Novel sulfamides as potential carbonic anhydrase isoenzymes inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.01.019

Sulfamides represent an important class of biologically active compounds. A series of novel sulfamides were synthesized from 1-aminoindanes, 1-aminotetralin, 2-aminoindanes and 2-aminotetralin via the reactions of free amines, benzyl alcohol and chlorosulfonyl isocyanate (CSI) followed by hydrogenolysis of the obtained sulfamoylcarbamates. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the new sulfamides have been investigated. The human (h) isozymes hCA I and hCA II have been investigated in this study by using an esterase assay with 4-nitrophenyl acetate as substrate. The new sulfamides showed inhibition constants in the micro-submicromolar range, with one compound (N-(indane-1-yl)sulfamide) showing a K_i of 0.45 μM against hCA I and of 1.07 μM against hCA II.

Full text of DOI:10.1016/j.bmc.2013.01.019

Bioorganic & Medicinal Chemistry 21 (2013) 1379–1385
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel sulfamides as potential carbonic anhydrase isoenzymes
inhibitors
a
c
Akın Akıncıog˘lu a, Yusuf Akbaba a, Hülya Göçer b, Süleyman Göksu a, , Ilhami Gülçin , Claudiu T. Supuran
⇑
_
a Faculty of Science, Department of Chemistry, Atatürk University, 25240 Erzurum, Turkey
b Agri Ibrahim Cecen University, Central Researching Laboratory, 04200 Agri, Turkey
c Università degli Studi di Firenze, Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, 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:
Available online 22 January 2013
Sulfamides represent an important class of biologically active compounds. A series of novel sulfamides
were synthesized from 1-aminoindanes, 1-aminotetralin, 2-aminoindanes and 2-aminotetralin via the
reactions of free amines, benzyl alcohol and chlorosulfonyl isocyanate (CSI) followed by hydrogenolysis
of the obtained sulfamoylcarbamates. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the new
sulfamides have been investigated. The human (h) isozymes hCA I and hCA II have been investigated
in this study by using an esterase assay with 4-nitrophenyl acetate as substrate. The new sulfamides
showed inhibition constants in the micro–submicromolar range, with one compound (N-(indane-1-
Keywords:
Aminoindane
Aminotetralin
Sulfamide
Carbonic anhydrase
Inhibitors
yl)sulfamide) showing a Ki of 0.45
lM against hCA I and of 1.07
lM against hCA II.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
In the hydration direction, the first step of the reaction involves
the binding of a CO2 molecule within the active site.13 The next
step is the nucleophilic attack of a Zn2+-bound hydroxide ion on
CO2 with consequent formation of HCOꢀ3 , which is then displaced
from the active site by a water molecule. In the second step, the
Zn2+–OHꢀ is regenerated as a result of the transfer of a proton from
the Zn2+–H2O generated in step one, to the bulk solvent.14 A Zn2+
hydroxide species (ꢁZn2+–OHꢀ) of the enzyme is the catalytically
active species, acting as a strong nucleophile on the CO2 molecule
bound in a hydrophobic pocket nearby.13 This metal hydroxide
species is generated from water coordinated to the metal ion,
which is found at the bottom of the active site cavity (Scheme 1).
Sulfonamides are the most important CA inhibitors, which bind
in a tetrahedral geometry of the Zn2+ ions, in deprotonated state
(Scheme 2).15,16
Sulfamides are prominent biologically active compounds.1
Most drugs contain this functional group in their structures.
Sulfamide drug quinagolide (1), commercially known as norprolac,
is a selective D2 receptor agonist and used in the treatment
of hyperprolactinemia.2 Sulfamide doripenem (2), tradename
doribax, is a broad-spectrum injectable antibiotic.3 Compound 3
(JNJ-26990990) is an anticonvulsants drug.4 It has also been
reported that this compound has potential use in the treatment of
inflammatory pain, neuropathic pain, and depression.5 Further,
norovirus inhibitory,6 renin inhibitory,7 mammalian 15-lipoxyge-
nase inhibitory,8 glycogen phosphorylase inhibitory,9 antitumor,10
cholesterol acyltransferase inhibitory11 and a lot of other activities
of sulfamides have been reported (Fig. 1).
