN), 7.16 (d, 1H,
Ar-CH3), 2.23 (s, 6H, Ar-CH3), 2.22 (s, 6H, Ar-CH3); 13C-NMR J ¼ 8.2, Ar-H), 6.94 (d, 1H, J ¼ 8.2 Hz, Ar-H), 5.81 (s, 2H,
(DMSO-d6, 75 MHz) ꢀ/ppm: 162.8, 162.4, 160.4, 155.3, 150.1, coumarin–CH2–N–), 5.76 (s, 1H, –C¼C–H), 4.07 (t, 2H,
142.7, 134.3, 133.5, 132.3, 132.0, 127.7, 127.4, 126.2, 126.1, J ¼ 7.2 Hz, NCH2CH2CH2CH3), 1.81 (five, 2H, J ¼ 7.2 Hz,
114.7, 114.4, 113.8, 109.5, 107.8, 103.0, 47.0, 17.5, 17.2, 16.9.
NCH2CH2CH2CH3), 1.26 (six, 2H, J ¼ 7.2 Hz, NCH2CH2
CH2CH3), 0.91 (t, 3H, J ¼ 7.2 Hz, NCH2CH2CH2CH3); 13C-
NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 160.4, 150.8, 150.1, 143.8,
137.7, 133.2, 123.7, 123.4, 115.0, 113.2, 110.4, 109.8, 49.4, 49.0,
31.6, 19.3, 13.8.
1-(Naphthalen-2-ylmethyl)-3-(4-methylene-7-hydroxy-2H-chro-
men-2-one)benzimidazolium chloride (2h). White solid, yield;
60%, m.p.: 270–274 ꢁC; Anal Calcd for C28H21ClO3N2; C: 71.72,
H: 4.51, N: 5.97, found: C: 70.89, H: 4.55, N: 5.65; IR (cmꢀ1):
1
3320 (Ar-OH), 1713 (–C¼O), 1615(–C¼C–); H-NMR (DMSO- 1-(Naphthalen-2ylmethyl)-3-(4-methylene-7,8-dihydroxy-2H-
d6, 300 MHz) ꢀ/ppm; 10.99 (s, 1H, Ar-OH), 10.07 (s, 1H, NHCN), chromen-2-one)benzimidazolium
chloride
(3j). White
6.96–8.14 (m, 14H, Ar-H), 6.16 (s, 2H, –CH2–N–), 5.99 (s, 2H, solid, yield; 60%, m.p.: 270–274 ꢁC; Anal Calcd for
–CH2–N–), 5.80 (s, 1H, –C¼C–H); 13C-NMR (DMSO-d6, C28H21ClO4N2; C: 69.35, H: 4.37, N: 5.78, found: C: 69.19, H:
75 MHz) ꢀ/ppm: 162.4, 160.3, 150.6, 149.6, 144.2, 143.9, 133.2, 4.55, N: 5.65; IR (cmꢀ1): 3320 (Ar-OH), 1713 (–C¼O), 1615
133.2, 133.1, 132.0, 131.7, 131.6, 129.3, 128.4, 128.2, 127.6, (–C¼C–); 1H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 10.07
127.4, 127.3, 126.3, 115.2, 114.6, 114.4, 113.1, 110.3, 109.1, (s, 1H, NHCN), 6.96–8.14 (m, 13H, Ar-H), 6.13 (s, 2H, –CH2–
56.5, 50.9.
N–), 5.99 (s, 2H, –CH2–N–), 5.82 (s, 1H, –C¼C–H); 13C-NMR
(DMSO-d6, 75 MHz) ꢀ/ppm: 160.3, 150.6, 149.6, 144.2, 143.9,
133.2, 133.2, 133.1, 132.0, 131.7, 131.6, 129.3, 128.4, 128.2,
127.6, 127.4, 127.3, 126.3, 115.2, 114.6, 114.4, 113.1, 110.3,
109.1, 56.5, 50.9.
Synthesis of 4-chloromethyl-7,8-dihydroxycoumarin and 7,8-
dihydroxycoumarin bearing imidazolium and benzimidazolium
salts (3a–c, 3j)
Compounds 3d–i were available from our previous study52
(Scheme 3). 4-chloromethyl-7,8-dihydroxycoumarin (3) was
synthesized according to procedure described by Gumus44.
Synthesis of benzoxazinone bearing imidazolium and benzimida-
zolium salts (4a–i)
Subsequently, imidazolium and benzimidazolium salts (3a–c, About 10 mmol 1-alkylbenzimidazole or 1-alkylimidazole was
3j) were synthesized. About 10 mmol 1-alkylbenzimidazole or 1- dissolved in 5 mL DMF and 10 mmol 6-chloroacetyl-2H-1,4-
alkylimidazole was dissolved in 5 mL DMF and 10 mmol benzoxazine-3(4H)-one was added to this solution and the
compound 3 was added to this solution and the resulting mixture resulting mixture was heated for 8 h at 80 ꢁC (Scheme 4). Later,
was heated for 48 h at 90 ꢁC. Later, the mixture was cooled to the mixture was cooled to room temperature. A 20 mL diethyl
room temperature. A 20 mL diethyl ether was added and ether was added and precipitate was collected by filtration. The
precipitate was collected by filtration. Crude product was crude product was washed with hexane (2 ꢂ 10 mL) and diethyl
washed with hexane (2 ꢂ 10 mL) and diethyl ether (10 mL) then ether (10 mL) then dried under reduced pressure.
dried under reduced pressure.
