Synthesis of novel acridine and bis acridine sulfonamides with effective inhibitory activity against the cytosolic carbonic anhydrase isoforms II and VII

Article abstract of DOI:10.1016/j.bmc.2013.07.014

4-Amino-N-(4-sulfamoylphenyl)benzamide was synthesized by reduction of 4-nitro-N-(4-sulfamoylphenyl)benzamide and used to synthesize novel acridine sulfonamide compounds, by a coupling reaction with cyclic-1,3-diketones and aromatic aldehydes. The new com

Full text of DOI:10.1016/j.bmc.2013.07.014

Bioorganic & Medicinal Chemistry 21 (2013) 5799–5805
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel acridine and bis acridine sulfonamides
with effective inhibitory activity against the cytosolic carbonic
anhydrase isoforms II and VII
a
a
b
a
a,
⇑
_
Ramazan Ulus , Ibrahim Yeßsildag˘ , Muhammet Tanç , Metin Bülbül , Muharrem Kaya
,
Claudiu T. Supuran b,
⇑
a Chemistry Department, Faculty of Arts and Science, Dumlupınar University, 43100 Kütahya, Turkey
b Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Amino-N-(4-sulfamoylphenyl)benzamide was synthesized by reduction of 4-nitro-N-(4-sulfamoylphe-
nyl)benzamide and used to synthesize novel acridine sulfonamide compounds, by a coupling reaction
with cyclic-1,3-diketones and aromatic aldehydes. The new compounds were investigated as inhibitors
of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely the cytosolic isoforms
hCA I, II and VII. hCA I was inhibited in the micromolar range by the new compounds (KIs of 0.16–
Received 19 May 2013
Revised 30 June 2013
Accepted 8 July 2013
Available online 17 July 2013
9.64 lM) whereas hCA II and VII showed higher affinity for these compounds, with KIs in the range of
Keywords:
Carbonic anhydrase
Acridine
15–96 nM for hCA II, and of 4–498 nM for hCA VII. The structure–activity relationships for the inhibition
of these isoforms with the acridine–sulfonamides reported here were also elucidated.
Ó 2013 Elsevier Ltd. All rights reserved.
Sulfonamide
Enzyme inhibition
Isoforms CA I, II and VII
1. Introduction
2. Results and discussion
Sulfonamide/sulfamate represents the main class of inhibitors
of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1).1–3 CA
inhibitors (CAIs) are in clinical use for decades as antiglaucoma,4
diuretic,5 antiobesity,6 and antitumor agents,7 targeting diverse
of the various 16 CA isoforms known to date in humans.1–3 Re-
cently, targeting CAs from parasites (bacteria, fungi and/or proto-
zoa among others) with specific inhibitors was proposed as an
alternative for designing antiinfective agents with a new mecha-
nism of action.8 There are several clinically used sulfonamides/sul-
famates which indiscriminately inhibit most of the mammalian CA
isoforms in the nanomolar range, and for this reason such drugs
possess significant side effects.1–3 There is thus a constant search
for new types of such derivatives with improved efficacy for the
different CA isoforms.9,10
2.1. Chemistry
The rationale of this work was to incorporate the polycyclic
acridine moiety in the tails9,10 of aromatic sulfonamides, as it has
been reported recently that these are the moieties mainly respon-
sible for effective inhibitory action as well as differentiation be-
tween the many CA isoforms, in inducing selectivity.11–13 The
following chemistry has been employed to achieve this goal
(Schemes 1 and 2)
The general synthetic method shown in Scheme 1 was used to
prepare the sulfonamide containing benzamide derivatives 3, 4.
The nitro derivative 3 was synthesized in THF at room temperature
in the presence of triethylamine (TEA).14 Compound 4 was then ob-
tained by reduction reaction of the nitro derivative 3 in the pres-
ence of aqueous sodium poly-sulfide.14c
The general synthetic methods shown in Schemes 1 and 2 were
employed to prepare the acridine sulfonamide derivatives 7–23
and the bis acridine sulfonamides 24 and 25, as reported earlier
for similar derivatives by one of these groups.15 The synthesis of
acridine sulfonamide compounds were realized in water in a single
process through three successive reactions (Aldol condensation,
Michael addition and cyclization) and using a phase transfer cata-
lyst-Bronsted acid as p-dodecylbenzenesulphonic acid (DBSA).
⇑
Corresponding authors. Tel.: +90 274 2652051; fax: +90 274 2652056 (M.K.);
tel.: +39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
E-mail addresses: mhrkaya@yahoo.com (M. Kaya), claudiu.supuran@unifi.it (C.T.
Supuran).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.014

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R. Ulus et al. / Bioorg. Med. Chem. 21 (2013) 5799–5805
NO2
NH2
All spectral data of compounds 7–25 are in agreement with
the assigned structures (see Experimental for details). The infra-
red (IR) spectra of all the dioxoacridine sulfonamide compounds
showed sharp peaks for the carbonyl groups in region between
1690 and 1625 cmꢀl. The compounds 8, 9 and 22 of belong to
cyano group vibration peak was observed 2228, 2240 and
2228 cmꢀ1, respectively. Besides, in the IR spectra of the com-
pounds, aliphatic C–H stretching bands at 2962–2934 cmꢀ1 and
aromatic C–H stretching bands at 3097–3036 cmꢀ1 were ob-
served. The NH2 vibrations of acridine sulfonamide compounds
were observed in the region between 3354 and 3183 cmꢀ1. The
1H NMR spectra of compounds 7–21 showed singlet peaks that
belong to protons of the methyl groups in position 3 and 6 be-
tween 0.70 and 0.94 ppm. The CH2 group protons of the cyclo-
hexene rings of the compounds 7–21 showed doublet peaks in
the 1.75–2.29 ppm range and compounds 22 and 23 showed
multiple peaks in the 1.60–2.30 ppm. The compound 17 showed
singlet peak that belongs to protons of the methyl groups
2.87 ppm. The compound 18 showed singlet peak that belongs
to protons of the methyl group 2.23 ppm. The compound 19 ob-
served belongs to protons of ethyl group triplet peak (3H) at
1.14 ppm and multiple peaks(2H) at 2.49–2.57 ppm. Signals for
the methoxy group protons for compounds 16 and 21 were
shown in the range of 3.70–3.71 ppm. The signals for the CH
protons at 4.90–5.30 ppm and signals for the aromatic protons
in the range of 6.62–8.19 ppm were observed. Hydroxyl group
proton of compound 15 and 21 were observed as broad signal
at 8.82–9.03 ppm. The broad singlet peaks between 6.62 and
7.34 ppm were assigned to sulfonamide (ꢀSO2NH2) groups pro-
tons of the compounds 7–25. Signals of –NH groups protons
for the compounds 7–25 showed in the range of 10.73–
13.40 ppm.