The carbonic anhydrases (CAs, E.C. 4.2.1.1) are a superfamily of
metalloenzymes, which catalyze the intercoꢀnversion of carbon
dioxide (CO2) and water to bicarbonate (HCO3 ) and protons (H+)
by using a metal hydroxide nucleophilic mechanism.12 CA follows
a ping–pong catalytic mechanism for the hydration and dehydra-
tion of CO2 and HCOꢀ, respectively:
There are five distinct carbonic anhydrases genetic families
known to date, the a-, b-, c-, d- and f-carbonic anhydrases, which
differ in their preference for metal ions used within the active site
for performing the catalysis. Zn2+ ions are used by all five classes
mentioned above.17 The b-carbonic anhydrases are predominantly
in eubacteria, algae, and chloroplasts of both mono- as well as
dicotyledons. The
c-carbonic anhydrases are mainly in Archaea
Enzyme ꢁ Zn2þ ꢀ OHꢀ þ CO2 () Enzyme ꢁ Zn2þ ꢀ HCO3
and some eubacteria.12,18–20 Up to now, in higher vertebrates,
including humans, 16 different CA isozymes or CA-related proteins
have been described, which possess different subcellular localiza-
tion and tissue distribution. Among CA isozymes, CA I, CA II, CA
III and CA VII are the cytosolic ones. CA I is found together with
CA-II in erythrocytes. CA-II is the most widely distributed CA in
the eye, kidney, central nervous system and inner ear. Also, there
H2
O
Enzyme ꢁ Zn2þ ꢀ HCO3ꢀ () Enzyme ꢁ Zn2þ ꢀ H2O þ HCOꢀ
3
Enzyme ꢁ Zn2þ ꢀ H2O þ B () Enzyme ꢁ Zn2þ ꢀ OHꢀ þ BHþ
⇑
Corresponding author.
E-mail address: sgoksu@atauni.edu.tr (S. Göksu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.019

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A. Akıncıog˘lu et al. / Bioorg. Med. Chem. 21 (2013) 1379–1385
OH
O
O
S
O
N
H
N
H
H
NH2
N
S
O
S
O
S
O
N
H
N
NH2
H
H
N
H
HO
O
S
N
H
O
OH
2
1
3
Figure 1. Some sulfamide drugs and agents in clinical development (3).
Hy
dro
pho
bic p
oce
t
(
Va
l 121
, Val
143
, Le
u 198
)
O
C
O
C
O
H
OH
O
O
Zn2+
Zn2+
His 94
His 119
His 94
His 119
His 96
His 96
B
C
O
2
H
+
Inh_
Inh-
H2O
Zn2+
OH
OH2
-H+
B
+InhH
-H2O
Zn2+
Zn2+
Zn2+
-BH+
InhH
His 94
His 119
His 94
His 94
His 119
His 119
His 94
His 119
His 96
D
His 96
A
His 96
F
His 96
E
Scheme 1. Catalytic and inhibition mechanisms of carbonic anhydrases.
group to coordinate the catalytic zinc.17,31,32 These compounds
are widely used clinically, mainly as antiglaucoma agents but also
for the therapy of other diseases such as increased intracranial
pressure, various neurological/neuromuscular pathologies such as
epilepsy, genetic hemiplegic migraine, and ataxia, tardive diskine-
sia, hypokalemic periodic paralysis, essential tremor and Parkin-
son’s disease, and mountain sickness. Accordingly, drugs of this
pharmacological class are under constant development.12,15,19,30,33
Because of the important biological activities of sulfamides, bio-
isosteres of the sulfonamides, the synthesis and CA inhibitory
properties of sulfamides 16–21 will be useful for further synthetic
and biological purposes. In this context, here we report the synthe-
sis of sulfamide derivatives 16–21. We also evaluate CA isoen-
zymes (hCA I and hCA II) inhibitory effects of these compounds.
O
His-119
2+
R
S
N
H
Zn
His-96
O
His-94
Enzyme-Zn2+-I + H2O
Enzyme-Zn2+-OH2 +I
Scheme 2. Binding of sulfamide and Zn2+ ion.
