Facile, highly efficient, and clean one-pot synthesis of acridine sulfonamide derivatives at room temperature and their inhibition of human carbonic anhydrase isoenzymes

Article abstract of DOI:10.1007/s00706-013-1145-x

Reaction of dimedone, 4-amino-N-(diaminomethylene)benzenesulfonamide, and aromatic aldehydes was successfully realized using sulfuric acid as a cheap catalyst. Synthesis of novel acridine sulfonamide compounds was performed providing high yields in water as the solvent at room temperature. This method has several advantages such as use of a green solvent, high yields, and efficient one-pot procedure. In addition, human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of acetazolamide and the newly synthesized acridine sulfonamides on hydratase and esterase activities of these isoenzymes was studied in vitro. The esterase IC ₅₀ values of the new compounds are 47.2-230.1 μM for hCA I and 50.1-275.0 μM for hCA II. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-013-1145-x

Monatsh Chem
DOI 10.1007/s00706-013-1145-x
ORIGINAL PAPER
Facile, highly efficient, and clean one-pot synthesis of acridine
sulfonamide derivatives at room temperature and their inhibition
of human carbonic anhydrase isoenzymes
˙
Ibrahim Yes¸ildag Ramazan Ulus Erhan Bas¸ar
•
•
•
˘
•
•
Melike Aslan Muharrem Kaya Metin Bu¨lbu¨l
Received: 23 October 2013 / Accepted: 26 December 2013
Ó Springer-Verlag Wien 2014
Abstract Reaction of dimedone, 4-amino-N-(diamino-
methylene)benzenesulfonamide, and aromatic aldehydes was
successfully realized using sulfuric acid as a cheap catalyst.
Synthesis of novel acridine sulfonamide compounds was
performed providing high yields in water as the solvent at
room temperature. This method has several advantages
such as use of a green solvent, high yields, and efficient
one-pot procedure. In addition, human carbonic anhydrase
isoenzymes (hCA I and hCA II) were purified from
erythrocyte cells by affinity chromatography. The inhibi-
tory effects of acetazolamide and the newly synthesized
acridine sulfonamides on hydratase and esterase activities
of these isoenzymes was studied in vitro. The esterase IC50
values of the new compounds are 47.2–230.1 lM for hCA
I and 50.1–275.0 lM for hCA II.
and H?, and they participate in many crucial physiological
processes concerned with pH homeostasis, electrolyte
secretion, respiration, CO2 and ion transport, some bio-
synthesis reactions (gluconeogenesis, lipogenesis etc.),
tumorigenicity, and so on [1–3]. There are five distinct
genetic families known to date, the a-, b-, c-, d-, and f-
carbonic anhydrases [4]. The a-carbonic anhydrases are
present in mammals. Sixteen different a-carbonic anhy-
drase isoenzymes have been described in humans [5].
Among these isozymes hCA I and hCA II are cytosolic,
and hCA IV is membrane bound. The hCA II and hCA IV
isoenzymes are present in human eyes [6, 7]. Glaucoma is a
group of diseases characterized by gradual loss of visual
field due to an elevation in intraocular pressure (IOP) [8,
9]. Carbonic anhydrase inhibitors reduce intraocular pres-
sure by lowering aqueous humor formation. So carbonic
anhydrase isoenzymes (hCA I, hCA II, and hCA IV) are
therapeutic targets for the treatment of glaucoma [10, 11].
In this study, novel acridine derivatives were synthe-
sized, characterized, and tested with respect to inhibition of
hCA isoenzymes that were purified from human
erythrocytes.
Keywords Green chemistry ꢀ Cyclization ꢀ
Three-component reaction ꢀ Aldol reactions ꢀ Enzymes ꢀ
Michael additions
Introduction
Carbonic anhydrases (EC 4.2.1.1) are Zn(II) ion-containing
metalloenzymes that catalyze simple but important physi-
Results and discussion
-
ological reactions, like the conversion of CO2 to HCO3
Chemistry
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-013-1145-x) contains supplementary
material, which is available to authorized users.
The general synthesis method shown in Scheme 1 was
employed to prepare the acridine compounds 4–17. The
syntheses of these compounds were realized in water in a
single process through three successive reactions (aldol
condensation, Michael addition, and cyclization) using
sulfuric acid. Acridine compounds were prepared by means
of a one-pot reaction producing high yields and providing a
˙
˘
I. Yes¸ildag ꢀ R. Ulus ꢀ E. Bas¸ar ꢀ M. Aslan ꢀ M. Kaya (&) ꢀ
M. Bu¨lbu¨l
Chemistry Department, Faculty of Arts and Science, Dumlupınar
University, Evliya C¸ elebi Campus, 43100, Kutahya, Turkey
e-mail: mhrkaya@yahoo.com
123

˙
I. Yes¸ildag et al.
˘
Scheme 1
Table 1 Human carbonic
anhydrase isozymes (hCA I and
hCA II) inhibition data with
acridine sulfonamide
Product
Ar
Esterase ICa5,0b/lM
Selectivity
ratio (hCA
II/hCA I)
hCA I
hCA II
derivatives 4–17 determined by
an esterase assay with
4-nitrophenylacetate as
substrate
AAZ
4
–
3.02 0.02
207.0 1.1
72.4 2.8
2.08 0.02
198.4 3.0
59.0 1.9
0.69
0.96
0.81
0.97
1.72
–
4-CNC6H4
5
4-NO2C6H4
3-NO2C6H4
4-FC6H4
6
230.1 3.2
160.2 1.3
No inhibition
145.6 5.2
182.4 3.9
66.3 4.4
224.2 3.0
275.0 1.7
No inhibition
122.3 4.3
210.5 4.4
66.0 5.0
7
8
4-BrC6H4
9
2,4-Cl2C6H3
5-Br-2-OHC6H3
4-OHC6H4
0.84
1.15
0.99
1.06
0.98
–
10
11
12
13
14
15
16
17
4-CH3C6H4
4-C2H5C6H4
4-CH3SC6H4
4-CH3OC6H4
2,4-(CH3O)2C6H3
3,4-(CH3O)2C6H3
47.2 1.7
50.1 1.0
103.3 2.4
No inhibition
No inhibition
No inhibition
No inhibition
101.2 2.1
No inhibition
No inhibition
No inhibition
No inhibition
AAZ (acetazolamide) was used
as reference compound
–
a
Mean standard error, from
three different assays
–
–
b
P \ 0.005 for all analysis
simple work-up procedure. Dimedone (1) was condensed
with sulfaguanidine 2 and aldehydes 3 in the molar ratio of
2:1:1 at room temperature, leading to the acridine sulfon-
amide-containing compounds 4–17. By changing the
nature of the substituents present in the aldehyde compo-
nents, a rather large chemical diversity can be incorporated
in the acridine sulfonamide reported (Scheme 1; Table 1).
