Synthesis and characterization of novel dioxoacridine sulfonamide derivatives as new carbonic anhydrase inhibitors

Article abstract of DOI:10.3109/14756366.2011.599029

Novel dioxoacridine sulfonamide compounds were synthesized from reaction of cyclic 1,3-diketones, sulfanilamide (4-amino benzene sulfonamide) and aromatic aldehydes. The structures of these compounds were confirmed by using spectral analysis (IR, H-NMR, <

Full text of DOI:10.3109/14756366.2011.599029

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(4): 509–514
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.599029
research artIcle
Synthesis and characterization of novel dioxoacridine
sulfonamide derivatives as new carbonic anhydrase inhibitors
Muharrem Kaya, Erhan Basar, Emrah Çakir, Ekrem Tunca, and Metin Bülbül
Department of Chemistry, Faculty of Arts and Science, Dumlupinar University, 43100 Kütahya, Turkey
abstract
Novel dioxoacridine sulfonamide compounds were synthesized from reaction of cyclic 1,3-diketones, sulfanilamide
(4-amino benzene sulfonamide) and aromatic aldehydes. The structures of these compounds were confirmed by
using spectral analysis (IR, H-NMR, 13C-NMR, and mass). Human carbonic anhydrase isoenzymes (hCA I and hCA II)
were purified from erythrocyte cells by affinity chromatography.The inhibitory effects of sulfanilamide, acetazolamide
(AAZ), and newly synthesized sulfonamides on hydratase and esterase activities of these isoenzymes have been
studied in vitro. The IC50 values of compounds for esterase activity are 0.71–0.11 µM for hCA I and 0.45–0.12 µM for
hCA II, respectively. The Ki values of these inhibitors were determined as 0,38–0,008 µM for hCA I and 0,19–0,001 µM
for hCA II, respectively.
Keywords: Dioxoacridine sulfonamide, carbonic anhydrase, inhibition, hydratase activity, esterase activity
Introduction
clinical medicine4,5,15. ese inhibitors are very effective
in the treatment by reducing elevated IOP. Acetazolamide
(AAZ) was the first compound used as the inhibitor of this
potent hCA II. en, it was suggested that reducing aque-
ous humor secretion might provide an effective means of
lowering IOP to treat the disease17. Afterwards some sys-
temic sulfonamide drugs were mainly used clinically for
a long time as anti-glaucoma agents17. e orally admin-
istered drugs had affected various CA isozymes present
in other tissues and led to an entire range of side effects.
To decrease systemic side effects of oral CA inhibitors,
dorzolamide (DZA) and brinzolamide (BRZ) ophthalmic
suspension approved for the treatment of glaucoma have
been used as the topical CA inhibitors7,11. Each drug may
be an effective anti-glaucoma agent, but these drugs tend
to pose tolerability problems in many patients because
of local side effects18. us, it is required to develop new
compounds and new drugs.
Carbonic anhydrases (CAs) (E.C. 4.2.1.1) are catalytic
reversible hydration of carbon dioxide in a two-step
reaction to yield bicarbonate and proton1–5. Sixteen
different CA isoenzymes were described in higher ver-
tebrates, including humans6. e isoenzymes relevant
to the human eye are hCA I, hCA II and hCA IV. e
isoenzymes of hCA I and hCA II are cytosolic, whereas
hCA IV is membrane-bound7–9. e CA inhibitors,
which reduce aqueous production with a correspond-
ing decrease in intraocular pressure (IOP), have been
used as ocular hypotensive agents for the treatment of
glaucoma10,11. e disease is the second leading cause of
blindness worldwide12,13. e risk factors for glaucoma
disease include age, race, ocular hypertension, severe
myopia and a family history of glaucoma. Here, the
strongest risk factors are age, race and ocular hyperten-
sion. An elevated IOP was formerly synonymous with
glaucoma7.