are membrane-bound (CA IV, CA IX, CA XII, CA XIV), mitochondrial
(CA V), secretory forms (CA VI) and several acatalytic forms (CA
VIII, CA X, CA XI).21–23 Other CA isoforms are found in a variety
of tissues where they participate in several important biologic pro-
cesses.24–28 As mentioned above, these enzymes catalyze a very
simple physiological reaction, the interconversion between CO2
and HCOꢀ3 HCOꢀ3 , and are involved in crucial physiological processes
2. Results and discussion
2.1. Chemistry
connected with respiration and transport of CO2/HCOꢀ between
3
metabolizing tissues and the lungs, pH and CO2 homeostasis, elec-
trolyte secretion in a variety of tissues or organs, biosynthetic reac-
tions such as gluconeogenesis, lipogenesis, and ureagenesis, bone
resorption, calcification, tumorigenicity, and many other physio-
logic or pathologic processes.12,18,20,29 Thus, it is not surprising that
many of these isozymes have been discovered as important targets
for inhibitors with clinical applications.19,28,30 Almost all of the
most potent inhibitors of carbonic anhydrases isoenzymes contain
a terminal sulfonamide, sulfamide and phenolic as the anchoring
Amines 4–9 were prepared according to the literature proce-
dure described previously.34,35 Sulfamides can be synthesized from
the reaction of amines with CSI,9,36 ClSO2NH237 or other methods.4
Here, the first synthetic methodology was chosen for the synthesis
of sulfamides. For this purpose, sulfamide carbamates 10–15 were
obtained in moderate yields from the reactions of 1 equiv amines
4–9 and 1.4 equiv PhCH2OH with CSI at 0–25 °C for 4 h. Pd-C cata-
lyzed hydrogenolysis of carbamates containing benzyl groups to

A. Akıncıog˘lu et al. / Bioorg. Med. Chem. 21 (2013) 1379–1385
1381
O
S
O
O
O
S
O
H
N
R1
R1
R4
R1
R4
HN
( )
NH2
NH
NH2
OCH2Ph
R2
R3
R2
R3
R2
R3
i
ii
( )
( )
n
n
n
R4
10
11
4 R1=R4=H, R2=R3 =OMe, n=1
5 R1=R2=R3 = R4=H, n=1
R1=R4=H, R2=R3 =OMe, n=1, 59% 16
R1=R4=H, R2=R3 =OMe, n=1, 71%
R1=R2=R3 = R4=H, n=1,
58% 17
R1=R2=R3 = R4=H, n=1,
70%
6
12 R1=R2=R3 =H, R4=OMe, n=2, 64%
R1=R2=R3 =H, R4=OMe, n=2
18 R1=R2=R3 =H, R4=OMe, n=2, 76%
R1
R1
R2
O
S
O
R1
R2
O
S
O
ii
O
H
N
H
N
H
N
i
NH2
NH2
R2
n
OCH2Ph
n
n
7 R1=R2=H, n=2
13 R1=R2=H, n=2,
59%
R1=R2=OMe, n=1, 57%
15 R1=R2=H, n=1, 58%
19 R1=R2=H, n=2,
80%
R1=R2=OMe, n=1, 76%
21 R1=R2=H, n=1, 82%
8
R1=R2=OMe, n=1
14
20
9 R1=R2=H, n=1
Scheme 3. Synthesis of sulfamides: (i) CSI/NEt3/PhCH2OH, CH2Cl2, 0–25 °C, 4 h; (ii) H2/Pd-C, MeOH, 25 °C, 4 h.
give amine related compounds has been given clearly in the liter-
ature.38–40 By a similar approach, hydrogenolysis of sulfamoyl car-
bamates 10–15 afforded sulfamides 16–21 in good yields
(Scheme 3). The structures of all synthesized compounds were
characterized by 1H, 13C NMR.
of 1285.4 EU mgꢀ1 and an overall yield of 27.1%. We report
here the first study on the inhibitory effects of novel sulfamides
16–21 on the esterase activity of hCA I and hCA II. The inhibitor
concentration that caused 50% inhibition (IC50) was determined
from activity versus (%)–[sulfamides] plots and the average inhibi-
tion constant (Ki values) were calculated from Lineweaver–Burk
plots (Table 1). The data of Table 1 shows the following inhibition
of hCA I and hCA II with sulfamides 16–21 by an esterase assay,
with 4-NPA as substrate.