NH2
NO2
THF, rt
TEA
Na2S.9H2O,S8
EtOH-H2O
HN
O
O
HN
O
S
O
Cl
O
NH2
2
1
O
O
S
O
S
O
NH2
4
NH2
3
NH2
O
S
O
O
NH
NH2
O
O
O
O
S
R2
R1
R2
R1
N
DBSA
R1 R2
5
H2O, reflux
+
O
HN
O
O
R3
O
R5
R4
R4
R3
R5
NH2
4
6a-t
7-23
Scheme 1. Synthesis of sulfonamides (containing benzamide derivatives 3, 4 and
acridine moieties incorporating 7–23).
2.2. Carbonic anhydrase inhibition
The new compounds reported here and the standard drug acet-
azolamide were assayed as inhibitors of three cytosolic human iso-
forms, hCA I, II and VII (Table 1).16 As seen from data of Table 1,
these sulfonamides show effective inhibitory activity against all
three tested isoforms. The following structure activity relationship
(SAR) could be observed:
The slow cytosolic isoform hCA I was inhibited with inhibition
constants in the micromolar range by most of the newly synthe-
sized sulfonamides 7–25, which showed KIs of 0.16–9.64 lM (Ta-
Sulfonamide compounds were prepared by one pot reaction in pro-
cessing high yields and simple work-up procedure. Amino-deriva-
tive 4 was condensed with substituted 1,3-diketones 5 and
substituted benzaldehydes 6, in the molar range of 1:2:1, at reflux,
leading to the acridine-incorporating compounds 7–23 (Scheme 1).
By changing the nature of the substituents present in the diketone
and/or aldehyde components, a rather large chemical diversity can
be incorporated in the acridine sulfonamides reported here
(Scheme 1 and Table 1). Indeed, 5,5-dimethyl-substituted 1,3-
cyclohexanedione and 1,3-cyclohexanedione have been employed
in the synthesis, together with substituted benzaldehydes incorpo-
rating a range of substituents in the ortho-, meta and para positions
with respect to the CHO moiety. These various substituents in-
cluded cyano, nitro, halogen (F, Cl, Br), methoxy, dimethylamino,
methyl, ethyl and formyl moieties (Table 1).
ble 1). Compound 7, possessing no substituent (except hydrogen)
at the part of the molecule originally found in the aldehyde compo-
nent, was the best hCA I inhibitor (KIs 0.16
compound possessing OH and methyl moieties in the aromatic part
of the molecule (21, KIs of 0.26 M). The remaining derivatives
were less effective as hCA I inhibitors, with KIs of 1.58–9.64 M.
lM) together with the
l
l
The moiety which seems to influence significantly the enzyme
inhibitory activity is the one coming from the aldehyde component
in the synthesis of these derivatives. Indeed, both the positions of
the substituents R3–R4, as well as their nature, influence
By working at molar ratios of 4: 1,3-diketone: 1,4-benzene-dial-
dehyde or 1,3-benzene-dialdehyde of 2:2:1, the bis acridine disulf-
onamides 24, 25 were prepared (Scheme 2).
O
O
H
O
N
N
N
H
N
S
H2N
O
O
O
O
O
S
NH2
O
O
24, 25
Scheme 2. Structures of the bis acridine sulfonamide derivatives (24 and 25).

R. Ulus et al. / Bioorg. Med. Chem. 21 (2013) 5799–5805
5801
Table 1
Inhibition data of sulfonamides 7–25 and acetazolamide (AAZ, as standard drug) against hCA I, hCA II and hCA VII isoenzymes by a stopped flow CO2 hydrase assay
Inhibitor
R1
R2
R3
R4
R5
Ki valuesa
(lM)
CA I
CA II
CA VII
AAZ
7
8
0.25
0.16
4.26
1.58
7.34
3.51
6.22
8.85
8.26
2.13
9.64
9.49
5.33
1.89
9.34
0.26
2.14
3.47
4.33
0.85
0.012
0.021
0.047
0.015
0.029
0.023
0.041
0.023
0.046
0.049
0.065
0.096
0.059
0.051
0.023
0.033
0.038
0.067
0.053
0.030
0.028
0.004
0.055
0.065
0.199
0.041
0.245
0.368
0.264
0.152
0.127
0.498
0.334
0.225
0.375
0.005
0.014
0.022
0.142
0.046
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
OH
H
Cl
H
H
CN
H
NO2
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
CN
H
NO2
H
F
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Cl
Br
OH
OCH3
N(CH3)2
CH3
C2H5
Cl
H
CN
Cl
H
H
1,4-Phenylene bis acridine sulfonamide
1,3-Phenylene bis acridine sulfonamide
a
Mean from 3 different assays. Errors were in the range of 10% of the reported data (not shown).
significantly the inhibition of hCA I. For example compounds pos-
sessing such a group in the meta position (9, 11 and 21) have a
good inhibitory activity, whereas such a group in the para position,
such as in 8 as in 12, 13, 14, 16 and 17, led to less effective inhib-
itors. However some p-substituted compounds, such as 15, 19 and
22 showed effective hCA I inhibition, as the meta-substituted ones
mentioned above (Table 1).
hCA II, the physiologically dominant CA isoform was also effec-
tively inhibited by the new sulfonamides 7–25, with KIs in the range
of 15–96 nM. In this case the SAR shows a more compact behavior, of
potent inhibitor for most of the synthesized compounds. The best
hCA II inhibitors in this series were derivatives 7, 9, 11, 13, and 20
which showed KIs of 15–23 nM. They incorporate methyl moiety
in the part of the molecule coming from the 1,3-diketone, as well
as meta- and para- substituents (e.g., no substituent at all apart H
in 7, and cyano, nitro and chloro in the remaining compounds) in
the part of the molecule originary present in the aldehyde compo-
nent for the synthesis of Schemes 1 (Table 1). However, there are
no important differences of activity between these compounds with
the least effective ones (16, 17 and 23) possessing anyhow KIs of 67–
96 nM. Interestingly, the very bulky bis sulfonamides 24 and 25 was
also a rather effective hCA II inhibitor.
were identified. As sulfonamide CAIs have applications in treat-
ment of some diseases, such as glaucoma, obesity and as diuretics,
exploring diverse sulfonamide chemotypes targeting various such
enzymes is of interest for many medicinal chemists. Furthermore,
antimicrobial, antimalarial, antitrypanosomal and anticancer ef-
fects were also recently reported for many CAIs targeting parasite
or human (hCA IX and XII) enzymes.17,18 As shown in a recent spe-
cial issue of this journal,19 at the 80th anniversary of the discovery
of this enzyme, there are still many important drug design aspect
to be addressed for obtaining isoform-selective and more effective
sulfonamide inhibitors.