In this study, novel dioxoacridine derivatives have
been synthesized and characterized by IR, 1H and
13C-NMR data and satisfactory mass spectral analy-
ses, respectively. e synthesis of new CA inhibitors
by using sulfanilamide and their effects on human
Sulfonamides, which are powerful CA inhibitors, are
now widely used as the drug for the treatment or preven-
tion of a variety of diseases4,5,14–16. ey are the best known
inhibitors of CA enzyme in the treatment of glaucoma in
Address for Correspondence: Muharrem Kaya, Department of Chemistry, Faculty of Arts and Science, Dumlupinar University,
43100 Kütahya, Turkey. Tel: + 90-274-2652051. Fax: + 90-274-2652056. E-mail: mhrkaya@yahoo.com
(Received 05 May 2011; revised 14 June 2011; accepted 14 June 2011)
509

510 M. Kaya et al.
CA isoenzymes (hCA I, hCA II) purified from human
erythrocytes are reported. Further, the potential use
of these compounds as new inhibitors of hCA I and
hCA II isoenzymes in the treatment of glaucoma are
investigated.
4-(9-(4-Methoxyphenyl)-3,3,6,6-tetramethyl-1,8-
dioxo-1,2,3,4,5,6,7,8-octahydro acridine-10(9H)-yl)
benzenesulfonamide (5)
As yellow crystals, (0.481 mg, 90%), mp 227°C (ethanol–
1
H2O). H NMR (400 MHz, CDCl3) δ (ppm): 0.85 (s, 6H,
2 × CH3), 0.98 (s, 6H, 2 × CH3), 1.75–1.81 (m, 2H, −CH2),
1.99–2.23 (m, 6H, −CH2), 3.78 (s, 3H, −OCH3), 5.06 (s,
2H, SO2NH2), 5.24 (s, 1H, −CH), 6.82 (d, 2H, J = 8.65 Hz
Ar-H), 7.34 (d, 2H, J = 8.65 Hz Ar-H), 7.44 (d, 2H, J = 8.40
Hz Ar-H), 8.16 (d, 2H, J = 8.40 Hz Ar-H); 13C NMR (100
MHz, CDCl3) δ (ppm): 26.87, 28.99, 31.68, 32.64, 42.05,
51.10, 55.13, 113.29, 115.23, 123.05, 128.47, 130.88,
131.35, 138.15, 146.57, 157.61, 157.89, 195.68. IR (cm−1):
3303 and 3222 w (NH2), 3093 and 3071 w (Ar-H), 2997
w (C-H), 1633 s (C = O), 1626 and 1570 m (C = C), 1386,
1221, 1166, 1136, 1032; MS(CI) m/z 535.10 (M + 1),
428.00 (M-PhOCH3).
Material and methods
Materials
e chemicals used in the synthesis of dioxoacridine
sulfonamide derivatives were provided from Merck
and Aldrich Chemical Company and Sepharose 4B
for affinity column and electrophoresis reagents were
obtained from Sigma Chem. Co. All chemicals and
solvents used for the synthesis were spectroscopic
reagent grade. Melting points were measured on a
Bibby Stuart Scientific apparatus. Fourier Transform
Infrared (FT-IR) spectra were recorded from Bruker
Optics, Andrtex 70 FT-IR spectrometer using ATR dia-
4-(3,3,6,6-tetramethyl-1,8-dioxo-9-(pyridin-2-
yl)-1,2,3,4,5,6,7,8-octahydroacridine-10(9H)-yl)
1
mond crystal. e H-NMR, and 13C-NMR spectra were
obtained with a Bruker DPX-400 FT-NMR instrument
in CDCl3 and DMSO-d6 as solvent with trimethylsilane
as the internal reference, at 400 and 100 MHz, respec-
tively. Chemical shifts are expressed in δ units (ppm).
e mass analyses were performed on Waters 2695
Alliance Micromass ZQ instrument LC/MS.