2.2. CA isoenzymes inhibition studies
2.2.1. CA isoenzymes purification and activity assay
Inhibition of the carbonic anhydrase has pharmacologic appli-
cations in the field of antiglaucoma, anticonvulsant and anticancer
agents. It is well known that sulfamides is the main class of CA
inhibitors, which bind to the metal ion from the enzyme active
site.17 In order to remind CA inhibition mechanism by sulfamide
inhibitors (Scheme 1) should be noted that the active domain of
CA isoenzymes contains an active zinc ion (Zn2+) site; a strong Le-
wis acid that binds to, and activates a substrate H2O molecule to
catalyze the reversible hydration reaction of carbon dioxide. The
metal ion is situated at the bottom of active site, being coordinated
by three histidine residues (His 94, 96 and 119) and water mole-
cule/hydroxide ion (Scheme 1).41 Also, It was known that sulfa-
mides were one of the most important CA inhibitors, bind in a
tetrahedral geometry of the Zn2+ ion, in deprotonated state
(Scheme 2).
The metal-complexing anions and the sulfamides with a termi-
nal –SO2NH2 group that coordinate to the Zn2+ ion. Simple anions
such as HS–, CN–, NCO–, N3–, HSOꢀ3 , Iꢀ and HCOOꢀ may bind with
tetrahedral geometry or form trigonal–bipyramidal coordina-
tion,20,29 whereas the sulfamides replace the water coordinated
to zinc and the ‘deep-water’ hydrogen-bonded to Thr199-NH. Both
waters are present in the uncomplexed state.42
2.2.2. CA isoenzymes inhibition effects
There are many studies in the literature on the interactions of dif-
ferent compounds and carbonic anhydrase isoenzymes. The interac-
tion of dantrolene,22 melatonin,30 vitamin E,33 morphine,31
ethanol,43 sildenafil,32 antioxidant phenols,18 phenolic acids,24,26
natural polyphenols products,25,41 phenolic compounds,27
synthetic methanon derivatives28 with two human isozymes, hCA I
and hCA II, has recently been investigated, evidencing several inhib-
itors. Indeed, the inhibition profiles of hCA I and hCA II with novel
sulfamide compounds 16–21 are very variable, with inhibition con-
stants ranging from the nanomolar to the micromolar level for novel
sulfamides. For this purpose, hCA I and hCA II inhibitory effects of the
novel sulfamide compounds 16–21 were tested under in vitro condi-
tions and IC50 and Ki values were calculated and given in Table 1.
We report here the initial study on the inhibitory effects of syn-
thesised sulfamides 16–21 and on the hydratase activity of hCA I
and hCA II. The data in Table 1 shows the following regarding the
inhibition of hCA I and hCA II activity by sulfamides 16–21. The
strongest inhibitory activity has been observed with compound
17, investigated here for the inhibition of the rapid cytosolic iso-
In the present study, both hCA I and hCA II were purified from
human erythrocytes. The purification of both CA isozymes was
performed using a simple one step method with a Sepharose-
4B-L
-Thyrosine-sulfanilamide affinity column chromatography.32
Human erythrocyte CA I isoenzyme was purified, 336.1-fold with
a specific activity of 1445.2 EU mgꢀ1 and overall yield of 62.4%;
CA II isoenzyme was purified, 298.9-fold with a specific activity
Table 1
Human carbonic anhydrase isoenzymes (hCA I and hCA II) inhibition data with some
novel sulfamides 16–21 by an esterase assay with 4-nitrophenylacetate as substrate
Compounds
Ki (lM)
zymes hCA I (Ki: 0.45 0.23
l
M, r2: 0.993) and hCA II (Ki:
hCA-I
r2
hCA-II
r2
1.07 0.22
l
M, r2: 0.984). We speculated that aromatic groups of
16
17
18
19
9.31 4.67
0.45 0.23
6.72 2.33
8.30 0.61
10.23 4.27
1.54 0.37
36.2b
0.993
0.982
0.986
0.994
0.987
0.992
—
8.86 1.06
1.07 0.22
3.09 0.22
6.75 1.31
13.44 3.25
1.28 0.22
3.70b
0.985
0.984
0.981
0.993
0.982
0.992
—
this molecule sterically facilitated the inhibition of hCA I and hCA II.
It has been reported that sulfamide and sulfamic acid act as
moderate hCA II inhibitors, with inhibition constants of 1130 lM
20
for sulfamide and 390 lM for sulfamic acid at the physiological
pH (7.4), respectively.17 In contrast to these literature, in the pres-
ent study, we observed strong inhibitory effect of synthesized
sulfamide compounds (16–21) on hCA I and hCA II. Ki values
of these sulfamide compounds (16–21) are in the range of
21
AZAa
a
Acetazolamide (AZA) was used as positive standard of CA I and CA II inhibitor.