4. Materials and methods
4.1. Materials
All chemicals and solvents used for the synthesis were spectro-
scopic reagent grade. Melting points were measured on a Bibby
Stuart Scientific apparatus. FT-IR spectra were recorded from a
Bruker Optics, Andrtex 70 FT-IR spectrometer with an ATR dia-
mond crystal. 1H NMR, and 13C NMR spectra were obtained with
a Bruker DPX-300 FT-NMR and Bruker DPX-400 FT-NMR instru-
The brain-associated cytosolic isoform CA VII was inhibited by
sulfonamides 7–25 with KIs in the range of 4–498 nM (Table 1).
SAR is more complicated for the inhibition of this isoform with re-
spect to the other two discussed above. Indeed, compound 7 was a
very effective hCA VII inhibitor (KI of 4 nM) being much more effec-
tive compared to acetazolamide, a clinically used drug.1–3 Other
effective hCA VII inhibitors were 11, 21, 22, and 23, with inhibition
constants in the range of 5–41 nM. These compounds incorporate
3-nitro, 3-methoxy, 4-cyano and 4-chloro moieties in the fragment
of the molecule coming from the aldehyde component. Other sub-
stitution patterns, as those found in the remaining derivatives, led
to a loss of activity, with these derivatives having KIs in the range of
55–498 nM. The bis-sulfonamide 25 was also a rather effective hCA
VII inhibitor.
ment in CDCl3 or DMSO-d6 as
a solvent, at 300 MHz and
400 MHz, respectively. Chemical shifts are expressed in d units
(ppm). The mass analyses were performed on Waters 2695 Alli-
ance Micromass ZQ instrument LC/MS.
4.1.1. General procedure for preparation of 4-nitro-N-(4-
sulfamoylphenyl)benzamide compound (3)
4-Aminobenzenesulfonamide (1.739 g, 10.1 mmol), p-nitro ben-
zoyl chloride (1.856 g, 10 mmol), 3 mL dry triethylamine (TEA) and
in dry 30 mL THF were stirred for 5 h at room temperature. After the
solvent was removed in vacuo and washed with H2O. The raw prod-
uct was purified by recrystallization from ethanol.
4.1.2. General procedure for preparation of 4-amino-N-(4-
sulfamoylphenyl)benzamide compound (4)
3. Conclusions
Na2Sꢁ9H2O (1 mmol) and sulfur (2 mmol) were dissolved by
boiling in 20 ml of water. This solution (sodium poly-sulfur) was
then added dropwise to a stirred and warm solution of compound
3 (1 mmol) in ethanol–water. The progress of the reaction was
A series of novel sulfonamides incorporating acridine and bis
acridine moieties were synthesized and their structural features

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R. Ulus et al. / Bioorg. Med. Chem. 21 (2013) 5799–5805
monitored by TLC. Once the reaction is completed, the mixture was
cooled to room temperature and solid filtered off and washed with
H2O. The sulfonamide product was purified and recrystallized from
the ethanol (90%).
2H, –CH2), 2.22 (d, 4H, 2ꢂ–CH2), 5.03 (s, 1H, –CH), 7.32 (s, 2H, –
NH2), 7.52 (t, 3H, J = 7.5 Hz, Ar-H), 7.61 (d, 2H, J = 8.0 Hz, Ar-H),
7.68 (s, 1H, Ar-H), 7.84 (d, 2H, J = 8.0 Hz, Ar-H), 7.99 (d, 2H,
J = 8.0 Hz, Ar-H), 8.18 (d, 2H, J = 8.0 Hz, Ar-H), 10.79 (s, 1H, –NH);
13C NMR (75 MHz, DMSO-d6) d (ppm): 26.64, 29.51, 32.57, 33.18,
41.47, 49.81, 111.04, 112.65, 119.54, 120.41, 127.06, 127.20,
130.01, 130.25, 130.35, 131.80, 133.11, 135.72, 139.44, 141.60,
142.38, 148.00, 150.99, 165.48, 195.64; IR (cmꢀ1): 3272 w (–NH2),
2959 w (C–H), 2240 (C„N), 1678 and 1642 s (C@O), 1595 m
(C@C), 1361 s and 1222 s (SO2); MS(CI) m/z 648 [M]+ (65%).
As white crystals, (0.25 g, 86%), mp 313 °C (ethanol–water). 1H
NMR (400 MHz, DMSO-d6) d (ppm): 5.85 (s, 2H, –NH2), 6.63 (d,
2H, J = 8.3 Hz, Ar-H), 7.26 (s, 2H, –NH2), 7.77 (t, 4H, J = 8.3 Hz, Ar-
H), 7.95 (d, 2H, J = 8.6 Hz, Ar-H), 10.08 (s, 1H, –NH); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 112.54, 119.39, 120.41, 126.40,
129.58, 137.83, 142.84, 152.50, 165.56; IR (cmꢀ1): 3448 w (–NH)
3382 and 3328 w (–NH2), 3035 w (Ar-H), 2953 w (C–H), 1650 s
(C@O), 1589 and 1500 m (C@C), 1285 s and 1144 s (SO2); MS(CI)
m/z 292 [M+1]+ (100%).