benzenesulfonamide (6)
As yellow crystals, (0.429 mg, 85%), mp 170°C (etha-
nol–H2O). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.01
(s, 6H, 2 × CH3), 1.11 (s, 6H, 2 × CH3), 2.14–2.28 (m, 4H,
−CH2), 2.43–2.58 (m, 4H, −CH2), 4.94 (s, 1H, −CH), 5.35
(s, 2H, SO2NH2), 7. 19 (qxd, 2H, J = 2.29 Hz, J = 1.44 Hz
Ar-H), 7.53–7.64 (m, 4H, Ar-H), 8.27 (dxd, 1H, J = 7.41
Hz, J = 1.39 Hz Ar-H), 8.39 (dxq, 1H, J = 4.80 Hz, J = 0.64
Hz Ar-H); 13C NMR (100 MHz, CDCl3) δ (ppm): 27.15,
29.30, 32.30, 34.45, 40.86, 50.75, 114.33, 121.40, 122.10,
124.96, 130.95, 131.77, 135.71, 145.02, 148.89, 161.72,
163.37, 196.98. IR (cm−1): 3300 w (NH2), 3065 w (Ar-H),
2960 w (C–H), 1652 s (C = O), 1621 and 1570 m (C = C),
1360, 1223, 1196, 1165, 1001; MS(CI) m/z 506.78 (M +
1).
General procedure for preparation of dioxoacridine
sulfonamide derivatives (4–12).
A mixture of a 5,5-dimethylcyclohexane-1,3-dione 1
(280 mg, 2 mmol), sulfanilamide 2 (172 mg, 1 mmol),
4-cyanobenzaldehyde 3 (131 mg, 1 mmol), and DBSA
(420 mg, 10 mol%) in H2O (40 mL) was stirred at refluxing
for 4 h (6 h for cyclohexane-1,3-dione derivatives; 10 h
for 4,4-dimethylcyclohexane-1,3-dione derivatives). e
progress 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.
e acridine-1,3-dion sulfonamide products were puri-
fied and recrystallized from the following solvent mixture
for each compound (76%–91%).
4-(9-(2,4-dimethoxyphenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridine-10(9H)-yl) benzenesulfonamide (7)
As yellow crystals, 396 mg, 78%, mp 243°C (chloroform).
1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.60–1.91 (m,
6H, 3 × CH2), 2.19–2.46 (m, 6H, 3 × CH2), 3.72 (s, 3H,
−OCH3), 3.80 (s, 3H, −OCH3), 5.18 (s, 1H, −CH), 6.44
(dxd, 1H, J = 8.53 Hz, J = 2.60 Hz Ar-H), 6.50 (d, 1H,
J = 2.54 Hz Ar-H), 7.24 (d, 1H, J = 8.57 Hz Ar-H), 7.62 (s,
2H, Ar-H), 7.69 (s, 2H, SO2NH2), 8.06 (d, 2H, J = 8.50 Hz
Ar-H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 21.40,
27.94, 31.96, 36.51, 55.73, 55.91, 99.03, 103.14, 116.90,
122.08, 124.54, 130.66, 131.13, 133.46, 144.80, 156.09,
159.48, 161.29, 196.08. IR (cm−1): 3310 w (NH2), 3171
and 3031 w (Ar-H), 2971 w (C–H), 1641 s (C=O), 1608
and 1568 m (C=C), 1362, 1234, 1089, 1042; MS(CI) m/z
509.65 (M + 1).
4-(9-(4-Cyanophenyl)-3,3,6,6-tetramethyl-1,8-
dioxo-1,2,3,4,5,6,7,8-octahydroacridine-10(9H)-yl)
benzenesulfonamide (4)
As yellow crystals, (471 mg, 87%), mp 215°C (ethanol–
1
H2O). H NMR (400 MHz, CDCl3) δ (ppm): 0.84 (s, 6H,
2 × CH3), 0.96 (s, 6H, 2 × CH3), 1.76–1.81 (m, 2H, −CH2),
1.99–2.23 (m, 6H, −CH2), 3.75 (s, 3H, −OCH3), 5.05 (s,
2H, SO2NH2), 5.29 (s, 1H, −CH), 7.41 (d, 2H, J = 8.72 Hz
Ar-H), 7.44 (m, 4H, Ar-H), 8.20 (d, 2H, J = 8.38 Hz Ar-H);
13C NMR (100 MHz, CDCl3) δ (ppm): 27.23, 28.96, 32.36,
33.96, 40.53, 50.04, 112.61, 113.89, 120.93, 122.58,
127.38, 130.40, 131.59, 134.18, 143.56, 150.72, 161.14,
197.03. IR (cm−1): 3298 and 3172 w (NH2), 3052 w (Ar-H),
2960 w (C–H), 2229 (CN), 1641 s (C = O), 1628 and 1575
m (C = C), 1361, 1220, 1170, 1145, 1019; MS(CI) m/z
530.90 (M + 1).