1-Methyl-3-(6-acetyl-2H-1,4-benzoxazine-3(4H)-one)imidazolium
chloride (4a). White solid, yield; 89%, m.p.: 291–294 ꢁC; Anal
dazolium chloride (3a). Yellow solid, yield; 47%, m.p.: 245– Calcd for C14H14ClO3N3; C: 54.64, H: 4.59, N: 13.65, found: C:
1-Methyl-3-(4-methylene-7,8-dihydroxy-2H-chromen-2-one)imi-
1
248 ꢁC; Anal Calcd for C14H13ClO4N2; C: 54.47, H: 4.24, N: 54.79, H: 4.73, N: 13.60; IR (cmꢀ1): 1672 (–C¼O); H-NMR
9.07, found: C: 54.32, H: 4.58, N: 8.93; IR (cmꢀ1): 3150 (Ar-OH), (DMSO-d6, 300 MHz) ꢀ/ppm; 11.16 (s, 1H, –N–H), 9.20 (s, 1H,
1
1724 (–C¼O), 1615 (–C¼C–); H-NMR (DMSO-d6, 300 MHz) –NCHN–), 7.14–7.80 (m, Ar-H, NCH ¼ CHN), 6.06 (s, 2H,
ꢀ/ppm; 9.32 (s, 1H, –NCHN–), 7.88 (s, 1H, NCH ¼ CHN), 7.82 benzoxazinone–CH2–N–), 4.74 (s, 2H, –C(O)–CH2–O–), 3.96 (s,
(s, 1H, NCH ¼ CHN), 7.18 (d, 1H, J ¼ 8.5 Hz, Ar-H), 6.95 (d, 1H, 3H, –N–CH3); 13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 190.2,
J ¼ 8.5 Hz, Ar-H), 5.81 (s, 1H, –C¼C–H), 5.76 (s, 2H, coumarin– 164.4, 148.6, 138.3, 128.4, 128.1, 124.9, 124.4, 123.7, 116.8,
CH2–N–), 3.90 (s, 3H, –N–CH3); 13C-NMR (DMSO-d6, 75 MHz) 115.7, 67.2, 55.4, 36.4.
ꢀ/ppm: 160.4, 150.6, 150.4, 143.8, 138.2, 133.1, 124.7, 123.5
115.1, 113.1, 109.5, 48.9, 36.6.
1-Methyl-2-methyl-3-(6-acetyl-2H-1,4-benzoxazine-3(4H)-one)i-
midazolium chloride (4b). White solid, yield; 91%, m.p.: 288–
291 ꢁC; Anal Calcd for C15H16ClO3N3; C: 55.99, H: 5.01, N:
1-Methyl-2-methyl-3-(4-methylene-7,8-dihydroxy-2H-chromen-2-
one)imidazolium chloride (3b). Yellow solid, yield; 48%, m.p.: 13.06, found: C: 56.16, H: 4.73, N: 13.19; IR (cmꢀ1): 1688
1
324–326 ꢁC; Anal Calcd for C15H15ClO4N2; C: 55.82, H: 4.68, N: (–C¼O); H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 11.17 (s, 1H,
8.68, found: C: 55.99, H: 4.86, N: 8.83; IR (cmꢀ1): 3205 (Ar-OH), –N–H), 7.15–7.75 (m, 5H, Ar-H, NCH ¼ CHN), 6.05 (s, 2H,
1
1731 (–C¼O), 1660 (–C¼C–); H-NMR (DMSO-d6, 300 MHz) benzoxazinone–CH2–N–), 4.73 (s, 2H, –C(O)–CH2–O–), 3.86 (s,
ꢀ/ppm; 7.69 (d, 1H, J ¼ 1.9 Hz, NCH ¼ CHN), 7.65 (d, 1H, 3H, N–CH3), 2.52 (s, 3H, –NC(CH3)N–); 13C-NMR (DMSO-d6,

DOI: 10.3109/14756366.2015.1063624
Coumarin or benzoxazinone-based inhibitors
7
75 MHz) ꢀ/ppm: 190.0, 164.4, 148.6, 146.3, 128.5, 128.1, 125.2, 1-(3,4,5-Trimethoxybenzyl)-3-(6-acetyl-2H-1,4-benzoxazine-
122.8, 116.7, 115.9, 67.2, 54.5, 35.4, 9.7.
3(4H)-one)benzimidazolium chloride (4h). White solid, yield;
91%, m.p.: 174–177 ꢁC; Anal Calcd for C27H26ClO6N3; C: 61.89,
H: 5.00, N: 8.02, found: C: 61.62, H: 4.78, N: 8.38; IR (cmꢀ1):
1706 (–C¼O), 1680 (–C¼O); 1H-NMR (DMSO-d6, 300 MHz)
ꢀ/ppm; 11.10 (s, 1H, –N–H), 9.89 (s, 2H, NCHN), 6.96–8.18 (m,
9H, Ar-H), 6.37 (s, 2H, benzoxazinone–CH2–N–), 5.78 (s, 2H,
–N–CH2Ph), 4.76 (s, 2H, –C(O)–CH2–O–), 3.79 (s, 6H, Ar-
OCH3), 3.65 (s, 3H, Ar-OCH3); 13C-NMR (DMSO-d6, 75 MHz)
ꢀ/ppm: 190.0, 164.5, 162.9, 153.7, 148.8, 144.0, 138.1, 132.6,
131.0, 129.5, 128.5, 128.1, 127.4, 127.2, 125.3, 116.8, 115.8,
114.4, 106.7, 67.2, 60.5,50.7 36.3, 31.2.