These various substituents comprised cyano, nitro, halogen
(F, Cl, Br), methoxy, hydroxyl, methylthio, methyl, and
ethyl moieties (Table 1).
Table 2 Comparison of the efficiency of various acid catalysts used
in the synthesis of acridine sulfonamide derivatives in water
Entry
Catalyst
Time/h
Yield/%
1
2
3
4
5
6
H2SO4
DBSA
HI
48
48
48
48
48
48
79.8
78.2
73.6
73.1
72.9
67.2
HCl
HBr
p-TsOH
The
reaction
of
dimedone,
4-amino-N-
[amino(imino)methyl]benzenesulfonamide (2) and 4-cya-
nobenzaldehyde (3a) in the presence of an acid catalyst
tested in water was selected as a model reaction. However,
the catalysts we tested are outlined in Table 2. The reaction
in the presence of p-toluenesulfonic acid (TsOH) alone
afforded the desired product in a very low yield (entry 6).
H2SO4 was found to be a good catalyst for this reaction
(entry 1), whereas p-dodecylbenzenesulfonic acid (DBSA)
(entry 2), hydroiodic acid (HI) (entry 3), hydrochloric acid
(HCl) (entry 4), and hydrobromic acid (HBr) (entry 5) were
much less effective than H2SO4, suggesting that the strong
acidity of H2SO4 is essential for the catalysis.
123

One-pot synthesis of acridine sulfonamide derivatives
were observed at 2.43 ppm. Compound 13 displayed a triplet
peak (3H) at 1.15 ppm and a multiplet (2H) at 1.05 ppm
assigned to an ethyl group [15]. The signals for the methoxy
group protons for compounds 15, 16, and 17 were observed
in the range of 3.67–3.69 ppm. The signals for the CH pro-
tons were observed at 4.92–5.19 ppm and the signals for the
aromatic protons were observed in the range of
6.41–8.15 ppm. Hydroxyl group protons of compound 11
and 10 were observed as a broad signal at 9.09 and
10.61 ppm. The broad peaks between 6.84 and 7.13 ppm
were assigned to sulfonamide (–SO2NC(NH2)2) group pro-
tons of all the compounds 4–17. The 13C NMR (APT) spectra
of compound 4 displayed a signal for the cyano group carbon
at 118.95 ppm. All the compounds 4–17 showed carbonyl
carbons peaks at 195.08–195.69 ppm. The mass spectra of
all the acridines showed molecular ion isotope [M ? 1]? or
[M - 1]- peaks.
Table 3 Optimization of conditions for preparation of acridine sul-
fonamide derivatives using sulfuric acid as a catalyst and in water at
room temperature
Entry
1
2
3
4
Concentration/%
Yield/%
0.5
77.6
1
2
10
78.3
79.8
79.8
Table 4 Comparison of the efficiency of various solvents used in the
synthesis of acridine sulfonamide derivatives
Entry
Solvent
Yield/%
1
2
3
4
5
Water
79.8
71.3
72.2
70.4
69.1
Ethanol
Methanol
Chloroform
Isopropyl alcohol
The reactions were performed within 48 h
Carbonic anhydrase inhibition
The catalyst plays a crucial role in the success of the
reactions in terms of the rate and the yields, and these exper-
iments are summarized in Table 3. For example,
4-cyanobenzaldehyde reacted with dimedone and sulfagua-
nidine in the presence of 0.5 % H2SO4 to give the product 4 in
modest yield (77.6 %) in water atroom temperature after 48 h
of reaction time. Increasing the catalyst to 1, 2, and 10 %
resulted in increasing the reaction yields to 78.3, 79.8, and
79.8 %. Use of just 2 % H2SO4 in water at room temperature
is enough to afford an efficient synthesis. Higher amounts of
sulfuric acid did not improve the results significantly.
Carbonic anhydrase (CA) inhibition has important phar-
macologic
applications
such
as
antiglaucoma,
anticonvulsant, and anticancer agents. It is well known that
sulfonamides are the main class of CA inhibitors, which
bind to the zinc(II) ion active site of the enzyme [4]. Some
sulfonamide drugs, such as acetazolamide (AAZ), dorzo-
lamide (DZA), and brinzolamide (BRZ) are used in
glaucoma therapy. These compounds are quite powerful
inhibitors but they have some side effects [16]. So syn-
theses of new CA inhibitors are necessary (Scheme 2).
In the present study, firstly hCA I and hCA II were
purified from human erythrocytes. The purification of these
isoenzymes was performed using a simple one-step method
We also investigated the effects of the solvent on a
model reaction, namely, between 4-cyanobenzaldehyde
and dimedone catalyzed by H2SO4 in various solvents.
From the results shown in Table 4, it is obvious that water
is the best choice. In addition, water is a green solvent; thus
we prefer it for this reaction.
Scheme 2
The infrared (IR) spectra of all the acridine compounds
showed sharp peaks for the carbonyl groups in the region
between1,616 and 1,643 cm-1 [12]. Compounds 4 exhibited
a peak at 2,229 cm-1 that belongs to a CN group [13].
Besides, in the IR spectra of the compounds, aliphatic C–H
stretching bands at 2,928–2,964 cm-1 and aromatic C–H
stretching bands at 3,101–3,028 cm-1 were observed. The
NH2 vibrations of acridine sulfonamide compounds were
observed in the region between 3,458 and 3,305 cm-1 [14].