4-(9-(4-methoxyphenyl)-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridine-10(9H)-yl)benzene sulfonamide (8)
As yellow crystals, 0.397 mg, 83%, mp 181°C (ethanol–
H2O). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.54–1.93
(m, 6H, 3 × CH2), 1.96-2.30 (m, 6H, 3 × CH2), 3.52 (s, 3H,
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of dioxoacridine sulfonamide derivatives 511
−OCH3), 5.11 (s, 1H, −CH), 6.64 (d, 2H, J= 8.80 Hz Ar-H),
6.77 (s, 2H, SO2NH2), 7.13 (d, 2H, J= 8.51 Hz Ar-H), 7.30
(d, 2H, J= 8.51 Hz Ar-H), 7.96 (d, 2H, J= 8.80 Hz Ar-H);
13C NMR (100 MHz, CDCl3) δ (ppm): 21.36, 27.95, 31.23,
36.48, 55.48, 113.70, 116.54, 122.16, 126.53, 130.69, 131.02,
138.29, 144.53, 150.63, 156.52, 196.43. IR (cm−1): 3312
w (NH2), 3125 and 3061 w (Ar-H), 2940 w (C–H), 1638 s
(C = O), 1595 and 1539 m (C = C), 1385, 1131, 1084, 1011;
MS(CI) m/z 479.22 (M + 1).
3096 w (Ar-H), 2963 w (C–H), 1631 s (C = O), 1579 and
1510 (C = C), 1362, 1263, 1227, 1159, 1027; MS(CI) m/z
565.88 (M + 1).
4-(9-(2,4-Dichlorophenyl)-2,2,7,7-tetramethyl–1,8-
dioxo–1,2,3,4,5,6,7,8-octahydro acridine–10(9H)-yl)
benzenesulfonamide (12)
As yellow crystals, 521 mg, 91%, mp 270°C (decompoze)
1
(ethanol–H2O). H NMR (400 MHz, DMSO-d6) δ (ppm):
0.81 (s, 6H, 2 × CH3), 0.97 (s, 6H, 2 × CH3), 1.52–1.58 (m,
2H, −CH2), 1.60–1.70 (m, 2H, −CH2), 1.84–1.95 (m, 2H,
−CH2), 2.19–2.23 (m, 2H, −CH2), 5.20 (s, 1H, −CH), 7.30
(dxd, 1H, J= 6.00 Hz, J= 2.31 Hz, Ar-H), 7.38 (d, 1H,
J= 2.77 Hz, Ar-H), 7.51 (d, 1H, J= 8.50 Hz, Ar-H), 7.61 (s,
2H, SO2NH2), 7.71 (m, 1H, Ar-H), 7.86 (m, 1H, Ar-H), 8.02
(d, 2H, J= 7.83 Hz, Ar-H); 13C NMR (100 MHz, DMSO-d6)
δ (ppm): 24.13, 24.20, 24.30, 33.01, 33.69, 39.90, 114.88,
122.45, 127.04, 130.32, 130.44, 131.18, 131.27, 134.56,
137.71, 140.83, 144.20, 161.04, 204.51. IR (cm−1): 3384
and 3319 w (NH2), 3088 w (Ar-H), 2963 w (C–H), 1642
s (C = O), 1620 and 1573 (C = C), 1332, 1221, 1198, 1160,
1013; MS(CI) m/z 573.75 (M + 1).
4-(9-(4-Cyanophenyl)-2,2,7,7-tetramethyl–1,8-
dioxo–1,2,3,4,5,6,7,8-octahydroacridine–10(9H)-yl)
benzenesulfonamide (9).
As yellow crystals, 471 mg, 89%, mp 299°C (ethanol–H2O).