1-Butyl-3-(6-acetyl-2H-1,4-benzoxazine-3(4H)-one)imidazolium
chloride (4c). White solid, yield; 88%, m.p.: 213–215 ꢁC; Anal
Calcd for C17H20ClO3N3; C: 58.37, H: 5.76, N: 12.01, found: C:
58.50, H: 5.87, N: 11.93; IR (cmꢀ1): 1697 (–C¼O); H-NMR
1
(DMSO-d6, 300 MHz) ꢀ/ppm; 11.18 (s, 1H, –N–H), 9.29 (s, 1H,
–NCHN–), 7.14–7.9 (m, 5H, Ar-H, NCH ¼ CHN), 6.05 (s, 2H,
benzoxazinone–CH2–N–), 4.74 (s, 2H, –C(O)–CH2–O–), 4.30 (t,
2H, J ¼ 7.0 Hz, NCH2CH2CH2CH3), 1.81 (five, 2H, J ¼ 7.4 Hz,
NCH2CH2CH2CH3), 1.29 (six, 2H, J ¼ 7.5 Hz, NCH2CH2
CH2CH3), 0.93 (t, 3H, J ¼ 7.4 Hz, NCH2CH2CH2CH3);
13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 190.2, 164.4, 148.6,
137.9, 128.4, 128.1, 124.9, 124.6, 122.4, 116.8, 115.7, 67.2, 55.5,
49.1, 31.9, 19.2, 13.8.
1-(Naphthalen-2-ylmethyl)-3-(6-acetyl-2H-1,4-benzoxazine-
3(4H)-one)benzimidazolium chloride (4i). White solid, yield;
86%, m.p.: 243–246 ꢁC; Anal Calcd for C28H22ClO3N3; C: 69.49,
H: 4.58, N: 8.68, found: C: 69.35, H: 4.63, N: 8.61; IR (cmꢀ1):
3410 (–N–H), 1687 (–C¼O); 1H-NMR (DMSO-d6, 300 MHz)
ꢀ/ppm; 11.14 (s, 1H, –N–H), 10.00 (s, 1H, NCHN), 7.19–8.15 (m,
14H, Ar-H), 6.40 (s, 2H, benzoxazinone–CH2–N–), 6.09 (s, 2H,
–N–CH2Ph), 4.76 (s, 2H, –C(O)–CH2–O–); 13C-NMR (DMSO-
d6, 75 MHz) ꢀ/ppm: 189.5, 163.9, 148.2, 143.7, 132.7, 132.2,
131.3, 130.6, 128.9, 128.0, 127.9, 127.7, 127.6, 126.8, 126.7,
125.6, 124.8, 116.3, 115.4, 114.1, 113.9, 66.8, 53.0, 50.2.
1-Methyl-3-(6-acetyl-2H-1,4-benzoxazine-3(4H)-one)benzimida-
zolium chloride (4d). White solid, yield; 84%, m.p.: 263–
264 ꢁC; Anal Calcd for C18H16ClO3N3; C: 60.43, H: 4.51, N:
11.74, found: C: 60.55, H: 4.43, N: 11.70; IR (cmꢀ1): 1681
1
(–C¼O); H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 11.15 (s, 1H,
–N–H), 9.83 (s, 1H, NCHN), 7.16–8.10 (m, 7H, Ar-H), 6.51 (s,
2H, benzoxazinone–CH2–N–), 4.75 (s, 2H, –C(O)CH2–O–), 4.21
(s, 3H, –N–CH3); 13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 190.1,
164.5, 148.7, 144.2, 132.3, 131.9, 128.5, 128.1, 127.2, 126.9,
125.4, 116.8, 115.9, 114.3, 114.1, 67.3, 53.3, 33.9.
Carbonic anhydrase inhibition
Preparation of hemolysate and purification from blood red cells
1-Allyl-3-(6-acetyl-2H-1,4-benzoxazine-3(4H)-one)benzimidazo-
lium chloride (4e). White solid, yield; 81%, m.p.: 263–265 ꢁC;
Anal Calcd for C20H18ClO3N3; C: 62.58, H: 4.73, N: 10.95,
found: C: 62.78, H: 4.60, N: 10.81; IR (cmꢀ1): 1694–1676
Blood samples (25 mL) were taken from healthy volunteers.
They were anticoagulated with acid-citrate-dextrose, centrifuged
at 1000 ꢂ g for 20 min at 4 ꢁC and the supernatant was
removed. The packed erythrocytes were washed three times
with 0.9% NaCl and then haemolysed in cold water. The ghosts
and any intact cells were removed by centrifugation at 3100 ꢂ g
for 25 min at 4 ꢁC, and the pH of the hemolysate was adjusted
to pH 8.5 with solid Tris-base. The 25 mL hemolysate was
applied to an affinity column containing L-tyrosine-sulfona-
mide-Sepharose-4B53 equilibrated with 25 mM Tris–HCl/0.1 M
Na2SO4 (pH 8.5). The affinity gel was washed with 50 mL of
25 mM Tris–HCl/22 mM Na2SO4 (pH 8.5). The human CA
(hCA) isozymes were then eluted with 0.1 M NaCl/25 mM
Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH
5.6), which recovered hCA-I and hCA-II, respectively. Fractions
of 3 mL were collected and their absorbance measured at
280 nm.