4.1.7. N-(4-Sulfamoylphenyl)-4-(3,3,6,6-tetramethyl-9-(4-
nitrophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-
yl)benzamide (10)
4.1.3. General procedure for preparation of acridine
sulfonamide derivatives (7–23)
As brown crystal, (0.44 g, 66%), mp 237 °C (decomposition) (eth-
anol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.75 (s, 6H, 2ꢂ–
CH3), 0.90 (s, 6H, 2ꢂ–CH3), 1.82 (d, 2H, –CH2), 2.03 (d, 2H, –CH2), 2.22
(d, 2H, –CH2), 2.27 (d, 2H, –CH2), 5.14 (s, 1H, –CH), 7.31 (s, 2H, –NH2),
7.62 (d, 2H, J = 8.0 Hz, Ar-H), 7.70 (d, 2H, J = 8.0 Hz, Ar-H) 7.85 (d, 2H,
J = 8.0 Hz, Ar-H), 8.00 (d, 2H, J = 8.0 Hz, Ar-H), 8.17 (t, 4H, Ar-H),
10.76 (s, 1H, –NH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 26.60,
29.63, 32.52, 33.49, 41.52, 49.86, 112.57, 120.46, 123.77, 127.08,
129.49, 130.12, 130.49, 135.79, 139.49, 141.61, 142.40, 146.18,
151.10, 154.04, 165.49, 195.53; IR (cmꢀ1): 3236 w (–NH2), 3069
(Ar-H), 2954 (C–H), 1633 s (C@O), 1515 m (C@C), 1339 s and 1149
s (SO2); MS(CI) m/z 667 [Mꢀ1]+ (100%).
A mixture of a 4-amino-N-(4-sulfamoylphenyl)benzamide 4
(0.291 g, 1 mmol), 5,5-dimethylcyclohexane-1,3-dione 5 (0.280 g,
2 mmol), benzaldehyde 6a (0.106 g, 1 mmol) and DBSA (0.420 g,
10 mol %) in 40 mL H2O was stirred and refluxed for 24 h. The pro-
gress of the reaction was monitored by TLC. Once the reaction is
completed, the mixture was cooled to room temperature and solid
filtered off and washed with H2O. The acridine sulfonamide prod-
ucts were purified and recrystallized from the following solvent
mixture for each compound (42–96%).
4.1.4. N-(4-sulfamoylphenyl)-4-(3,3,6,6-tetramethyl-1,8-dioxo-
9-phenyl-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)benzamide
(7)
4.1.8. N-(4-Sulfamoylphenyl)-4-(3,3,6,6-tetramethyl-9-(3-
nitrophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-
yl)benzamide (11)
As yellow crystal, (0.45 g, 72%), mp 216 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.75 (s,
6H, 2ꢂ–CH3), 0.90 (s, 6H, 2ꢂ–CH3), 1.80 (d, 2H, –CH2), 2.02 (d,
2H, –CH2), 2.20 (d, 2H, –CH2), 2.25 (d, 2H, –CH2), 5.10 (s, 1H, –
CH), 7.12 (s, 2H, –NH2), 7.24–7.36 (m, 5H, Ar-H), 7.62 (d, 2H,
J = 8.0 Hz, Ar-H) 7.84 (d, 2H, J = 8.0 Hz, Ar-H), 8.00 (d, 2H,
J = 8.0 Hz, Ar-H), 8.17 (d, 2H, J = 8.0 Hz, Ar-H), 10.75 (s, 1H, –NH);
13C NMR (75 MHz, DMSO-d6) d (ppm): 26.56, 29.73, 32.47, 32.51,
41.50, 50.04, 113.60, 120.43, 126.30, 127.07, 128.06, 128.40,
130.11, 130.43, 135.66, 139.48, 141.87, 142.40, 146.61, 150.36,
165.52, 195.56; IR (cmꢀ1): 3229 w (–NH2), 3036 w (Ar-H), 2953
w (C–H), 1666 and 1641 s (C@O), 1534 m (C@C), 1319 s and
1154 s (SO2); MS(CI) m/z 622 [Mꢀ1]+ (100%).
As yellow crystal, (0.51 g, 76%), mp 240 °C (decomposition) (eth-
anol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.70 (s, 6H, 2ꢂ–
CH3), 0.89 (s, 6H, 2ꢂ–CH3), 1.84 (d, 2H, –CH2), 2.04 (d, 2H, –CH2), 2.23
(d, 2H, –CH2), 2.29 (d, 2H, –CH2), 5.16 (s, 1H, –CH), 7.31 (s, 2H, –NH2),
7.59–7.64 (m, 4H, Ar-H), 7.83 (t, 2H, J = 7.5 Hz, Ar-H) 8.01 (t, 2H,
J = 8.6 Hz, Ar-H), 8.19 (t, 4H, J = 7.5 Hz, Ar-H), 10.78 (s, 1H, –NH);
13C NMR (75 MHz, DMSO-d6) d (ppm): 26.53, 29.62, 32.59, 32.98,
41.45, 49.83, 112.79, 120.42, 120.46, 121.54, 122.58, 127.07,
130.22, 130.26, 134.80, 135.81, 139.49, 141.56, 142.38, 147.93,
148.60, 151.17, 165.44, 195.63; IR (cmꢀ1): 3205 w (–NH2), 3092 w
(Ar-H), 2962 w (C–H), 1631 s (C@O), 1523 m (C@C), 1365 s and
1153 s (SO2); MS(CI) m/z 667 [Mꢀ1]+ (100%).
4.1.5. 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-(4-
4.1.9. 4-(9-(4-Fluorophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
sulfamoylphenyl)benzamide (8)
sulfamoylphenyl)benzamide (12)
As yellow crystal, (0.61 g, 94%), mp 228 °C (decomposition) (eth-
anol–water). 1H NMR (400 MHz, DMSO-d6) d (ppm): 0.73 (s, 6H, 2ꢂ–
CH3), 0.90 (s, 6H, 2ꢂ–CH3), 1.84 (d, 2H, –CH2), 2.02–2.10 (m, 2H, –
CH2), 2.21–2.28 (m, 4H, 2ꢂ–CH2), 5.10 (s, 1H, –CH), 7.55 (d, 2H,
J = 8.3 Hz, Ar-H), 7.69 (d, 2H, J = 8.6 Hz, Ar-H), 7.34 (s, 2H, –NH2),
7.75 (d, 2H, J = 8.3 Hz, Ar-H), 7.86 (d, 2H, J = 9.0 Hz, Ar-H), 8.01 (d,
2H, J = 9.0 Hz, Ar-H), 8.19 (d, 2H, J = 8.8 Hz, Ar-H), 13.40 (s, 1H, –
NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 26.12, 29.13, 32.05,
33.07, 41.02, 49.37, 108.65, 112.11, 118.97, 119.95, 126.59, 128.82,
129.63, 129.95, 132.00, 135.28, 138.99, 141.13, 141.91, 150.51,
151.46, 165.00, 195.06; IR (cmꢀ1): 3229 w (–NH2), 3095 w (Ar-H),
2954 w (C–H), 2228 s (C„N), 1677 and 1633 s (C@O), 1524 m
(C@C), 1362 s and 1150 s (SO2); MS(CI) m/z 647 [Mꢀ1]+ (65%).