1
The H NMR spectra of compounds 4–17 showed singlet
peaks that belong to protons of the methyl groups in positions
3 and 6 between 0.71 and 0.91 ppm. The CH2 group protons
of the cyclohexene rings of compounds 4–17 showed doublet
peaks at 1.67–2.23 ppm [15]. Compound 12 showed a sin-
glet peak that belongs to protons of the methyl group at
2.23 ppm [15]. The methylthio protons of compound 14
123

˙
I. Yes¸ildag et al.
˘
with Sepharose-4B–L-tyrosine-p-aminobenzene sulfon-
amide affinity chromatography. For purity control, SDS-
PAGE was performed. Then the newly synthesized acri-
dine compounds 4–17 as well as the reference compound
AAZ were tested under in vitro conditions for their enzyme
inhibition activities against human CA isoforms hCA I and
hCA II. The inhibitor concentration that caused 50 %
inhibition (IC50) was determined from activity (%) versus
[I] plots [17].
In conclusion, compounds 4–13 have a moderate inhi-
bition effect on human CA isoforms (hCA I and hCA II).
However, these newly synthesized CA inhibitors could be
evaluated in further studies.
Experimental
The chemicals usedinthe synthesis ofacridinesulfaguanidine
derivatives were obtained from Merck and Aldrich Chemical
Company, and Sepharose 4B for affinity column and elec-
trophoresisreagentswereobtainedfromSigmaChem. Co. All
chemicals and solvents used for the synthesis were of spec-
troscopic reagent grade. Melting points were measured on a
Bibby Stuart Scientific apparatus. Fourier Transform Infrared
(FT–IR) spectra were recorded on a Bruker Optics, ALPHA
In the in vitro studies all the newly synthesized com-
pounds did not inhibit the hydratase activities of hCA I and
hCA II. The effects of the compounds on the esterase
activities of hCA I and hCA II are described below. Some
of the tested compounds (8, 14–17) have no inhibitory
effect on hCA I and hCA II. However, other compounds
(4–13) have IC50 values in the millimolar region. Overall
differences between the highest and lowest IC50 values are
about sixfold (i.e., IC50 value of compound 7 for hCA
II = 275.0 lM; IC50 value of compound 12 for hCA
I = 47.2 lM; Table 1).
The slow cytosolic isoform, hCA I, was moderately
inhibited by 4–13. Compounds 14 and 15 have –OCH3 and
–SCH3 groups on the para position. These compounds did
not affect hCA I activity. Compounds 16 and 17 have
similar effects on this isozyme. Both of them incorporate
two methoxy groups (16: 2,4 positions; 17: 3,4 positions).
These functional groups (–OCH3 and –SCH3) are bigger
than others investigated in the present study. Therefore
steric effects of these groups might block the enzyme–
inhibitor interaction. Interestingly, the difference in inhi-
bition is threefold between 5 (IC50 value 72.4 lM) and 6
(IC50 value 230.1 lM). Both of them have a –NO2 group
on the aromatic ring. But the locations of –NO2 groups are
different. Compound 12 has a most powerful inhibition
effect on hCA I. Compound 12 contains a methyl group,
whereas 13 has an ethyl group. Obviously, alkyl group
elongation leads to a decrease in inhibition potential of the
compounds (IC50 value of 12 = 47.2 lM, IC50 value of
13 = 103.3 lM). The inhibition effects of the compounds
1
FT–IR spectrometer. The H NMR and 13C NMR spectra
were obtained with Bruker DPX-300 and DPX-400 FT-NMR
instruments in CDCl3 and DMSO-d6 as solvents with tri-
methylsilane as the internal reference. Chemical shifts are
expressedindunits(ppm). Themassanalyseswereperformed
on a Waters 2695 Alliance Micromass ZQ instrument LC/MS
and Agilent 1100 Series LC/MSD Trap VL&SL. The ele-
mentalanalyses(C, H, N) wereconductedusingtheElemental
Analyser LECO CHNS-932.
General procedure for preparation of acridine
sulfonamide derivatives 4–17
A mixture of 0.280 g dimedone (1, 2 mmol), 0.214 g
4-amino-N-(diaminomethylene)benzenesulfonamide
(2,
1 mmol), 0.131 g 4-cyanobenzaldehyde (3a, 1 mmol), and
0.50 g H2SO4 (2 %) in 25 cm3 water was stirred continuously
for 48 h at room temperature. The reaction progress was
monitored by TLC. The solid product was filtered, washed
with 500 cm3 water, and recrystallized from ethanol/water.
4-[9-(4-Cyanophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl]-N-
(diaminomethylene)benzenesulfonamide
against to hCA
I isoform are in the order of
(4, C31H33N5O4S)
12 [ 11 [ 5 [ 13 [ 9 [ 7 [ 10 [ 4 [ 6.
Yellow solid; 80 % yield; m.p.: 292–293 °C (EtOH/water);
1H NMR (400 MHz, DMSO-d6): d = 0.73 (s, 6H, 2 CH3),
0.91 (s, 6H, 2 CH3), 1.77 (d, 2H, J = 16.5 Hz, CH2), 2.02
(d, 2H, J = 16.5 Hz, CH2), 2.21 (m, 4H, 2 CH2), 5.09 (s,
1H, CH), 6.87 (s, 4H, 2 NH2), 7.51 (d, 2H, J = 8.5 Hz, Ar–
H), 7.62 (d, 2H, J = 8.5 Hz, Ar–H), 7.73 (d, 2H,
J = 8.5 Hz, Ar–H), 7.97 (d, 2H, J = 8.5 Hz, Ar–H)
ppm; 13C NMR (100 MHz, DMSO-d6): d = 26.12 (CH3),
29.05 (CH3), 32.05 (C), 32.96 (CH), 40.93 (CH2), 49.36
(CH2), 108.65 (CN), 112.14 (C), 118.95 (C), 127.16 (CH),
128.76 (CH), 129.22 (CH), 132.00 (CH), 140.33 (C),
145.35 (C), 150.50 (C), 151.38 (C), 158.20 (C), 195.09 (C)
The human hCA II isozyme showed a similar inhibition
profile for the tested compounds. No inhibition was
observed for 8 or 14–17. The differences in inhibition
potentials of 5 and 6 are similar to those for the hCA I
isoform. Compound 12 is the most powerful inhibitor
(IC50 = 50.1 lM), whereas 7 is the weakest inhibitor
(IC50 = 275.0 lM) for hCA II. The inhibition effects of
the newly synthesized compounds against the hCA II iso-
zyme are in the order of 12 [ 5 [ 11 [ 13 [
9 [ 4 [ 10 [ 6 [ 7. hCA I and hCA II have almost equal
selectivity towards the novel compounds (see selectivity
ratio in Table 1).