1
(–C¼O); H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 11.14 (s, 1H,
–N–H), 9.84 (s, 1H, NCHN), 7.19–8.08 (m, 7H, Ar-H), 6.40 (s,
2H, beznoxazinone–CH2–N–), 6.18 (m, 1H, –CH2–CH ¼ CH2),
5.32–5.45 (m, 4H, –CH2–CH¼CH2), 4.76 (s, 2H, –C(O)–CH2–
O_); 13C-NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 190.0, 164.5, 148.7,
144.0, 132.5, 131.5, 131.1, 128.9, 128.5, 128.1, 127.3, 127.1,
125.3, 120.9, 116.8, 115.9, 114.5, 114.3, 67.3, 53.4,49.4.
1-Butyl-3-(6-acetyl-2H-1,4-benzoxazine-3(4H)-one)benzimidazo-
lium chloride (4f). White solid, yield; 88%, m.p.: 236–238 ꢁC;
Anal Calcd for C21H22ClO3N3; C: 63.08, H: 5.55, N: 10.51,
found: C: 63.35, H: 5.40, N: 10.44; IR (cmꢀ1): 1686–1673
1
(–C¼O); H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 11.13 (s, 1H,
–N–H), 9.88 (s, 1H, NCHN), 7.19–8.18 (m, 7H, Ar-H), 6.38 (s,
2H, benzoxazinone–CH2–N–), 4.76 (s, 2H, –C(O)–CH2–O–), 4.63
(t, 2H, J ¼ 7.2 Hz, NCH2CH2CH2CH3), 1.92 (five, 2H, J ¼ 7.5 Hz,
NCH2CH2CH2CH3), 1.36 (six, 2H, J ¼ 7.6 Hz, NCH2CH2
CH2CH3), 0.94 (t, 3H, J ¼ 7.4 Hz, NCH2CH2CH2CH3); 13C-
NMR (DMSO-d6, 75 MHz) ꢀ/ppm: 190.0, 164.5, 148.7, 143.9,
132.5, 131.1, 128.5, 128.1, 127.2, 127.1, 125.4, 116.8, 115.9,
114.5, 114.2, 67.3, 53.3, 47.0, 31.0, 19.5, 13.8.
CA enzyme assay
Carbonic anhydrase activity was measured by the Maren method
which is based on determination of the time required for the pH to
decrease from 10.0 to 7.4 due to CO2 hydration54. The assay
solution was 0.5 M Na2CO3/0.1 M NaHCO3 (pH 10.0) and Phenol
Red was added as the pH indicator. CO2-hydratase activity
[enzyme units (EU)] was calculated using the equation t0 ꢀ tc/tc
where t0 and tc are the times for pH change of the non-enzymatic
and the enzymatic reactions, respectively.
1-Benzyl-3-(6-acetyl-2H-1,4-benzoxazine-3(4H)-one)benzimida-
zolium chloride (4g). White solid, yield; 94%, m.p.: 222–
226 ꢁC; Anal Calcd for C24H20ClO3N3; C: 66.44, H: 4.65, N:
9.68, found: C: 66.67, H: 4.48, N: 9.60; IR (cmꢀ1): 3385 (–N–H),
In vitro inhibition studies
1
1689 (–C¼O); H-NMR (DMSO-d6, 300 MHz) ꢀ/ppm; 11.15 (s,
For the inhibition studies of coumarin different concentrations of
these compounds were added to the enzyme. Activity percentage
values of CA for different concentrations of each coumarin were
determined by regression analysis using Microsoft Office 2000
Excel. CA enzyme activity without a coumarin solution was
accepted as 100% activity.
1H, –N–H), 10.02 (s, 1H, –NCHN–), 7.19–8.11 (sm, 12H, Ar-H),
6.41 (s, 2H, benzoxazinone–CH2–N–), 5.93 (s, 2H, –N–CH2Ph),
4.76 (s, 2H, –C(O)–CH2–O–); 13C-NMR (DMSO-d6, 75 MHz)
ꢀ/ppm: 189.5, 163.9, 148.2, 143.7, 133.9, 132.1, 130.5, 129.0,
129.3, 128.8, 128.3, 128.1, 127.6, 126.8, 126.7,124.8 116.3,
115.4, 114.1, 113.9, 66.8, 53.0, 49.9.

8
M. O. Karatas¸ et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Anticonvulsant activity
Results and discussion
This study was approved by the Ethics Committee of Inonu Synthesis and characterization of compounds
University (Date: 13/01/2011, Number: 2011/A-09).