As yellow crystal, (0.61 g, 95%), mp 218 °C (ethanol-water). 1H
NMR (300 MHz, DMSO-d6) d (ppm): 0.75 (s, 6H, 2ꢂ–CH3), 0.89 (s,
6H, 2ꢂ–CH3), 1.80 (d, 2H, –CH2), 2.03 (d, 2H, –CH2), 2.19 (d, 2H,
–CH2), 2.25 (d, 2H, –CH2), 5.04 (s, 1H, –CH), 7.08 (t, 2H, J = 8.8 Hz,
Ar-H), 7.31 (s, 2H, –NH2), 7.33–7.38 (m, 2H, Ar-H), 7.63 (d, 2H,
J = 8.1 Hz, Ar-H) 7.84 (d, 2H, J = 8.7 Hz, Ar-H), 7.98 (d, 2H,
J = 8.7 Hz, Ar-H), 8.17 (d, 2H, J = 8.3 Hz, Ar-H), 10.77 (s, 1H, –NH);
13C NMR (75 MHz, DMSO-d6) d (ppm): 26.58, 29.69, 31.98, 32.51,
41.49, 50.00, 113.49, 114.87, 120.44, 127.06, 129.76, 130.42,
135.69, 139.50, 141.79, 142.40, 142.87, 150.41, 159.33, 162.52,
165.49, 195.55; IR (cmꢀ1): 3183 w (–NH2), 3096 w (Ar-H), 2955
w (C–H), 1681 s (C@O), 1594 m (C@C), 1362 s (C–N), 1362 s and
1151 s (SO2); MS(CI) m/z 642 [M+1]+ (100%).
4.1.6. 4-(9-(3-Cyanophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
4.1.10. 4-(9-(4-Chlorophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
sulfamoylphenyl)benzamide (9)
sulfamoylphenyl)benzamide (13)
As yellow crystal, (0.58 g, 89%), mp 294 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.73 (s,
6H, 2ꢂ–CH3), 0.89 (s, 6H, 2ꢂ–CH3), 1.85 (d, 2H, –CH2), 2.04 (d,
As yellow crystal, (0.59 g, 90%), mp 230 °C (decomposition) (eth-
anol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.76 (s, 6H, 2ꢂ–
CH3), 0.91 (s, 6H, 2ꢂ–CH3), 1.80 (d, 2H, –CH2), 2.02 (d, 2H, –CH2), 2.19

R. Ulus et al. / Bioorg. Med. Chem. 21 (2013) 5799–5805
5803
(d, 2H, –CH2), 2.25 (d, 2H, –CH2), 5.00 (s, 1H, –CH), 7.31 (s, 2H, –NH2),
7.33–7.37 (m, 4H, Ar-H), 7.64 (d, 2H, J = 8.6 Hz, Ar-H), 7.84 (d, 2H,
J = 8.6 Hz, Ar-H) 7.98 (d, 2H, J = 8.6 Hz, Ar-H), 8.16 (d, 2H,
J = 8.6 Hz, Ar-H), 10.76 (s, 1H, –NH); 13C NMR (75 MHz, DMSO-d6)
d (ppm): 26.59, 29.67, 32.30, 32.51, 41.49, 49.96, 113.19, 120.43,
127.06, 128.36, 129.97, 130.44, 130.78, 130.60, 135.70, 139.48,
141.74, 142.39, 145.57, 150.57, 165.49, 195.54; IR (cmꢀ1): 3250 w
(–NH2), 3096 w (Ar-H), 2955 w (C–H), 1680 and 1630 s (C@O),
1595 m (C@C), 1362 s and 1152 s (SO2); MS(CI) m/z 658 [M]+ (100%).
6H, 2ꢂ–CH3), 0.88 (s, 6H, 2ꢂ–CH3), 1.77 (d, 2H, –CH2), 2.01 (d,
2H, –CH2), 2.18 (d, 2H, –CH2), 2.24 (d, 2H, –CH2), 2.87 (s, 6H, 2ꢂ–
CH3), 4.92 (s, 1H, –CH), 6.62 (d, 2H, J = 8.7 Hz, Ar-H), 7.14 (d, 2H,
J = 8.6 Hz, Ar-H), 7.31 (s, 2H, –NH2) 7.55–7.65 (m, 2H, Ar-H), 7.84
(d, 2H, J = 8.8 Hz, Ar-H), 7.98 (d, 2H, J = 8.8 Hz, Ar-H), 8.16 (d, 2H,
J = 8.6 Hz, Ar-H), 10.76 (s, 1H, –NH); 13C NMR (75 MHz, DMSO-
d6) d (ppm): 26.63, 29.79, 31.14, 32.50, 40.78, 41.47, 50.11,
112.57, 114.13, 120.41, 127.06, 128.57, 130.07, 131.05 134.94,
135.59, 139.44, 142.04, 142.41, 149.10, 149.75, 165.53, 195.60; IR
(cmꢀ1): 3206 w (–NH2), 3040 w (Ar-H) 2959 w (C–H), 1634 s
(C@O), 1534 m (C@C), 1366 s and 1155 s (SO2); MS(CI) m/z 667
[M+1]+ (100%).
4.1.11. 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-(4-
sulfamoylphenyl)benzamide (14)
As brown crystal, (0.60 g, 85%), mp 233 °C (ethanol–water). 1H
NMR (300 MHz, DMSO-d6) d (ppm): 0.73 (s, 6H, 2ꢂ–CH3), 0.89 (s,
6H, 2ꢂ–CH3), 1.80 (d, 2H, –CH2), 2.02 (d, 2H, –CH2), 2.19 (d, 2H,
–CH2), 2.25 (d, 2H, –CH2), 5.00 (s, 1H, –CH), 7.28 (s, 2H, Ar-H),
7.30 (s, 2H, –NH2), 7.45 (d, 2H, J = 8.4 Hz, Ar-H), 7.64 (d, 2H,
J = 8.4 Hz, Ar-H) 7.84 (d, 2H, J = 8.4 Hz, Ar-H), 7.98 (d, 2H,
J = 8.4 Hz, Ar-H), 8.17 (d, 2H, J = 8.4 Hz, Ar-H), 10.78 (s, 1H, –NH);
13C NMR (75 MHz, DMSO-d6) d (ppm): 26.61, 29.67, 32.39, 32.52,
41.49, 49.97, 113.14, 119.29, 120.44, 127.06, 130.07, 130.21,
130.40, 131.28, 135.71, 139.49, 141.74, 142.39, 145.99, 150.58,
165.49, 195.54; IR (cmꢀ1): 3263 w (–NH2), 3066 w (Ar-H), 2956
w (C–H), 1631 s (C@O), 1596 m (C@C), 1363 s and 1152 s (SO2);
MS(CI) m/z 702 [M]+ (100%).