123

One-pot synthesis of acridine sulfonamide derivatives
ppm; FT–IR: mꢀ ¼ 3,453 and 3,343 (NH2), 2,964 (C–H),
2,229 (C:N), 1,616 (C=O), 1,557 and 1,494 (C=C), 1,361
and 1,220 (SO2) cm-1; MS (ESI): m/z = 572 ([M ? 1]?).
H), 7.97 (d, 2H, J = 8.0 Hz, Ar–H) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 26.06 (CH3), 29.14 (CH3),
31.39 (CH), 32.04 (C), 40.90 (CH2), 49.46 (CH2), 112.99
(C), 114.44 (CH), 114.65 (CH), 127.17 (CH), 129.25
(CH), 140.50 (C), 142.30 (C), 145.26 (C), 149.91 (C),
N-(Diaminomethylene)-4-[3,3,6,6-tetramethyl-9-
(4-nitrophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl]benzenesulfonamide
(5, C30H33N5O6S)
ꢀ
158.19 (C), 162.96 (C), 195.11 (C) ppm; FT–IR: m ¼
3,442 and 3,367 (NH2), 2,958 (C–H), 1,641 (C=O),
1,541 (C=C), 1,362 and 1,221 (SO2) cm-1; MS (ESI): m/
z = 565 ([M ? 1]?).
1
Yellow solid; 76 % yield; m.p.: 250 °C (EtOH/water); H
NMR (300 MHz, DMSO-d6): d = 0.71 (s, 6H, 2 CH3),
0.89 (s, 6H, 2 CH3), 1.76 (d, 2H, J = 17.4 Hz, CH2), 2.01
(d, 2H, J = 16.0 Hz, CH2), 2.21 (d, 4H, J = 16.6 Hz, 2
CH2), 5.15 (s, 1H, CH), 6.85 (s, 4H, 2 NH2), 7.60 (d, 2H,
J = 8.8 Hz, Ar–H), 7.64 (d, 2H, J = 8.5 Hz, Ar–H), 7.96
(d, 2H, J = 9.0 Hz, Ar–H), 8.15 (d, 2H, J = 8.8 Hz, Ar–
H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 27.01
(CH3), 29.55 (CH3), 32.53 (C), 33.36 (CH), 41.41 (CH2),
49.85 (CH2), 112.49 (C), 123.77 (CH), 127.65 (CH),
129.43 (CH), 130.86 (CH), 140.80 (C), 145.88 (C), 146.17
(C), 151.07 (C), 153.95 (C), 158.70 (C), 195.53 (C) ppm;
4-[9-(4-Bromophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl]-N-
(diaminomethylene)benzenesulfonamide
(8, C30H33BrN4O4S)
1
Yellow solid; 76 % yield; m.p.: 240 °C (EtOH/water); H
NMR (400 MHz, DMSO-d6): d = 0.72 (s, 6H, 2 CH3), 0.88
(s, 6H, 2 CH3), 1.77 (d, 2H, J = 16.0 Hz, CH2), 2.02 (d, 2H,
J = 16.0 Hz, CH2), 2.21 (d, 4H, J = 16.0 Hz, 2 CH2), 5.02
(s, 1H, CH), 7.13 (s, 4H, 2 NH2), 7.28 (d, 2H, J = 4.0 Hz, Ar–
H), 7.45 (d, 2H, J = 8.0 Hz, Ar–H), 7.58 (d, 2H, J = 4.0 Hz,
Ar–H), 8.15 (d, 2H, J = 8.0 Hz, Ar–H) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 26.11 (CH3), 29.13 (CH3), 31.81
(C), 32.04 (CH), 40.89 (CH2), 49.44 (CH2), 112.65 (C),
113.87 (C), 118.82 (C), 127.15 (CH), 129.87 (CH), 130.33
(CH), 130.81 (CH), 140.44 (C), 145.29 (C), 150.08 (C),
ꢀ
FT–IR: m ¼ 3,367 and 3,334 (NH2), 2,954 (C–H), 1,639
(C=O), 1,555 and 1,513 (C=C), 1,336 and 1,218 (SO2)
cm-1; MS (ESI): m/z = 592 ([M ? 1]?).
N-(Diaminomethylene)-4-[3,3,6,6-tetramethyl-9-
(3-nitrophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl]benzenesulfonamide
(6, C30H33N5O6S)
ꢀ
158.19 (C), 195.09 (C) ppm; FT–IR: m ¼ 3,446 and 3,371
(NH2), 2,957 (C–H), 1,637 (C=O), 1,540 (C=C), 1,360 and
1,221 (SO2) cm-1; MS (ESI): m/z = 625 ([M ? 1]?).