1H NMR (400 MHz, CDCl3) δ (ppm): 0.82 (s, 6H, 2 × CH3),
0.93 (s, 6H, 2 × CH3), 1.63–1.72 (m, 2H, −CH2), 1.78–1.92
(m, 2H, −CH2), 1.99–2.10 (m, 2H, −CH2), 2.28–2.47 (m,
2H, −CH2), 5.11 (s, 1H, −CH), 6.80 (s, 2H, SO2NH2),
7.33–7.46 (m, 6H, Ar-H), 8.01 (d, 2H, J= 8.30 Hz Ar-H);
13C NMR (100 MHz, CDCl3) δ (ppm): 24.08, 24.16, 24.35,
32.57, 33.84, 39.91, 112.08, 114.50, 120.54, 122.46, 127.45,
130.27, 131.65, 133.71, 144.10, 151.03, 160.23, 204.60. IR
(cm−1): 3356 and 3280 w (NH2), 3076 w (Ar-H), 2957 w
(C–H), 2222 (CN), 1643 s (C = O), 1619 and 1566 (C = C),
1349, 1220, 1198, 1160, 1010; MS(CI) m/z 530.93 (M + 1).
Purification of carbonic anhydrase I and II from human
erythrocytes
Erythrocytes were purified from human blood. e
blood samples were centrifuged at 1500 rpm for 20 min
and 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. e pH of hemolysate was adjusted to 8.7 with
solid Tris. e hemolysate was applied to affinity col-
umn with a structure of Sepharose-4B-L-tyrosine-p-
aminobenzenesulfonamide and equilibrated with 25 mM
Tris–HCl/0.1 M Na2SO4 (pH 8.7). e affinity gel was
washed with solution of 25 mM Tris–HCl/22 mM Na2SO4
(pH 8.7). e hCA-I and hCA-II isozymes were diluted
with the solution of 1 M NaCl/25 mM Na2HPO4 (pH 6.3)
and 0.1 M NaCH3COO/0.5 M NaClO4 (pH 5.6), respec-
tively19. For protein content estimation, Bradford method
4-(9-(4-Hydroxyphenyl)-2,2,7,7-tetramethyl–1,8-
dioxo–1,2,3,4,5,6,7,8-octahydro acridine–10(9H)-yl)
benzenesulfonamide (10)
As yellow crystals, 442 mg, 85%, mp 280°C (decompoze)
1
(ethanol–H2O). H NMR (400 MHz, DMSO-d6) δ (ppm):
0.91 (s, 6H, 2 × CH3), 1.00 (s, 6H, 2 × CH3), 1.60–1.71 (m,
4H, −CH2), 1.88–1.92 (m, 2H, −CH2), 2.28–2.33 (m, 2H,
−CH2), 4.98 (s, 1H, −CH), 7.61 (d, 2H, J= 8.45 Hz Ar-H),
7.05 (d, 2H, J= 8.45 Hz Ar-H), 7.55 (d, 2H, J= 7.62 Hz
Ar-H), 8.02 (d, 2H, J= 7.62 Hz Ar-H), 8.25 (s, 1H, Ar-OH),
9.02 (s, 2H, SO2NH2); 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 24.12, 24.23, 24.38, 32.69, 33.81, 39.93, 114.56,
115.38, 122.49, 129.06, 130.50, 131.23, 139.51, 144.17, 154.
91, 160.31, 204.56. IR (cm−1): 3353 and 3278 w (NH2), 3066
and 3029 w (Ar-H), 2971 w (C–H), 1631 s (C = O), 1610 and
1586 m (C = C), 1362, 1222, 1161, 1145, 1016; MS(CI) m/z
521.83 (M + 1).
was used with bovine serum albumin as a standard20,21
.
SDS polyacrylamide gel electrophoresis was performed
after the purification of the enzyme (see Figure 1)22.
4-(9-(3,4-Dimethoxyphenyl)-2,2,7,7-tetramethyl–1,8-
dioxo–1,2,3,4,5,6,7,8-octahydro acridine–10(9H)-yl)
benzenesulfonamide (11)
Determination of hydratase and esterase activities of
hCA I and hCA II
As yellow crystals, 429 mg, 76%, mp 190°C (ethanol–H2O).