4.1.15. N-(4-Sulfamoylphenyl)-4-(3,3,6,6-tetramethyl-1,8-
dioxo-9-(p-tolyl)-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-
yl)benzamide (18)
As yellow crystal, (0.61 g, 96%), mp 225 °C (ethanol-water). 1H
NMR (300 MHz, DMSO-d6) d (ppm): 0.72 (s, 6H, 2ꢂ–CH3), 0.86 (s,
6H, 2ꢂ–CH3), 1.79 (d, 2H, –CH2), 2.01 (d, 2H, –CH2), 2.17-2.26
(m, 4H, 2ꢂ–CH2), 2.23 (s, 3H, –CH3), 5.00 (s, 1H, –CH), 7.06 (d,
2H, J = 7.9 Hz, Ar-H), 7.14 (d, 2H, J = 7.9 Hz, Ar-H), 7.31 (s, 2H, –
NH2) 7.58-7.63 (m, 2H, Ar-H), 7.84 (d, 2H, J = 8.8 Hz, Ar-H), 7.98
(d, 2H, J = 8.8 Hz, Ar-H), 8.17 (d, 2H, J = 8.6 Hz, Ar-H), 10.73 (s,
1H, –NH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 21.08, 26.59,
29.75, 31.97, 32.50, 50.06, 56.50, 113.75, 120.43, 127.06, 127.96,
128.98, 129.40, 130.10, 135.13, 135.64, 139.48, 141.91, 142.40,
143.74, 150.19, 165.51, 195.52; IR (cmꢀ1): 3198 w (–NH2), 3066
w (Ar-H), 2958 w (C–H), 1679 and 1626 s (C@O), 1596 m (C@C),
1365 s and 1156 s (SO2); MS(CI) m/z 638 [M+1]+ (100%).
4.1.12. 4-(9-(4-Hydroxyphenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
sulfamoylphenyl)benzamide (15)
As yellow crystal, (0.60 g, 94%), mp 308 °C (decomposition)
(ethanol-water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.71 (s,
6H, 2ꢂ–CH3), 0.86 (s, 6H, 2ꢂ–CH3), 1.75 (d, 2H, –CH2), 2.01 (s,
2H, –CH2), 2.17 (s, 4H, 2ꢂ–CH2), 4.90 (s, 1H, –CH), 6.62 (s, 2H,
–NH2), 7.00–7.40 (m, 4H, Ar-H), 7.50–7.65 (m, 2H, Ar-H)
7.75–8.30 (m, 6H, Ar-H), 9.03 (s, 1H, –OH) 10.74 (s, 1H, –NH);
13C NMR (75 MHz, DMSO-d6) d (ppm): 26.57, 29.76, 31.30, 32.50,
41.48, 50.11, 114.03, 115.13, 120.42, 127.06, 128.91, 130.10,
130.44, 135.60, 137.33, 139.46, 141.97, 142.40, 149.92, 155.76,
165.53, 195.58; IR (cmꢀ1): 3260 br (Ar-OH), 3259 w (–NH2),
2957 w (C–H), 1667 and 1624 s (C@O), 1574 m (C@C), 1365 s
and 1151 s (SO2); MS(CI) m/z 640 [M]+ (100%).
4.1.16. 4-(9-(4-Ethylphenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
sulfamoylphenyl)benzamide (19)
As yellow crystal, (0.60 g, 92%), mp 231 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.72 (s,
6H, 2ꢂ–CH3), 0.94 (s, 6H, 2ꢂ–CH3), 1.14 (t, 3H, –CH3), 1.79 (d,
2H, –CH2), 2.02 (d, 2H, –CH2), 2.19 (d, 2H, –CH2), 2.24 (d, 2H,
–CH2), 2.49–2.57 (m, 2H, –CH2), 5.00 (s, 1H, –CH), 7.09 (d, 2H,
J = 8.1 Hz, Ar-H), 7.24 (d, 2H, J = 8.1 Hz, Ar-H), 7.31 (s, 2H, –NH2)
7.57–7.65 (m, 2H, Ar-H), 7.84 (d, 2H, J = 8.9 Hz, Ar-H), 7.98 (d,
2H, J = 8.9 Hz, Ar-H), 8.17 (d, 2H, J = 8.7 Hz, Ar-H), 10.80 (s, 1H,
–NH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 15.90, 26.65, 28.17,
29.71, 32.00, 32.50, 41.49, 50.07, 113.76, 120.42, 127.06, 127.74,
127.99, 130.10, 130.44, 135.64, 139.48, 141.43, 141.91, 142.40,
143.99, 150.20, 165.50, 195.54; IR (cmꢀ1): 3251 and 3209 w
(–NH2), 3068 w (Ar-H), 2958 w (C–H), 1679 and 1629 s (C@O),
1596 m (C@C), 1363 s and 1153 s (SO2); MS(CI) m/z 652 [M+1]+
(100%).
4.1.13. 4-(9-(4-Methoxyphenyl)-3,3,6,6-tetramethyl-1,8-dioxo-
1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
sulfamoylphenyl)benzamide (16)
As yellow crystal, (0.61 g, 93%), mp 244 °C (decomposition)
(ethanol-water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.79 (s,
6H, 2ꢂ–CH3), 0.88 (s, 6H, 2ꢂ–CH3), 1.79 (d, 2H, –CH2), 2.02 (d,
2H, –CH2), 2.19 (d, 2H, –CH2), 2.24 (d, 2H, –CH2), 3.70 (s, 3H,
–OCH3), 4.98 (s, 1H, –CH), 6.79 (d, 2H, J = 8.0 Hz, Ar-H), 7.24 (d,
2H, J = 8.0 Hz, Ar-H) 7.06 (s, 2H, –NH2), 7.61 (d, 2H, J = 8.0 Hz,
Ar-H), 7.84 (d, 2H, J = 8.0 Hz, Ar-H), 7.98 (d, 2H, J = 8.0 Hz, Ar-H),
8.17 (d, 2H, J = 8.0 Hz, Ar-H) 10.78 (s, 1H, –NH); 13C NMR
(75 MHz, DMSO-d6) d (ppm): 26.60, 29.75, 31.53, 32.50, 41.49,
50.07, 55.33, 113.73, 113.87, 120.43, 127.07, 129.02, 130.09,
130.43, 135.64, 138.97, 139.48, 141.93, 142.40, 150.06, 157.79,
165.51, 195.51; IR (cmꢀ1): 3189 w (–NH2), 3067 w (Ar-H), 2957
w (C–H), 1691 and 1627 s (C@O), 1529 m (C@C), 1367 s and
1150 s (SO2); MS(CI) m/z 652 [Mꢀ1]+ (100%).