Twenty four compounds were synthesized according to the
The compounds were tested for anticonvulsant activity accord-
procedures in the literature and characterized by H-NMR, 13C-
ing to MES and ScMet tests23,24. Phase I evaluation was designed
NMR, FT-IR spectroscopy and elemental analysis (Schemes 1–4).
to identify anticonvulsant activity and neurotoxicity with MES,
Among these, two 7-hydroxycoumarin (2g, 2h) and four
ScMet and rotorod tests. Dual Impedance Stimulator (Harvard
7,8-dihydroxycoumarin derivatives (3a–c, 3j) were synthesized
Apparatus 6020), Linear Isolated Stimulator (Biopac) and corneal
by the reaction of 1-alkyl imidazoles/1-alkyl benzimidazoles
electrodes were used for the evaluation of anticonvulsant activity.
with 7-hydroxy/7,8-dihydroxy coumarin derivatives. 6,8-
Suspensions of the compounds in 30% aqueous of PEG 400 were
Dimethylcoumarin derivatives (1a–i) were obtained through
administered intraperitoneally at two dose levels (30 and 100 mg/
the reaction of 4-chloromethyl-6,8-dimethylcoumarin with
kg) 30 min and 4 h after the administration. Four Balb-C mice (20–
1-alkylimidazole/1-alkylbenzimidazole derivatives in DMF at
24 g) were used for each compound. The mice were obtained from
80 ꢁC for 24 h.
1
the Laboratory Animals Research Center of Inonu University.
Fourteen compounds (2a–f, 2i, 2j, 3d–i) which bear
Pentylenetetrazole was supplied by Sigma Chemical Co. and
7-hydroxycoumarin or 7,8-dihydroxycoumarin were available
administered subcutaneously over the back of the neck. The rotorod
from the previous studies. In addition to these compounds, two
toxicity test was performed for neurological deficits.
novel 7-hydroxycoumarin bearing compounds (2g and 2h) and four
novel 7,8-dihydroxycoumarin bearing compounds (3a–c, 3j) were
synthesized. These six novel compounds were synthesized accord-
MES test
MES seizures were elicited with a 60 Hz alternating current of 50 ing to the previously described procedures51,52. Target compounds
mA intensity delivered for 0.2 s via corneal electrodes. A drop of were synthesized by the reaction of 1-alkyl imidazoles or 1-alkyl
0.9% saline was instilled in the eyes prior to the application of the benzimidazoles with 7-hydroxy or 7,8-dihydroxy coumarin deriva-
electrodes in order to prevent the animal death. Abolition of the tives. 1-Alkylbenzimidazoles were synthesized by the procedure
hind limb tonic extension component of the seizure was defined described by Ozdemir49 and 4-chloromethyl-7,8-dihidroxycou-
1
as protection.
marin was synthesized procedure described by Gumus55. In H-
NMR spectra of compounds 2g and 2h, signals for –NCHN–
protons were located at 9.23 and 10.07 ppm, while signals of
hydroxide protons were located at 11.02 and 10.99 ppm, respect-
ively. Signal of olefinic proton is a characteristic one for coumarin
derivatives. For compounds 2g and 2h, these signals were located
at 5.58 and 5.80 ppm, respectively. 13C-NMR, IR spectra and
elemental analysis results also supported the structures the
compounds. In 1H-NMR spectra of compounds 3a, 3c and 3j,
signals of –NCHN– protons were located at 9.32, 9.46 and
10.07 ppm, respectively. Signals of free hydroxide protons for
Subcutaneous pentylenetetrazole (metrazol) (ScM) test
Pentylenetetrazole (85 mg/kg) which produces seizures in greater
than 95% of mice was administered as a 0.5% solution subcuta-
neously. The animals were observed for 30 min, failure to observe
even a threshold seizure (a single episode of clonic spasms of at
least 5 s duration) was defined as protection.
Neurotoxicity test
The rotorod test was used to evaluate neurotoxicity. The animals compound 3j were located at 10.53 and 9.51 ppm but they were not
were placed on a 1-inch-diameter knurled wooden rod rotating at observed for compounds 3a–c. Signals of olefinic protons for four
6 rpm. Normal mice remain on a rod rotating at this speed compounds were located in the range of 5.51–5.82 ppm. 13C-NMR,
indefinitely. Neurotoxicity was indicated by the inability of the IR spectra and elemental analyses were supportive of the structures
animal to maintain equilibrium on the rod for at least 1 min in of these compounds too. Along with these compounds, nine novel
each of the three trials. In rats, neurological deficit is indicated by 6,8-dimethylcoumarin bearing (1a–i) imidazolium and benzimi-
ataxia and loss of placing response and muscle tone.
dazolium salts were synthesized. For synthesis of compounds 1a–i,
first, 4-chloromethyl-6,8-dimethylcoumarin (compound 1) was
synthesized according to procedure described by Frasinyuk50.
Compounds 1a–i were than synthesized by the reaction of
compound 1 with three different 1-alkylimidazole and six different
1-alkylbenzimidazole derivatives in DMF, at 80 ꢁC for 24 h. In 1H-
NMR spectra of these compounds (except 1b), signals of –NCHN–
protons are located in the range of 9.41–10.24 ppm. Signals of
olefinic protons are located in the range of 5.78–6.13 ppm. In
13C-NMR, signals of carbonyl carbons belong to coumarin were
located in the range of 159.9–162.4 ppm. IR spectrum and
elemental analyses results were supportive of the structures of
compounds 1a–i. For all synthesized compounds, 1H-NMR
In silico docking studies
Enzyme setup
Determination of the consistent receptor was based on previous
studies32. Macromolecule file (PDB code: 3EFT) was modified
using the ADT package version 1.5.6rc3 (Ankara, Turkey). All
water molecules were deleted and polar hydrogens were added.