1H NMR (400 MHz, CDCl3) δ (ppm): 1.04 (s, 6H, 2 × CH3),
1.11 (s, 6H, 2 × CH3), 1.67–1.72 (m, 4H, 2 × CH2), 1.85–2.03
(m, 2H, −CH2), 2.15–2.28 (m, 2H, −CH2), 3.86 (s, 3H,
−OCH3), 3.92 (s, 3H, −OCH3), 5.01 (s, 2H, SO2NH2), 5.29
(s, 1H, −CH), 6.78 (m, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 7.47
(d, 2H, J= 8.20 Hz, Ar-H), 8.14 (d, 2H, J= 7.50 Hz, Ar-H);
13C NMR (100 MHz, CDCl3) δ (ppm): 24.17, 24.20, 24.33,
32.98, 33.73, 39.88, 55.23, 55.29, 112.55, 114.30, 114.65,
122.41, 122.49, 130.53, 131.24, 137.62, 144.26, 156.49,
157.20, 160.72, 204.61. IR (cm−1): 3306 and 3239 w (NH2),
e CO2-hydratase activity of the enzyme was deter-
mined at 0°C in a veronal buffer (pH= 8.15) with pH-state
method as indicator and saturated carbon dioxide solu-
tion as substrate in a final volume of 4.2 mL. e time (in
seconds) taken for the solution to change from pH 8.15 to
pH 6.5 was measured by pH meter. e enzyme unit (EU)
is the enzyme amount that reduces the non-enzymatic
reaction time by 50%. Activity of an enzyme unit was cal-
culated by using the equation (t0−tc/tc) where to and tc are
times for pH change of the non-enzymatic and enzymatic
reactions, respectively23–25. Esterase activities of hCA I
© 2012 Informa UK, Ltd.

512 M. Kaya et al.
and hCA II isoenzymes, eluted from affinity column,
were determined by hydrolysis of p-nitrophenylacetate.
e change of absorbance was determined at 348nm
after 3 min26.
e infrared (IR) spectra of all the dioxoacridine
sulfonamide compounds showed sharp peaks for the
carbonyl groups in region between 1650 and 1631 cm−l 31
.
e compounds 4, 9 exhibited peaks that belong to
CN group 2229 and 2222 cm−1, respectively32. Besides,
in the IR spectra of the compounds, aliphatic C–H
stretching bands at 2997–2940 cm−1 and aromatic C–H
stretching bands at 3171–3029 cm−1 were observed.
e NH2 vibrations of dioxoacridine sulfonamide com-
pounds were observed in the region between 3384
Determination of IC50 and Ki values of compounds
e study consists of two parts. In the first part, IC50
values have been obtained as in vitro. e IC50 values
of inhibitors (4–12) were assayed by the hydrolysis of
p-nitrophenyl acetate on the esterase activities of CA
isoenzymes in the presence of various inhibitor concen-
trations. e absorbance was determined at 348 nm after
3 min27. Regression analysis graphs were drawn by plot-
ting inhibitor concentrations versus enzyme activity.
To determine Ki value as well as the inhibition type,
three different inhibitor concentrations giving 30%,
50% and 70% inhibition were selected. At each of these
inhibitor concentrations, enzyme activity was mea-
sured in the presence of various substrate concentra-
tions (0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM and 0.7 mM)
and the data were linearized with Lineweaver–Burke
plot for Vmax and the Ki determination. Enzyme activity
was also measured in the presence of the same sub-
1
and 3172 cm−1 33. e H-NMR spectra of compounds
4–6 showed singlet peaks that belong to protons of the
methyl groups in position 3 and 6 between 0.84 and 1.11
ppm. e compounds 9–12 showed singlet peaks that
belong to protons of the methyl groups in position 2
and 7 between 0.81 and 1.11 ppm30. e CH2 group pro-
tons of the cyclohexene rings of the compounds 4–12
showed multiple peaks in the 1.54–2.58 ppm range30.
Signals for the methoxy group protons for compounds
5, 7, 8, 11 were shown in the range of 3.52–3.92 ppm. e
signals for the CH protons at 4.98–5.35 ppm and signals
for the aromatic protons in the range of 6.44–8.39 ppm
were observed. Signals of pyridine ring protons for the
compound 6 showed in the range of 7.01–8.39 ppm.