4.1.17. 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)-N-(4-
sulfamoylphenyl)benzamide (20)
As brown crystal, (0.46 g, 71%), mp 310 °C (decomposition)
(ethanol–water). 1H NMR (400 MHz, DMSO-d6) d (ppm): 0.77 (s,
6H, 2ꢂ–CH3), 0.88 (s, 6H, 2ꢂ–CH3), 1.79 (d, 2H, –CH2), 1.98 (d,
2H, –CH2), 2.19 (d, 4H, 2ꢂ–CH2), 5.26 (s, 1H, –CH), 7.34 (s, 2H,
–NH2), 7.37 (d, 1H, J = 4.0 Hz, Ar-H), 7.43 (d, 1H, J = 4.0 Hz, Ar-H),
7.55 (d, 1H, J = 8.0 Hz, Ar-H), 7.66 (s, 2H, Ar-H), 7.86 (d, 2H,
J = 8.0 Hz, Ar-H), 8.00 (d, 2H, J = 8.0 Hz, Ar-H), 8.19 (d, 2H,
J = 4.0 Hz, Ar-H) 10.82 (s, 1H, –NH); 13C NMR (100 MHz,
DMSO-d6) d (ppm): 26.03, 29.21, 31.87, 32.88, 41.21, 49.38,
111.49, 119.92, 126.59, 128.63, 129.47, 129.89, 130.29, 130.63,
130.92, 133.48, 135.23, 138.96, 141.33, 141.92, 142.08, 150.67,
165.02, 194.94; IR (cmꢀ1): 3225 w (–NH2), 3068 w (Ar-H), 2960
w (C–H), 1638 s (C@O), 1537 m (C@C), 1362 s and 1154 s (SO2);
MS(CI) m/z 692 [M]+ (100%).
4.1.14. 4-(9-(4-(Dimethylamino)phenyl)-3,3,6,6-tetramethyl-
1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
sulfamoylphenyl)benzamide (17)
As brown crystal, (0.60 g, 90%), mp 197 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.75 (s,

5804
R. Ulus et al. / Bioorg. Med. Chem. 21 (2013) 5799–5805
4.1.18. 4-(9-(2-Hydroxy-3-methoxyphenyl)-3,3,6,6-tetramethyl-
1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl)-N-(4-
sulfamoylphenyl)benzamide (21)
4.1.22. 4,40-(9,90-(1,3-Phenylene)bis(3,3,6,6-tetramethyl-1,8-
dioxo-1,2,3,4,5,6,7,8-octahydroacridine-10,9(9H)-diyl))bis(N-(4-
sulfamoylphenyl)benzamide) (25)
As brown crystal, (0.59 g, 88%), mp 235 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.75 (s,
6H, 2ꢂ–CH3), 0.89 (s, 6H, 2ꢂ–CH3), 1.76 (d, 2H, –CH2), 2.00 (d,
2H, –CH2), 2.21 (d, 4H, 2ꢂ–CH2), 3.74 (s, 3H, –OCH3), 5.01 (s, 1H,
–CH), 6.65–6.73 (m, 2H, Ar-H), 6.83 (dd, 1H, J1 = 7.2 Hz,
J2 = 2.1 Hz, Ar-H), 7.31 (s, 2H, –NH2) 7.71 (s, 2H, Ar-H), 7.84 (d,
2H, J = 8.8 Hz, Ar-H), 7.98 (d, 2H, J = 8.8 Hz, Ar-H), 8.15 (d, 2H,
J = 8.7 Hz, Ar-H), 8.82 (s, 1H, –OH) 10.79 (s, 1H, –NH); 13C NMR
(75 MHz, DMSO-d6) d (ppm): 26.49, 29.34, 29.78, 32.41, 41.59,
49.89, 56.00, 110.16, 112.15, 118.80, 120.41, 122.80, 127.06,
129.92, 130.63, 132.67, 135.61, 139.43, 142.17, 142.42, 144.61,
148.69, 151.50, 165.1, 196.43; IR (cmꢀ1): 3200 br (Ar-OH), 3200
w (–NH2), 3097 w (Ar-H) 2954 w (C–H), 1671 s (C@O), 1591 m
(C@C), 1367 s and 1151 s (SO2); MS(CI) m/z 670 [M+1]+ (100%).
As yellow crystal, (0.500 g, 42%), mp 295 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.71 (s,
12H, 4ꢂ–CH3), 0.86 (s, 12H, 4ꢂ–CH3), 1.80 (d, 4H, 2ꢂ–CH2), 1.96
(d, 4H, 2ꢂ–CH2), 2.24 (t, 8H, 4ꢂ–CH2), 5.04 (s, 2H, 2ꢂ–CH), 7.07–
7.17 (m, 3H, Ar-H), 7.30 (s, 4H, 2ꢂ–NH2) 7.49 (s, 1H, Ar-H), 7.84
(d, 8H, J = 8.8 Hz, Ar-H), 8.00 (d, 4H, J = 8.8 Hz, Ar-H), 8.19 (d, 4H,
J = 8.5 Hz, Ar-H), 10.77 (s, 2H, 2ꢂ–NH); 13C NMR (75 MHz,
DMSO-d6) d (ppm): 26.52, 30.07, 32.14, 32.43, 41.57, 50.14,
113.58, 120.40, 120.45, 125.38, 127.06, 127.95, 127.97, 130.07,
130.60, 135.58, 139.42, 142.12, 142.30, 142.43, 146.18, 150.22,
165.54, 195.42; IR (cmꢀ1): 3311 and 3262 w (–NH2), 3060 w (Ar-
H), 2956 w (C–H), 1631 s (C@O), 1596 m (C@C), 1363 s and 1151
s (SO2); MS(CI) m/z 1169 [M]+ (100%).