Subsequently, Gasteiger charges were calculated and the gener-
ated pdbqt files were saved.
Ligands
and 13C-NMR were consistent with the literature51,52,56
.
Energy minimization of compounds 1i, 2a, 3g and 3j were carried
out using GAMESS module for ChemOffice version Ultra 8.0.3
(Ankara, Turkey). All data were saved as pdb with the aid of
Molegro Molecular Viewer version 2.5 (Ankara, Turkey). Further
modification of these partial charges of pdb files was carried out
through the ADT package so that the charges of the non-polar
hydrogen atoms allocated to the atom to which the hydrogen is
attached. These modified pdb files saved as pdbqt files.
Appropriate grid box points were determined by centering on
ligand separately for each compound.
Compounds 4a–i were synthesized by the reaction of 6-
(chloroacetyl)-2H-1,4-benzoxazine-3(4H)-one (compound 1)
with three different 1-alkylimidazole and six different 1-alkylben-
zimidazole derivatives in DMF, at 80 ꢁC for 8 h. In 1H-NMR
spectras of compounds 4a–i (except 4b), signals of –NCHN–
protons were located in the range of 9.20–9.97 ppm. Free –N–H
protons were located in the range of 11.10–11.86 ppm. 13C-NMR,
IR spectrum and elemental analysis were also matching with the
structures of compounds 4a–i. For all synthesized compounds,
1H-NMR and 13C-NMR were consistent with the literature56.

DOI: 10.3109/14756366.2015.1063624
Coumarin or benzoxazinone-based inhibitors
9
Table 2. IC50 values of synthesized and previously reported compounds Table 3. ScMet and neurotoxicity screening data in mice dosed ip with
against hCA I and II inhibition.
Compounds
the compounds.
hCA I (mM)
hCA II (mM)
ScMet
Toxicity
1a
1b
1c
1d
11.76
16.41
10.11
12.80
7.79
17.17
23.00
14.52
15.52
9.91
½ h
4 h
½ h
4 h
mg/kg
mg/kg
mg/kg
mg/kg
Compounds
30
100
30
100
30
100
30
100
1e
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2h
2i
*
*
0/1
*
0/1
*
0/1
1/1
0/1
1/1
*
*
*
*
*
*
0/1
*
0/1
*
0/1
0/1
0/1
0/1
*
*
*
*
1f
1g
5.34
6.01
0/1
0/1
0/1
0/1
0/1
1/1
0/1
1/1
*
0/1
0/1
0/1
*
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
*
0/1
0/1
0/1
*
15.62
12.57
9.46
19.67
16.90
8.92
0/1
0/1
*
0/1
1/1
0/1
*
*
*
*
0/1
*
0/1
0/1
*
0/1
1/1
0/1
*
1h
1i
2a51
264.00
218.00
138.00
147.00
153.00
79.00
11.76
16.41
81.00
99.00
10.98
11.72
9.48
28.55
49.32
48.06
25.63
22.09
33.10
4.99
8.99
18.09
9.06
15.49
6.86
14.71
9.90
7.61
6.49
3.30
327.00
188.00
137.00
221.00
154.00
88.00
17.17
23.00
94.00
89.00
56.82
26.81
36.98
49.40
51.45
29.70
40.01
28.90
20.33
6.01
2b51
*
*
2c51
0/1
0/1
1/1
0/1
*
0/1
0/1
*
1/1
0/1
0/1
0/1
*
0/1
0/1
*
2d51
2e51
2f51
2g
*
*
0/1
0/1
0/1
1/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
1/1
1/1
1/1
0/1
0/1
0/1
0/1
1/1
*
*
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
*
*
1/1
1/1
1/1
1/1
0/1
1/1
1/1
*
1/1
*
0/1
*
1/1
*
1/1
*
1/1
0/1
*
0/1
1/1
1/1
*
0/1
0/1
0/1
0/1
0/1
1/1
1/1
0/1
0/1
*
0/1
*
0/1
*
0/1
*
0/1
0/1
*
0/1
0/1
0/1
*
0/1
0/1
0/1
0/1
0/1
1/1
*
0/1
0/1
0/1
*
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
*
2h
2i51
2j51
3a
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
1/1
0/1
0/1
0/1
0/1
1/1
1/1
0/1
1/1
0/1
1/1
1/1
0/1
0/1
*
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
3b
3c
3d52
3e52
2j
1/1
1/1
0/1
0/1
0/1
1/1
1/1
1/1
0/1
1/1
1/1
0/1
0/1
0/1
*
3f52
3a
3b
3c
3d
3e
3f
3g
3h
3i
3g52
3h52
3i52
3j
4a
4b
4c
4d
4e
4f
4g
14.34
20.16
11.20
22.92
10.73
24.28
18.42
9.88
3j
4a
4b
4c
4d
4e
4f
4g
4h
4i
4h
4i
8.15
2.40
Acetazolamide57
*
1/1
1/1
0/1
1/1
1/1
1/1
0/1
1/1
0/1
0/1
1/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
CA inhibition, anticonvulsant activity and structure–
activity relationship
Compounds were administered to mice intraperitoneally at 30 and
100 mg/kg; seizure protection and neurotoxicity were measured 0.5 and
4 h after administration. ScMet: Subcutaneous metrazole test (number
of animals protected/ number of animals tested). Toxicity: Rotarod test
(number of animals exhibiting toxicity/number of animals tested). 0/1:
no activity or toxicity at dose level. 1/1: noticeable activity or toxicity at
dose level (activity given in bold). *Animal death.