From Ref. 26.
b

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A. Akıncıog˘lu et al. / Bioorg. Med. Chem. 21 (2013) 1379–1385
0.45–10.23
l
M for hCA I and 1.07–13.44
lM for hCA II (Table 1).
4.1.1. Standard procedure for the synthesis of
The arithmetic average Ki values of novel synthesized sulfamides
16–21 is 6.72 2.08 M for hCA I and 5.75 1.05 M for hCA II.
sulfamoylcarbamates: benzyl N-(5,6-dimethoxyindane-1-
yl)sulfamoylcarbamate (10)
l
l
The half maximal inhibitory concentration (IC50) is a measure of
the effectiveness of novel sulfamides 16–21 in inhibiting CA
isoenzyme function. A lower IC50 and Ki value reflects strong CA
isoenzyme inhibition effect. It is widely known that sulfamides
compounds are CA isoenzymes.44–47 The results obtained from
Table 1 clearly showed that the physiologically dominant cytosolic
isozymes hCA I and hCA II were effectively inhibited by sulfamide
compounds 16–21, with KIs within the range of 0.45 0.23 to
Benzylalcohol (0.65 g, 6.01 mmol) was added to a solution of
CSI (0.61 g, 4.30 mmol) in CH2Cl2 (10 mL) at 0 °C. A solution of
amine 4 (0.83 g, 4.30 mmol) in CH2Cl2 (30 mL) and NEt3 (0.48 g,
4.72 mmol) were added to the solution of CSI drop wise and stirred
at 0 °C for 1 h. then at room temp for 3 h. The reaction mixture was
cooled to 0 °C and to this mixture was added a solution of 0.1 N HCl
(50 mL). Organic phase was separated and H2O phase was ex-
tracted with CH2Cl2 (2 ꢂ 30 mL). Combined organic layers were
dried over Na2SO4 and the solvent was evaporated. Column chro-
matography of the residue on silica gel (30 g) with 30% EtOAc/hex-
ane yielded carbamate 10 (2.52 g, 59%) as white solid.
13.44 3.25 lM. The structure–activity relationship and inhibition
properties of hCA II isoenzyme is particularly comparable to that
what is outlined above for hCA I, due to the two isoenzymes having
a high sequence homology of amino acid present within the active
site.44,48 All synthesized sulfamide compounds were much stron-
ger CA I inhibition effects than the clinically used acetazolamide
(AZA) included in the assays as a standard inhibitor. On the other
hand, CA II inhibition effects of some sulfamides 17, 18 and 21
are stronger than that of AZA, but the inhibition effects of other
novel sulfamides (16, 19 and 20) is close to AZA (Table 1).
The synthesized sulfamides 16–21 have one aromatic ring.
These new synthesised compounds have been investigated as CA
isoenzymes inhibitors in this study. The rationale of investigating
these compounds as CA inhibitors exists in the fact that the com-
pounds with aromatic rings have been shown to be the only com-
petitive inhibitor with CO2 as the substrate for the main isoform of
CA, that is, human CA I and CA II.
Sulfamoylcarbamates 11–15 were also synthesized by this pro-
cedure with yields of 58%, 64%, 59%, 57% and 58%, respectively.
4.1.2. Benzyl N-(5,6-dimethoxyindane-1-yl)sulfamoylcarbamate
(10)
White solid. Mp 146–148 °C. 1H NMR (400 MHz, CDCl3) d 8.03
(br s, 1H, NH), 7.37–7.30 (m, 5H, Ph-H), 6.85 (s,1H, Ar-H), 6.70 (s,
1H, Ar-H), 5.54 (d, 1H, NH, J = 8.4 Hz), 5.17 (A part of AB, d, 1H,
Ha of CH2, J = 12.1 Hz), 5.12 (B part of AB, d, 1H, Hb of CH2,
J = 12.1 Hz), 4.84 (dd, 1H, CH–N, J = 7.3 and J = 14.9 Hz), 3.84 (s,
3H, OCH3), 3.79 (s, 3H, OCH3), 2.84 (A part of AB, ddd, 1H, Ha of
CH2, J = 4.0, J = 8.7 and J = 15.5 Hz), 2.73–2.65 (B part of AB, m,
1H, Hb of CH2), 2.43 (A part of AB, dddd, 1H, Ha of CH2, J = 4.1,
J = 7.8, J = 12.3 and J = 15.5 Hz), 1.88–1.81 (B part of AB, m, 1H,
Hb of CH2). 13C NMR (100 MHz, CDCl3) d 151.7 (CO), 150.0 (C)
148.8 (C), 135.0 (C), 134.8 (C), 132.6 (C), 129.1 (CH), 128.9 (2CH),
128.7 (2CH), 107.7 (CH), 107.6 (CH), 68.7 (OCH2), 60.1 (CH–N),
56.3 (OCH3), 56.2 (OCH3), 33.8 (CH2), 30.2 (CH2). IR (CH2Cl2,
cmꢀ1): 3579, 3501, 3264, 3064, 2939, 2857, 2829, 1717, 1607,
1504, 1453, 1355, 1308, 1258, 1222, 1158, 1096, 1032, 1001. Anal.