4.2. CA inhibition assay
4.1.19. 4-(9-(4-Cyanophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl)-N-(4-sulfamoylphenyl)benzamide
(22)
An applied photophysics stopped-flow instrument has been used
for assaying the CA catalysed CO2 hydration activity.16 Phenol red (at
a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5)
as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic
strength), following the initial rates of the CA-catalyzed CO2 hydra-
tion reaction for a period of 10–100 s. The CO2 concentrations ran-
ged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants. For each inhibitor at least six
traces of the initial 5–10% of the reaction have been used for deter-
mining the initial velocity. The uncatalyzed rates were determined
in the same manner and subtracted from the total observed rates.
Stock solutions of inhibitor (0.1 mM) were prepared in distilled–
deionized water and dilutions up to 0.01 nM were done thereafter
with the assay buffer. Inhibitor and enzyme solutions were preincu-
bated together for 15 min at room temperature prior to assay, in or-
der to allow for the formation of the E–I complex. The inhibition
constants were obtained by nonlinear least-squares methods using
PRISM 3, as reported earlier,8 and represent the mean from at least
three different determinations. All CA isofoms were recombinant
ones obtained in-house as reported earlier.11–13
As brown crystal, (0.40 g, 67%), mp 329 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 1.60–
2.30 (m, 12H, 6ꢂ–CH2), 5.20 (s, 1H, –CH), 7.30 (s, 2H, –NH2), 7.52
(d, 2H, J = 8.3 Hz, Ar-H), 7.68 (s, 2H, Ar-H), 7.72 (d, 2H, J = 8.3 Hz,
Ar-H), 7.83 (d, 2H, J = 8.8 Hz, Ar-H), 7.97 (d, 2H, J = 8.8 Hz, Ar-H),
8.15 (d, 2H, J = 8.8 Hz, Ar-H), 10.72 (s, 1H, –NH); 13C NMR
(75 MHz, DMSO-d6) d (ppm): 21.09, 28.31, 32.79, 36.60, 109.10,
113.43, 119.52, 120.44, 127.06, 129.18, 129.73, 130.49, 132.60,
135.70, 139.46, 141.66, 142.36, 152.40, 153.10, 165.46, 195.94; IR
(cmꢀ1): 3354 and 3204 w (–NH2), 3092 w (Ar-H), 2952 w (C–H),
2228 s (C„N), 1667 and 1630 s (C@O), 1599 m (C@C), 1363 s
and 1153 s (SO2); MS(CI) m/z 593 [M+1]+ (100%).
4.1.20. 4-(9-(2,4-Dichlorophenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl)-N-(4-sulfamoylphenyl)benzamide
(23)
As brown crystal, (0.46 g, 72%), mp 240 °C (decomposition) (eth-
anol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 1.63–2.16 (m,
12H, 6ꢂ–CH2), 5.30 (s, 1H, –CH), 7.31 (s, 2H, –NH2), 7.33–7.34 (m,
1H, Ar-H), 7.37–7.38 (m, 1H, Ar-H), 7.53 (d, 1H, J = 8.3 Hz, Ar-H),
7.67–7.78 (br, 2H, Ar-H), 7.84 (d, 2H, J = 8.7 Hz, Ar-H), 7.97 (d, 2H,
J = 8.7 Hz, Ar-H), 8.16 (d, 2H, J = 7.8 Hz, Ar-H), 10.72 (s, 1H, –NH);
13C NMR (75 MHz, DMSO-d6) d (ppm): 21.19, 28.46, 32.94, 36.64,
113.34, 120.43, 127.05, 128.93, 129.65, 130.29, 130.82, 131.36,
134.04, 134.24, 135.68, 139.46, 141.87, 142.37, 143.24, 152.96,
165.47, 195.59; IR (cmꢀ1): 3252 w (–NH2), 3091 (Ar-H), 2950 w
(C–H), 1665 and 1621 s (C@O), 1592 m (C@C), 1358 s and 1155 s
(SO2); MS(CI) m/z 636 [M]+ (100%).
Acknowledgements
This research was financed in part by an EU grant (Dynano) and
by Dumlupınar University Research Fund (Grant No. 2012-38) as
well as by the Dumlupınar University ILTEM research laboratory.
The authors are grateful to Professor Dr. Yılmaz Yıldırır, Gazi
University, Ankara, Turkey.
Supplementary data
4.1.21. 4,40-(9,90-(1,4-Phenylene)bis(3,3,6,6-tetramethyl-1,8-
dioxo-1,2,3,4,5,6,7,8-octahydroacridine-10,9(9H)-diyl))bis(N-(4-
sulfamoylphenyl)benzamide) (24)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.07.014.
As yellow crystal, (0.650 g, 56%), mp 307 °C (decomposition)
(ethanol–water). 1H NMR (300 MHz, DMSO-d6) d (ppm): 0.68 (s,
12H, 4ꢂ–CH3), 0.89 (s, 12H, 4ꢂ–CH3), 1.80 (d, 4H, 2ꢂ–CH2), 2.01
(d, 4H, 2ꢂ–CH2), 2.17 (d, 4H, 2ꢂ–CH2), 2.23 (d, 4H, 2ꢂ–CH2),
5.04 (s, 2H, 2ꢂ–CH), 7.21 (s, 4H, Ar-H), 7.30 (s, 4H, 2ꢂ–NH2) 7.61
(d, 4H, J = 7.6 Hz, Ar-H), 7.84 (d, 4H, J = 8.7 Hz, Ar-H), 7.98 (d, 4H,
J = 8.7 Hz, Ar-H), 8.17 (d, 4H, J = 8.0 Hz, Ar-H), 10.76 (s, 2H, 2ꢂ–
NH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 26.44, 29.70, 31.63,
32.52, 41.49, 50.04, 113.61, 120.43, 127.07, 127.48, 130.09,
130.44, 135.63, 139.44, 141.85, 142.40, 143.97, 150.32, 165.54,
195.64; IR (cmꢀ1): 3313 and 3258 w (–NH2), 3096 w (Ar-H),
2959 w (C–H), 1627 s (C@O), 1596 m (C@C), 1364 s and 1149 s
(SO2); MS(CI) m/z 1169 [M]+ (100%).
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