For evaluating the CA inhibitory activity, all synthesized com-
pounds were subjected to CA inhibition assay with CO2 as
substrate. The results showed that all synthesized compounds
(1–3j) inhibited hCA I and II enzyme activity. The inhibition
values of 1a–4i against CAs are summarized in Table 2. We
determined the IC50 values ranging between 4.99 and 18.09 mM
for hCA I and 6.01–56.82 mM for hCA II. Among them, 3j was
found to be the most active (IC50: 4.99 mM for hCA I and 6.01 mM
for hCA II). The synthesized compounds have a lower affinity to
CAI and II compared to acetazolamide (IC50: 3.30 mM for hCA I
2.40 mM for hCA II), which is used in the treatment of
glaucoma57.
Table 4. MES screening data of 1i, 2f, 3g and 3j in mice dosed ip.
MES
½ h
4 h
Compounds
30 mg/kg
100 mg/kg
30 mg/kg
100 mg/kg
The results showed that both coumarin and benzoxazinone
derivatives inhibited the hCA I and II enzyme activity.
1i
2f
3g
3j
4i
1/1
0/1
0/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
0/1
0/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
We investigated anticonvulsant activities of the synthesized
compounds considering the potential anticonvulsant activities of
CAIs. Anticonvulsant activity and neurotoxicity screening tests
(ScMet and rotorod) results of the synthesized compounds are
summarized in Table 3. The most active compounds of 1, 2, 3 and
4 series were also scanned with MES test (Table 4). Compounds
were tested at the dose of 30 mg/kg and 100 mg/kg at 0.5 and 4 h.
Seventeen compounds (1g, 1i, 2c–f, 2h, 3a, 3e, 3g–i, 4c, 4d, 4f,
4g and 4i) were active at 100 mg/kg at 0.5 h. Twelve compounds
Compounds were administered to mice intraperitoneally at 30 and
100 mg/kg; seizure protection was measured 0.5 and 4 h after admin-
istration. MES: Maximal electroshock test (number of animals
protected/number of animals tested). 0/1: no activity at dose level.
1/1: noticeable activity at dose level (given in bold).

10 M. O. Karatas¸ et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
(1g, 1i, 2e, 2f, 3d, 3g–i, 4d, 4f, 4g, 4i) showed protection at
From anticonvulsant activities of the compounds, some
30 mg/kg at 0.5 h. Animal deaths were observed with compounds opinions about structure–activity relationship have emerged. We
1a, 1c, 1e, 2a, 2b, 2j, 3b, 3d, 3f, 4a and 4e at 100 mg/kg at 0.5 h. can classify the compounds in two different ways: (i) imidazole or
Although compound 3d was active at 30 mg/kg at 0.5 h, animal benzimidazole bearing compounds, (ii) 6,8-dimethylcoumarin
death was observed at 100 mg/kg at 0.5 h. At 4 h at 100 mg/kg, (1a–i) or 7-hydroxycoumarin (2a–j) or 7,8-dihydroxycoumarin
only eleven compounds (1g, 1i, 2f, 3a, 3e, 3g, 3h, 3i, 3j, 4h (3a–j) or benzoxazinone (4a–i) bearing compounds. When
and 4i) continued protection. When we decreased the dose to compared, benzimidazolium derivatives are much more
30 mg/kg at 4 h, eight compounds (1i, 3g–i, 4d, 4e, 4g and 4i) active than imidazolium derivatives. With the imidazolium
showed activity. Compounds 1i and 4d were active at 30 mg/kg at derivatives no activity was observed, except compound 3a
4 h although they caused animal death at 100 mg/kg at 4 h.
(active at 100 mg/kg at 0.5 and 4 h). According to these results,
Table 5. Molecular docking binding scores and binding interactions of compounds 1i, 2a, 3g and 3j within the hCA II
active site.
Docking score
(kcal/mol)
Compounds
H-bonds
Close van der Waals contacts
3g
2a
3j
1i
ꢀ5.9
ꢀ4.2
ꢀ9.7
ꢀ9.5
–
–
–
–
Lys170, Phe231, Glu239
Lys170, Gly171
Gln92, Leu198, Val121, Phe 131, Pro202, Thr199, Zn262
Gln92, Leu198, Val121, Phe131, Asn67, Leu60, Asn62, Gly171
˚
Residues participating in hydrogen bonds and close van der Waals contacts (54 A) with the inhibitors are shown.
Figure 1. Docking of compound 3j within the
hCA II active site. (A) Discovery Studio 4.0
Client images and (B) ADT images.

DOI: 10.3109/14756366.2015.1063624
Coumarin or benzoxazinone-based inhibitors 11
addition of a benzene ring to the imidazolium derivatives showed anti ScMet and MES at any dose and time levels and
increased lipophilicity of the compounds, making them more toxicity was not observed for this compound.
active than those with lower lipophilicity. In addition to these
results, many imidazolium derivatives caused animal deaths.