Calcd for (C19H22N2O6S): C, 56.15; H, 5.46; N, 6.89; S, 7.89. Found:
C, 56.03; H, 5.49; N, 6.85; S, 7.92.
3. Conclusions
In summary, a series of sulfamides 16–21 were synthesized
starting from their corresponding free amines. These novel sulfa-
mides and their starting reagents sulfamoylcarbamates may be
important synthons for further synthetic and biological purposes.
In addition, carbonic anhydrase inhibitory properties of 16–21
have also been evaluated. Some of the compounds investigated
here showed effective CA inhibitory activity, in the low-micromo-
lar range, by the esterase method, which usually gives Ki values
and order of magnitude higher as compared to the CO2 hydratase
assay. These compounds 16–21 are selective hCA I and hCA II
inhibitors with selectivity ratios in the range of 0.45 0.23 to
4.1.3. Benzyl N-(indane-1-yl)sulfamoylcarbamate (11)
White solid. Mp 136–138 °C. 1H NMR (400 MHz, CDCl3) d 7.42–
7.37 (m, 5H, Ph-H), 7.35–7.16 (m, 5H, Ar-H and NH), 5.36 (br s, 1H,
NH), 5.21 (A part of AB, d, 1H, Ha of CH2, J = 12.0 Hz), 5.18 (B part of
AB, d, 1H, Hb of CH2, J = 12.0 Hz), 4.89–4.87 (m, 1H, CH–N), 2.93–
2.90 (m, 1H, CH2), 2.89–2.76 (m, 1H, CH2), 2.47–2.43 (m, 1H,
CH2), 1.91–1.88 (m, 1H, CH2). 13C NMR (100 MHz, CDCl3) d 151.5
(CO), 143.2 (C) 141.1 (C), 134.8 (C), 129.2 (2CH), 129.0 (CH),
128.9 (2CH), 128.87 (2CH), 127.2 (2CH), 125.1 (CH), 124.5 (CH),
68.9 (OCH2), 59.8 (CH–N), 33.8 (CH2), 30.3 (CH2). IR (CH2Cl2,
cmꢀ1): 3512, 3274, 3064, 3030, 2945, 2851, 1721, 1458, 1356,
1231, 1162, 1102, 1074. Anal. Calcd for (C17H18N2O4S): C, 58.94;
H, 5.24; N, 8.09; S, 9.26. Found: C, 58.90; H, 5.22; N, 8.11; S, 9.28.
13.44 3.25 lM. Also, these findings point out that novel sulfa-
mides 16–21 may be used as leads for generating potent hCA I
and hCA II inhibitors eventually targeting other isoforms, which
have not been assayed yet for their interactions with such agents.
4. Experimental
All chemicals and solvents are commercially available and were
used after distillation or treatment with drying agents. Melting
points were determined on a capillary melting apparatus (BUCHI
530) and are uncorrected. IR spectra were obtained from solutions
in 0.1 mm cells with a Perkin–Elmer spectrophotometer. The 1H
and 13C NMR spectra were recorded on a 400 (100)-MHz Varian
and 400 (100)-MHz Bruker spectrometer; d in ppm, Me4Si as the
internal standard. Elemental analyses were performed on a Leco
CHNS-932 apparatus. All column chromatography was performed
on silica gel (60-mesh, Merck). PLC is preparative thick-layer chro-
matography: 1 mm of silica gel 60 PF (Merck) on glass plates.