1
Yellow solid; 77 % yield; m.p.: 295 °C (EtOH/water); H
NMR (300 MHz, DMSO-d6): d = 0.71 (s, 6H, 2 CH3) 0.89
(s, 6H, 2 CH3), 1.81 (d, 2H, J = 17.4 Hz, CH2), 2.02 (d,
2H, J = 16.1 Hz, CH2), 2.22 (d, 4H, J = 17.3 Hz, 2 CH2),
5.13 (s, 1H, CH), 6.85 (s, 4H, 2 NH2), 7.61 (d, 2H,
J = 7.8 Hz, Ar–H), 7.79 (d, 2H, J = 7.7 Hz, Ar–H), 8.00
(t, 3H, J = 8.5 Hz, Ar–H), 8.13 (s, 1H, Ar–H) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 26.53 (CH3), 26.56
(CH3), 32.59 (C), 32.96 (CH), 41.28 (CH2), 49.82 (CH2),
112.79 (C), 121.53 (CH), 123.05 (CH), 127.73 (CH),
130.00 (CH), 130.23 (CH), 134.71 (CH), 140.77 (C),
145.91 (C), 147.87 (C), 148.55 (C), 151.13 (C), 158.69 (C),
N-(Diaminomethylene)-4-[9-(2,4-dichlorophenyl)-3,3,6,6-
tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-
10(9H)-yl]benzenesulfonamide (9, C30H32Cl2N4O4S)
1
Yellow solid; 75 % yield; m.p.: 250 °C (EtOH/water); H
NMR (300 MHz, DMSO-d6): d = 0.74 (s, 6H, 2 CH3), 0.86
(s, 6H, 2 CH3), 1.71 (d, 2H, J = 17.3 Hz, CH2), 1.94 (d, 2H,
J = 16.0 Hz, CH2), 2.11 (m, 4H, 2 CH2), 5.19 (s, 1H, CH),
6.84 (s, 4H, 2 NH2), 7.32 (d, 2H, J = 10.3 Hz, Ar–H), 7.40
(s, 1H, Ar–H), 7.51 (d, 1H, J = 8.4 Hz, Ar–H), 7.58 (d, 2H,
J = 8.1 Hz, Ar–H), 7.95 (d, 1H, J = 8.1 Hz, Ar–H) ppm;
13C NMR (75 MHz, DMSO-d6): d = 26.49 (CH3), 29.63
(CH3), 32.34 (C), 33.37 (CH), 41.63 (CH2), 49.87 (CH2),
111.99 (C), 126.99 (CH), 127.20 (CH), 127.68 (CH), 131.05
(CH), 131.39 (C), 133.96 (C), 134.46 (CH), 141.02 (C),
142.49 (C), 145.79 (C), 151.09 (C), 158.69 (C), 195.40 (C)
ꢀ
195.64 (C) ppm; FT–IR: m ¼ 3,458 and 3,414 (NH2), 2,959
(C–H), 1,636 (C=O), 1,557 and 1,521 (C=C), 1,360 and
1,221 (SO2) cm-1; MS (ESI): m/z = 592 ([M ? 1]?).
N-(Diaminomethylene)-4-[9-(4-fluorophenyl)-3,3,6,6-
tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-
10(9H)-yl]benzenesulfonamide (7, C30H33FN4O4S)
ꢀ
ppm; FT–IR: m ¼ 3,451 and 3,334 (NH2), 2,960 (C–H),
1,626 (C=O), 1,541 (C=C), 1,360 and 1,223 (SO2) cm-1
;
MS (ESI): m/z = 615 ([M ? 1]?).
Yellow solid; 75 % yield; m.p.: 290–291 °C (EtOH/
water); 1H NMR (400 MHz, DMSO-d6): d = 0.73 (s,
6H, 2 CH3), 0.89 (s, 6H, 2 CH3), 1.75 (d, 2H,
J = 16.0 Hz, CH2), 2.02 (d, 2H, J = 16.0 Hz, CH2),
2.20 (d, 4H, J = 16.0 Hz, 2 CH2), 5.05 (s, 1H, CH),
6.88 (s, 4H, 2 NH2), 7.07 (t, 2H, J = 8.0 Hz, Ar–H),
7.35-7.45 (m, 2H, Ar–H), 7.60 (d, 2H, J = 8.0 Hz, Ar–
4-[9-(5-Bromo-2-hydroxyphenyl)-3,3,6,6-tetramethyl-1,8-
dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl]-N-
(diaminomethylene)benzenesulfonamide
(10, C30H33BrN4O5S)
1
Yellow solid; 74 % yield; m.p.: 312 °C (EtOH/water); H
NMR (300 MHz, DMSO-d6): d = 0.73 (s, 6H, 2 CH3),
123

˙
I. Yes¸ildag et al.
˘
0.88 (s, 6H, 2 CH3), 1.67 (d, 2H, J = 17.4 Hz, CH2), 1.98
(d, 2H, J = 16.0 Hz, CH2), 2.23 (m, 4H, 2 CH2), 4.92 (s,
1H, CH), 6.85 (s, 4H, 2 NH2), 6.94 (d, 2H, J = 8.0 Hz, Ar–
H), 7.03 (s, 1H, Ar–H), 7.10 (d, 1H, J = 8.0 Hz, Ar–H),
7.30 (d, 2H, J = 8.0 Hz, Ar–H), 7.95 (s, 1H, J = 8.0 Hz,
Ar–H), 10.61 (s, 1H, OH) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 29.74 (CH3), 31.43 (CH3), 32.08 (CH),
32.27 (C), 32.46 (CH2), 41.47 (CH2), 110.90 (C), 118.22
(CH), 127.58 (CH), 128.74 (C), 129.97 (CH), 131.02 (CH),
133.45 (C), 134.24 (CH), 141.53 (C), 145.69 (C), 151.63
N-(Diaminomethylene)-4-[9-(4-ethylphenyl)-3,3,6,6-tetra-
methyl-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-
yl]benzenesulfonamide (13, C32H38N4O4S)
1
Yellow solid; 75 % yield; m.p.: 180 °C (EtOH/water); H
NMR (400 MHz, DMSO-d6): d = 0.74 (s, 6H, 2 CH3),
0.89 (s, 6H, 2 CH3), 1.02–1.05 (m, 2H, CH2), 1.15 (t, 3H,
J = 8.0 Hz, CH3), 1.75 (d, 2H, J = 20.0 Hz, CH2), 2.02
(d, 2H, J = 16.0 Hz, CH2), 2.20 (d, 4H, J = 20.0 Hz, 2
CH2), 5.02 (s, 1H, CH), 6.87 (s, 4H, 2 NH2), 7.09 (d, 2H,
J = 8.0 Hz, Ar–H), 7.23 (d, 2H, J = 8.0 Hz, Ar–H), 7.57
(d, 2H, J = 8.0 Hz, Ar–H), 7.97 (d, 2H, J = 8.0 Hz, Ar–
H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 15.45
(CH3), 26.14 (CH3), 27.70 (CH2), 29.17 (CH), 31.42
(CH3), 32.04 (C), 40.90 (CH2), 49.55 (CH2), 113.27 (C),
127.28 (CH), 127.95 (CH), 128.57 (CH), 129.70 (CH),
140.61 (C), 143.44 (C), 145.22 (C), 149.70 (C), 158.18
ꢀ
(C), 155.70 (C), 158.68 (C), 196.15 (C) ppm; FT–IR: m ¼
3,448 and 3,346 (NH2), 2,958 (C–H), 1,617 (C=O), 1,542
(C=C), 1,370 and 1,234 (SO2) cm-1; MS (ESI): m/z = 641
([M ? 1]?).