Molecular docking studies
When we compare the activities of benzimidazolium derivatives,
In order to obtain more insights into the binding mode, molecular
docking studies were also performed for the coumarin derivative
compounds (1i, 2a, 3g and 3j) experimentally tested on animals
and hCA activity, then all data were compared. The rationale of
docking studies of coumarin derivatives is due to the fact that
simple coumarin and 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-
2H-chromen-2-one were shown to be competitive inhibitor with
CO2 as substrate for the main isoforms for CA, i.e. human CA
II26,31,32. Docking scores were obtained using Lamarckian
Genetic Algorithm and scoring function of AutoDock 4.
Afterwards, interactions were checked with the aid of ADT and
Discovery Studio 4.0 Client (Ankara, Turkey). In general, the
results achieved computationally were in good agreement with the
experimental values. Docking scores and binding interactions of
naphthalene bearing compounds (especially compounds 1i and 3j)
showed better activity than other benzimidazolium derivatives.
These results support the suggestion that lipophilicity increases
anticonvulsant activity. Another factor that affect anticonvulsant
activity is coumarin or benzoxazinone scaffold. 7-
Hydroxycoumarin bearing imidazolium and benzimidazolium
derivatives except 2f (active at 100 mg/kg) were inactive, whereas
7,8-dihydroxycoumarin bearing derivatives were active.
Compounds 3g–i were active at any dose level at 0.5 and 4 h.
Some of the compounds were also scanned with MES test, all
of which showed anti-MES activity at either dose level. 1i, 3j and
4i exhibited anti MES activity at 30 and 100 mg/kg at both time
intervals. Compounds 2f, 3j and 4i showed anti MES activity at
100 mg/kg (Table 4). In the synthesized compounds, only 4i
Figure 2. Docking of compound 1i within the
hCA II active site. (A) Discovery Studio 4.0
Client images and (B) ADT images.

12 M. O. Karatas¸ et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Carter ND, Edwards YH, eds. X-ray crystallographic studies on
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inhibitors: cloning, characterization and inhibition studies of the
cytosolic isoenzyme III with sulphonamides. Bioorg Med Chem
2007;15:7229–36.
compounds 1i, 2a, 3g and 3j with hCA II (PDB code: 3EFT) are
presented in Table 5. Final images of compounds 1i and 3j for
binding interactions are shown in Figures 1 and 2.
As seen from the data presented in Table 5, there are many
amino acid residues participating in hydrogen bonds or close van
˚
der Waals (54 A) contacts with the inhibitors 1i, 2a, 3g and 3j
14. Nishimori I, Minakuchi T, Onishi S, et al. Carbonic anhydrase
inhibitors. DNA cloning, characterization and inhibition studies of
human secretary isoform VI, a new target for sulfonamide and
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when bound to the active site of hCA II. Most of these amino
acids include Thr199 (one of the gate keeper residues of this
enzyme) as well as the residues lining the CO2 binding pocket,
namely, Val121, Val143, Leu198, Val207 and Trp20926,31,32. Only
compound 3j was found coordinated to Zn(II) ion in the hCA II
active site. Compound 3j has two hydroxy groups on the coumarin
scaffold. As mentioned in the ‘‘Introduction’’ section, phenols are
inhibitors of coumarin derivatives and compounds 3j may act in
the active site of hCA II like phenols for binding Zn(II) ion
besides non-Zinc binding interactions.
Conclusion
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20. Vullo D, Franchi M, Gallori E, et al. Carbonic anhydrase inhibitors:
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21. Vullo D, Innocenti A, Nishimori I, et al. Carbonic anhydrase
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sulphonamides – a new target for the design of antitumor and
antiglaucoma drugs? Bioorg Med Chem Lett 2005;15:963–9.
22. Lehtonen J, Shen B, Vihinen M, et al. Characterization of CA XIII, a
novel member of the carbonic anhydrase isozyme family. J Biol
Chem 2004;279:2719–27.
Coumarins are known as a novel type of hCA I. In this study, we
have synthesized novel coumarin and benzoxazinone derivatives.
All of the synthesized compounds inhibited hCA I and hCA II.
Docking studies suggest that the mechanism of action of the
compounds synthesized in this study is similar to that previously
described for coumarin derivatives. CAI are known to exhibit
anticonvulsant properties although their mechanisms of action
have not fully been deciphered. With the prospect of potential
anticonvulsant properties of CAIs, anticonvulsant activities of
synthesized and previously described compounds were tested.
Among the tested compounds, 4i exhibited anti-SCM and anti-
MES at either given dose level; neither animal death nor toxicity
was observed. Our results suggest that these coumarin and
benzoxazinone derivatives are likely to be adopted as candidates
to treat glaucoma and epilepsy.
23. Nishimori I, Vullo D, Innocenti A, et al. Carbonic anhydrase
inhibitors: inhibition of the transmembrane isozyme XIV with
sulphonamides. Bioorg Med Chem Lett 2005;15:3828–33.
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Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
25. Breton S. The cellular physiology of carbonic anhydrases.
J Pancreas 2001;2:159–64.
26. Durdagi S, Senturk M, Ekinci D, et al. Kinetic and docking studies
of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX
and XII, evidence a new binding mode within the enzyme active
site. Bioorg Med Chem 2011;19:1381–9.
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