4.1.4. Benzyl N-(5-methoxy-1,2,3,4-tetrahydronaphthalene-1-
yl)sulfamoylcarbamate (12)
White solid. Mp 152–154 °C. 1H NMR (400 MHz, CDCl3) d 7.54
(br s, 1H, NH), 7.37–7.32 (m, 5H, Ar-H), 7.09 (dd, 1H, Ar-H, J = 7.7
and J = 8.1 Hz), 6.90 (d, 1H, Ar-H, J = 7.7 Hz), 6.70 (d, 1H, Ar-H,
J = 8.1 Hz), 5.26 (d, 1H, NH, J = 7.3 Hz), 5.19 (s, 2H, CH2), 4.58–
4.54 (m, 1H, CHN), 3.80 (s, 3H, OCH3), 2.68 (A part of AB, dt, 1H,
Ha of CH2, J = 5.7 and J = 18.3 Hz), 2.52 (B part of AB, dt, 1H, Hb
of CH2, J = 7.2 and J = 18.3 Hz), 1.92–1.70 (m, 4H, 2CH2). 13C NMR
(100 MHz, CDCl3) d 157.3 (CO), 151.4 (C), 135.8 (C), 134.9 (C),
129.1 (C) 129.0 (2CH), 128.9 (2CH), 126.9 (CH), 121.1 (CH), 109.2
(CH), 68.8 (OCH2), 55.6 (OCH3), 53.3 (CH–N), 29.3 (CH2), 22.7
(CH2), 18.4 (CH2). IR (CH2Cl2, cmꢀ1): 3647, 3501, 3274, 3064,
3036, 2939, 2840, 1717, 1587, 1469, 1356, 1304, 1253, 1225,
4.1. Synthesis
Amines 4–9 were synthesized according to the procedure de-
scribed by us lately.34

A. Akıncıog˘lu et al. / Bioorg. Med. Chem. 21 (2013) 1379–1385
1383
1156, 1099, 1076. Anal. Calcd for (C19H22N2O5S): C, 58.45; H, 5.68;
N, 7.17; S, 8.21. Found: C, 58.43; H, 5.67; N, 7.19; S, 8.18.
108.6 (CH), 58.7 (CH–N), 56.30 (OCH3), 56.27 (OCH3), 34.8 (CH2),
30.1 (CH2). IR (CH2Cl2, cmꢀ1): 3366, 3282, 3014, 2935, 2901,
2834, 1605, 1560, 1504, 1460, 1440, 1418, 1331, 1317, 1264,
1218, 1191, 1147, 1093, 1032. Anal. Calcd for (C11H16N2O4S): C,
48.52; H, 5.92; N, 10.29; S, 11.77. Found: C, 48.49; H, 5.95; N,
10.25; S, 11.72.
4.1.5. Benzyl N-(1,2,3,4-tetrahydronaphthalene-2-
yl)sulfamoylcarbamate (13)
White solid. Mp 140–142 °C. 1H NMR (400 MHz, CDCl3) d 7.72
(br s, 1H, NH), 7.38ꢀ7.34 (m, 5H, Ar-H) 7.15–7.05 (m, 3H, Ar-H),
6.96 (d, 1H, Ar-H, J = 6.6 Hz), 5.36 (d, 1H, NH, J = 6.9 Hz), 5.17 (s,
2H, CH2), 3.78–3.70 (m, 1H, CH–N), 3.03 (A part of AB, dd, 1H, Ha
of CH2, J = 5.1 and J = 16.3 Hz), 2.90–2.76 (m, 2H, CH2), 2.70 (B part
of AB, dd, 1H, Hb of CH2, J = 8.4 and J = 16.3 Hz), 2.05–2.00 (m, 1H,
CH2), 1.82–1.73 (m, 1H, CH2). 13C NMR (100 MHz, CDCl3) d 151.6
(CO), 135.2 (C), 134.9 (C), 133.3 (C), 129.6 (CH), 129.1 (CH),
129.02 (2CH), 129.0 (2CH), 128.8 (CH), 126.7 (CH), 126.3 (CH),
68.8 (OCH2), 50.8 (CH–N), 36.1 (CH2), 29.3 (CH2), 27.2 (CH2). IR
(CH2Cl2, cmꢀ1): 3669, 3276, 3059, 3025, 2925, 2851, 1725, 1622,
1494, 1454, 1353, 1301, 1229, 1160, 1113, 1071. Anal. Calcd for
(C18H20N2O4S): C, 59.98; H, 5.59; N, 7.77; S, 8.90. Found: C,
59.93; H, 5.60; N, 7.76; S, 8.86.