N-(Diaminomethylene)-4-[9-(4-hydroxyphenyl)-3,3,6,6-
tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-
10(9H)-yl]benzenesulfonamide (11, C30H34N4O5S)
ꢀ
(C), 162.77 (C), 195.10 (C) ppm; FT–IR: m ¼ 3,427 and
3,337 (NH2), 2,957 (C–H), 1,623 (C=O), 1,537 (C=C),
1,361 and 1,221 (SO2) cm-1; MS (ESI): m/z = 575
([M ? 1]?).
Yellow solid; 76 % yield; m.p.: 225 °C (EtOH/water);
1H NMR (300 MHz, DMSO-d6): d = 0.73 (s, 6H, 2
CH3), 0.88 (s, 6H, 2 CH3), 1.71 (d, 2H, J = 17.3 Hz,
CH2), 2.00 (d, 2H, J = 16.0 Hz, CH2), 2.18 (d, 4H,
J = 16.7 Hz, 2 CH2), 4.92 (s, 1H, CH), 6.84 (s, 4H, 2
NH2), 6.61 (d, 2H, J = 8.5 Hz, Ar–H), 7.08 (d, 2H,
J = 8.5 Hz, Ar–H), 7.54 (d, 2H, J = 8.5 Hz, Ar–H),
7.94 (d, 2H, J = 8.5 Hz, Ar–H), 9.09 (s, 1H, OH) ppm;
13C NMR (75 MHz, DMSO-d6): d = 26.56 (CH3), 29.70
(CH3), 31.24 (CH), 32.31 (C), 41.40 (CH2), 50.08 (CH2),
114.05 (C), 115.13 (CH), 127.65 (CH), 128.88 (CH),
130.80 (CH), 137.27 (C), 141.17 (C), 145.67 (C), 149.89
(C), 155.78 (C), 158.67 (C), 195.60 (C) ppm; FT–IR:
N-(Diaminomethylene)-4-[3,3,6,6-tetramethyl-9-[4-
(methylthio)phenyl]-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl]benzenesulfonamide
(14, C31H36N4O4S2)
Yellow solid; 76 % yield; m.p.: 229–230 °C (EtOH/water);
1H NMR (400 MHz, DMSO-d6): d = 0.74 (s, 6H, 2 CH3),
0.89 (s, 6H, 2 CH3), 1.74 (d, 2H, J = 17.0 Hz, CH2), 2.02
(d, 2H, J = 16.0 Hz, CH2), 2.21 (d, 4H, J = 19.0 Hz, 2
CH2), 2.43 (s, 3H, -SCH3), 5.00 (s, 1H, CH), 6.89 (s, 4H, 2
NH2), 7.13 (t, 2H, J = 10.0 Hz, Ar–H), 7.27 (d, 2H,
J = 8.0 Hz, Ar–H), 7.59-7.70 (m, 2H, Ar–H), 7.97 (d, 2H,
J = 8.0 Hz, Ar–H) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 14.66 (CH3), 26.13 (CH3), 29.17 (CH3), 31.50 (CH),
32.04 (C), 40.91 (CH2), 49.50 (CH2), 113.01 (C), 123.53
(CH), 125.57 (CH), 127.18 (CH), 128.22 (CH), 135.01 (C),
140.56 (C), 142.93 (C), 145.24 (C), 149.82 (C), 158.18 (C),
ꢀ
m ¼ 3,435 and 3,321 (NH2), 2,955 (C–H), 1,622 (C=O),
1,527 (C=C), 1,365 and 1,223 (SO2) cm-1; MS (ESI):
m/z = 563 ([M ? 1]?).
N-(Diaminomethylene)-4-[3,3,6,6-tetramethyl-1,8-dioxo-9-
(p-tolyl)-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl]-
benzenesulfonamide (12, C31H36N4O4S)
ꢀ
195.11 (C) ppm; FT–IR: m ¼ 3,435 and 3,336 (NH2), 2,958
1
Yellow solid; 76 % yield; m.p.: 191 °C (EtOH/water); H
(C–H), 1,624 (C=O), 1,489 (C=C), 1,363 and 1,223 (SO2)
cm-1; MS (ESI): m/z = 593 ([M ? 1]?).