Hydroxyl group proton of compound 10 was observed
as broad signal at 8.25 ppm. e broad singlet peaks
between 4.94 and 9.02 ppm were assigned to sulfon-
amide (−SO2NH2) groups protons of all the compounds
4–12 (Scheme 2).
e concentration required inhibiting hCA I and hCA
II activities of the purified proteins by 50% (IC50) and
inhibition equilibrium constant (Ki) was determined for
each compound. Sulfanilamide and AAZ were used as
control compounds to compare inhibition potential of
newly synthesized compounds 4–12.
According to in vitro studies, any inhibition effects of
4–12 compounds were not observed on hydratase activ-
ity of hCA I and hCA II isoenzymes. e IC50 and Ki values
obtained from esterase activities of these compounds
were shown in Table 1.
strate concentrations but in the absence of any inhibi-
26
tor to determine the Vmax
.
result and discussion
e general synthetic method shown in Scheme 1 is
employed to prepare dioxoacridine sulfonamide deriva-
tives (4–12). All spectral data are in agreement with the
assigned structures.
e synthesis of dioxoacridine sulfonamide com-
pounds were realized in water in a single process through
twosuccessivereactions(AldolcondensationandMichael
addition) and using a phase transfer catalyst-Bronsted
acid as p-dodesilbenzensulphonic acid (DBSA). In recent
years, using DBSA as a combine catalyst (phase transfer
catalyst-Bronsted acid) has been a popular application
in organic chemistry28–30. Sulfonamide compounds were
prepared by one pot reaction in processing high yields
and simple work-up procedure.
All of the new inhibitor compounds are more effective
than control compounds (sulfanilamide and AAZ) for
Scheme 1. Synthesis of acridine-1,3-dion sulfonamides (4–12).
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of dioxoacridine sulfonamide derivatives 513
Scheme 2 . New CA inhibitors (4–12).
Table 1. e Ki values obtained from in vitro inhibition
experiments.
Ki Values (µM)
Inhibitor
hCA I
hCA II
2.2–2.1
AAZ
3.1–3.0
Sulfanilamide
4,6–4,4
3,9–3,8
4
5
6
7
8
9
10
11
12
0.38–0.66
0.08–0.07
0.05–0.06
0.01–0.02
0.008–0.009
0.13–0.14
0.04–0.05
0.19–0.22
0.21–0.23
0.19–0.31
0.18–0.12
0.12–0.14
0.001–0.002
0.006–0.008
0.09–0.10
0.11–0.14
0.11–0.13
0.03–0.04
Figure 1. SDS-PAGE analysis of CA isoenzymes: (a) Standard hCA
I, (b) isolated hCA I, (c) standard hCA II, (d) isolated hCA II.
hCA I and hCA II. Especially compounds 5, 6, 7, 8 and
10 have shown remarkable inhibition on hCA I and hCA
II isoenzymes.
e Ki values of all the novel compounds are lower
than control compounds for hCA I and hCA II.
In summary, these new compounds have potential
inhibitory effects; so these compounds can be candidates
for in vivo studies of glaucoma treatment.
Compounds 5, 7, 8 and 11 contain methoxy group,
while compound 10 contain hydroxyl group. ese com-
pounds are the most effective inhibitors in the newly
synthesized compounds. It is thought that these groups
in abovementioned compounds increase interactions of
inhibitor-zinc metal and inhibitor-histidine residues in
the active site of hCA I and hCA II isoenzymes34.
conclusion
A series of dioxoacridine sulfonamide containing nitrile,
methoxy, halogen, and hydroxyl groups were synthe-
sized. Synthesized compounds have inhibitory potential
© 2012 Informa UK, Ltd.

514 M. Kaya et al.
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acknowledgments
e authors are grateful to Prof. Dr. Yılmaz Yıldırır, Gazi
University, Ankara, Turkey.
Declaration of interest
e authors are very grateful to Dumlupinar University
Research Fund for providing financial support for this
project (Grant No. 2008-4).
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