NMR (400 MHz, DMSO-d6): d = 0.74 (s, 6H, 2 CH3),
0.89 (s, 6H, 2 CH3), 1.74 (d, 2H, J = 16.0 Hz, CH2), 2.01
(d, 2H, J = 16.0 Hz, CH2), 2.19 (d, 4H, J = 20.0 Hz, 2
CH2), 2.23 (s, 3H, CH3), 5.02 (s, 1H, CH), 6.85 (s, 4H, 2
NH2), 7.05 (d, 2H, J = 8.0 Hz, Ar–H), 7.21 (d, 2H,
J = 8.0 Hz, Ar–H), 7.57 (d, 2H, J = 8.0 Hz, Ar–H), 7.97
(d, 2H, J = 8.0 Hz, Ar–H) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 20.06 (CH3), 26.07 (CH3), 29.21 (CH),
31.41 (CH3), 32.03 (C), 40.91 (CH2), 49.54 (CH2), 113.25
(C), 127.20 (CH), 127.44 (CH), 128.51 (CH), 130.32
(CH), 134.65 (C), 140.62 (C), 143.19 (C), 145.21 (C),
N-(Diaminomethylene)-4-[9-(4-methoxyphenyl)-3,3,6,6-
tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-
10(9H)-yl]benzenesulfonamide (15, C31H36N4O5S)
1
Yellow solid; 75 % yield; m.p.: 203 °C (EtOH/water); H
NMR (300 MHz, DMSO-d6): d = 0.72 (s, 6H, 2 CH3),
0.87 (s, 6H, 2 CH3), 1.71 (d, 2H, J = 16.1 Hz, CH2), 1.99
(d, 2H, J = 16.0 Hz, CH2), 2.17 (d, 4H, J = 17.4 Hz, 2
CH2), 3.67 (s, 3H, OCH3), 4.98 (s, 1H, CH), 6.85 (s, 4H, 2
NH2), 6.79 (d, 2H, J = 8.5 Hz), 7.20 (d, 2H, J = 8.5 Hz,
Ar–H), 7.54 (d, 2H, J = 7.8 Hz, Ar–H), 7.94 (d, 2H,
J = 8.5 Hz, Ar–H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 26.59 (CH3), 29.15 (CH3), 30.75 (CH), 32.51 (C),
41.40 (CH2), 50.05 (CH2), 55.31 (OCH3), 113.72 (CH),
ꢀ
149.68 (C), 158.17 (C), 195.08 (C) ppm; FT–IR: m ¼
3,455 and 3,375 (NH2), 2,928 (C–H), 1,638 (C=O), 1,164
(C=C), 1,361 and 1,222 (SO2) cm-1
; MS (ESI):
m/z = 561 ([M ? 1]?).
123

One-pot synthesis of acridine sulfonamide derivatives
113.89 (C), 127.65 (CH), 128.98 (CH), 130.79 (CH),
138.89 (C), 141.12 (C), 145.72 (C), 150.02 (C), 157.77 (C),
plasma was removed. Later, red cells were washed with
NaCl (0.9 %), and the erythrocytes were hemolyzed with
1.5 volumes of ice-cold water. Cell membranes were
removed by centrifugation at 4 °C, 20,000 rpm for
30 min. The pH of hemolysate was adjusted to 8.7 with
solid TRIS [tris(hydroxymethyl)aminomethane]. The
hemolysate was applied to an affinity column (Sephar-
ose-4B–L-tyrosine-p-aminobenzene sulfonamide) and
equilibrated with 25 lM Tris–HCl/0.1 M Na2SO4 (pH
8.7). The affinity gel was washed with solution of 25 lM
Tris–HCl/22 mM Na2SO4 (pH 8.7). The hCA I and hCA
II isoenzymes were diluted with a solution of 1 M NaCl/
25 lM Na2HPO4 (pH 6.3) and 0.1 M NaCH3COO/0.5 M
NaClO4 (pH 5.6) [18]. For protein content estimation,
the Bradford method was used with bovine serum albu-
ꢀ
158.68 (C), 195.56 (C) ppm; FT–IR: m ¼ 3,415 and 3,305
(NH2), 2,956 (C–H), 1,635 (C=O), 1,542 (C=C), 1,362 and
1,229 (SO2) cm-1; MS (ESI): m/z = 577 ([M ? 1]?).
N-(Diaminomethylene)-4-[9-(2,4-dimethoxyphenyl)-
3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl]benzenesulfonamide
(16, C32H38N4O6S)
1
Yellow solid; 74 % yield; m.p.: 223 °C (EtOH/water); H
NMR (300 MHz, DMSO-d6): d = 0.71 (s, 6H, 2 CH3),
0.86 (s, 6H, 2 CH3), 1.69 (d, 2H, J = 17.1 Hz, CH2), 1.91
(d, 2H, J = 16.2 Hz, CH2), 2.06 (d, 4H, J = 12.0 Hz, 2
CH2), 3.69 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 5.06 (s, 1H,
CH), 6.85 (s, 4H, 2 NH2), 6.41 (t, 2H, J = 9.7 Hz, Ar–H),
7.21 (d, 1H, J = 8.5 Hz, Ar–H), 7.54 (d, 2H, J = 8.5 Hz,
Ar–H), 7.96 (d, 2H, J = 8.5 Hz, Ar–H) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 26.04 (CH3), 29.86 (CH3),
31.16 (CH), 32.44 (C), 41.79 (CH2), 50.12 (CH2), 55.34
(OCH3), 56.02 (OCH3), 98.66 (CH), 104.32 (CH), 112.27
(C), 125.51 (CH), 127.58 (CH), 130.90 (CH), 133.08 (C),
141.63 (C), 145.45 (C), 150.17 (C), 151.38 (C), 158.67 (C),
min as
a standard [19]. SDS–polyacrylamide gel
electrophoresis was performed after the purification of
the enzyme [20].
Determination of hydratase and esterase activities of hCA I
and hCA II
The CO2 hydratase activity of the enzyme was determined
at 0 °C in a veronal buffer (pH 8.15) with pH–stat method
as indicator and saturated carbon dioxide solution as sub-
strate in a final volume of 4.2 cm3. The time (in seconds)
taken for the solution to change from pH 8.15 to pH 6.5
was measured by the pH meter. The enzyme unit (EU) is
the amount of enzyme that reduces the non-enzymatic
reaction time by 50 %. The activity of an enzyme unit was
calculated by using the equation (t0 - tc/tc), where t0 and tc
are times for pH change of the non-enzymatic and enzy-
matic reactions, respectively [21].
ꢀ
159.15 (C), 195.36 (C) ppm; FT–IR: m ¼ 3,401 and 3,322
(NH2), 2,955 (C–H), 1,632 (C=O), 1,497 (C=C), 1,363 and
1,228 (SO2) cm-1; MS (ESI): m/z = 605 ([M - 1]-).
N-(Diaminomethylene)-4-[9-(3,4-dimethoxyphenyl)-
3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl]benzenesulfonamide
(17, C32H38N4O6S)
1
Yellow solid; 74 % yield; m.p.: 215 °C (EtOH/water); H
NMR (300 MHz, DMSO-d6): d = 0.78 (s, 6H, 2 CH3),
0.91 (s, 6H, 2 CH3), 1.73 (d, 2H, J = 17.3 Hz, CH2), 2.02
(d, 2H, J = 16.1 Hz, CH2), 2.20 (d, 4H, J = 18.0 Hz, 2
CH2), 3.69 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 5.00 (s, 1H,
CH), 6.85 (s, 4H, 2 NH2), 6.83 (d, 3H, J = 6.5 Hz, Ar–H),
7.51 (d, 2H, J = 7.6 Hz, Ar–H), 7.96 (d, 2H, J = 8.7 Hz,
Ar–H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 26.52
(CH3), 29.71 (CH3), 31.52 (CH), 32.52 (C), 41.42 (CH2),
50.04 (CH2), 55.74 (OCH3), 55.83 (OCH3), 111.69 (CH),
111.87 (CH), 113.75 (C), 119.85 (CH), 127.73 (CH),
130.69 (CH), 139.17 (C), 141.06 (C), 145.70 (C), 147.39
(C), 148.60 (C), 150.15 (C), 158.66 (C), 195.69 (C) ppm;
Esterase activities of hCA I and hCA II isoenzymes
were determined by hydrolysis of p-nitrophenyl acetate.
The change of absorbance was determined at 348 nm after
3 min [22].
Determination of IC50 values of the compounds
The IC50 values of inhibitors were determined by moni-
toring the hydrolysis of p-nitrophenyl acetate in the
presence of the CA isoenzymes and various concentrations
of inhibitors via the change in absorbance at 348 nm after
3 min [22]. Regression analysis graphs were drawn by
plotting inhibitor concentrations versus enzyme activity
and the IC50 values were determined.
ꢀ
FT–IR: m ¼ 3,443 and 3,344 (NH2), 2,954 (C–H), 1,643
(C=O), 1,464 (C=C), 1,363 and 1,263 (SO2) cm-1; MS
(ESI): m/z = 605 ([M - 1]-).
Statistical analysis
CA inhibition assay
All the presented data were confirmed in at least three inde-
pendent experiments and are expressed as the
mean standard deviation (SD). Data were analyzed by
using a one-way analysis of variance for multiple compari-
sons. P \0.005 was considered to be statistically significant.
Purification of carbonic anhydrase I and II isoenzymes
from human erythrocytes
Erythrocytes were purified from human blood. The blood
samples were centrifuged at 1,500 rpm for 20 min and
123

˙
I. Yes¸ildag et al.
˘
Acknowledgments This research was financed by Dumlupınar
University Research Fund (Grant No. 2012-38) as well as by the
Dumlupınar University ILTEM research laboratory. The authors are
grateful to Prof. Dr. Yılmaz Yıldırır, Gazi University, Ankara,
Turkey.
8. Scozzafava A, Banciu MD, Popescu A, Supuran CT (2000) J
Enzyme Inhib Med Chem 15:443
9. Schuman JS (2000) Clin Ther 22:167
´
10. Brzozowski Z, Sławinski J, Innocenti A, Supuran CT (2010) Eur
J Med Chem 45:3656
¨
11. Balaydın HT, S¸entu¨rk M, Goksu S, Menzek A (2012) Eur J Med
Chem 54:423
12. Kaya M, Yıldırır Y, Tu¨rker L (2009) J Heterocycl Chem 46:294
13. Kaya M, Yıldırır Y, Celik GY (2011) Med Chem Res 20:293
References
˙
14. Ulus R, Yes¸ildag I, Tanc¸ M, Bu¨lbu¨l M, Kaya M, Supuran CT
˘
(2013) Bioorg Med Chem 21:5799
1. Winum JY, Vullo D, Casini A, Montero JL, Scozzafava A,
Supuran CT (2003) J Med Chem 46:2197
¨ ˙
2. S¸entu¨rk M, Gu¨lc¸in I, Beydemir S¸, Ku¨frevioglu OI, Supuran CT
15. Kaya M, Basar E, Colak F (2012) Med Chem Res 27:509
˙
˙
˘
16. Ilkimen H, Yenikaya C, Sarı M, Bu¨lbu¨l M, Tunca E, Su¨zen Y
(2013) Polyhedron 61:56
(2011) Chem Biol Drug Des 77:494
¨
˙
3. Sarikaya SBO, Topal F, S¸entu¨rk M, Gu¨lc¸in I, Supuran CT (2011)
Bioorg Med Chem 21:4259
17. Kaya M, Bas¸ar E, C¸ akır E, Tunca E, Bu¨lbu¨l M (2012) J Enzyme
Inhib Med Chem 27:509
18. Rickli EE, Ghazanfar SA, Gibbons BH, Edsall JT (1964) J Biol
Chem 239:1065
˙
˘
¨
¨
¨
4. Akıncıoglu A, Akbaba Y, Goc¸er H, Goksu S, Gulc¸in I, Supuran
CT (2013) Bioorg Med Chem 21:1379
5. Supuran CT, Scozzafava A (2002) Exp Opin Ther Pat 12:217
6. Di Fiore A, Scozzafava A, Winum JY, Montero JL, Pedone C,
Supuran CT, De Simone G (2007) Bioorg Med Chem Lett
17:1726
7. Innocenti A, Vullo D, Pastorek J, Scozzafava A, Pastorekova S,
Nishimori I, Supuran CT (2007) Bioorg Med Chem Lett 17:1532
19. Bradford MM (1976) Anal Biochem 72:248
20. Laemmli UK (1970) Nature 227:680
21. Wilbur KM, Anderson NG (1948) J Biol Chem 176:147
22. Landolfi C, Marchetti M, Ciocci G, Milanese CJ (1997) Phar-
macol Toxicol Methods 38:169
123