4.1.9. N-(Indane-1-yl)sulfamide (17)
Standard procedure described above at 4.1.85 was applied to
sulfamoylcarbamate 11 to give 17 (70%). White solid. Mp 115–
117 °C (lit.49 Mp 115–116 °C). 1H NMR (400 MHz, CDCl3) d 7.42–
7.17 (m, 4H, Ar-H), 4.94–4.84 (m, 4H, CH–N, NH and NH2), 2.96
(A part of AB, ddd, 1H, Ha of CH2, J = 3.9, J = 8.6 and J = 15.9 Hz),
2.84–2.75 (B part of AB, m, 1H, Hb of CH2), 2.59 (A part of AB, dddd,
1H, Ha of CH2, J = 4.0, J = 7.6, J = 12.1 and J = 14.6 Hz), 2.00–1.91 (B
part of AB, m, 1H, Hb of CH2). 13C NMR (100 MHz, CDCl3) d 143.3
(C) 142.2 (C), 128.6 (CH), 127.1 (CH), 125.1 (CH), 124.6 (CH), 59.3
(CH–N), 34.7 (CH2), 30.3 (CH2). IR (CH2Cl2, cmꢀ1): 3361, 3272,
3019, 2941, 2845, 1569, 1476, 1445, 1334, 1317, 1295, 1136,
1104, 1079, 1037, 1001. Anal. Calcd for (C9H12N2O2S): C, 50.92;
H, 5.70; N, 13.20; S, 15.11. Found: C, 50.97; H, 5.66; N, 13.15; S,
15.16.
4.1.6. Benzyl N-(5,6-dimethoxyindane-2-yl)sulfamoylcarbamate
(14)
White solid. Mp 162–163 °C. 1H NMR (400 MHz, CDCl3) d 7.74
(br s, 1H, NH), 7.39–7.33 (m, 5H, Ph-H), 6.66 (s, 2H, Ar-H), 5.55
(d, 1H, NH, J = 7.9 Hz), 5.18 (s, 2H, CH2), 4.22–4.19 (m, 1H, CH–
N), 3.83 (s, 6H, 2OCH3), 3.12 (A part of AB, dd, 2Ha of CH2, J = 7.4
and J = 15.5), 2.76 (B part of AB, dd, 2Hb of CH2, J = 6.2 and
J = 15.5). 13C NMR (100 MHz, CDCl3) d 151.6 (CO), 148.8 (2C),
134.9 (2C), 131.5 (C), 129.1 (CH), 129.0 (2CH), 128.9 (2CH), 108.0
(2CH), 68.8 (OCH2), 56.3 (2OCH3), 55.9 (CH–N), 39.8 (2CH2). IR
(CH2Cl2, cmꢀ1): 3613, 3495, 3270, 3064, 3036, 2997, 2939, 2834,
1724, 1611, 1505, 1455, 1357, 1310, 1250, 1230, 1216, 1186,
1159, 1099, 1054, 1026. Anal. Calcd for (C19H22N2O6S): C, 56.15;
H, 5.46; N, 6.89; S, 7.89. Found: C, 56.16; H, 5.41; N, 6.84; S, 7.92.
4.1.10. N-(5-Methoxy-1,2,3,4-tetrahydronaphthalene-1-
yl)sulfamide (18)
Applying of the standard procedure described above at 4.1.8 to
sulfamoylcarbamate 12 gave 18 (76%). White solid. Mp 122–
124 °C. 1H NMR (400 MHz, CDCl3) d 7.15 (dd, 1H, Ar-H, J = 7.8
and J = 8.0 Hz), 7.06 (d, 1H, Ar-H, J = 7.8 Hz), 6.72 (d, 1H, Ar-H,
J = 8.0 Hz), 4.82 (br s, 2H, NH2), 4.67 (d, 1H, NH, J = 7.6 Hz), 4.61–
4.56 (m, 1H, CHN), 3.80 (s, 3H, OCH3), 2.69 (A part of AB, dt, 1H,
Ha of CH2, J = 5.6 and J = 17.9 Hz), 2.52 (B part of AB, dt, 1H, Hb
of CH2, J = 7.3 and J = 17.9 Hz), 2.01–1.96 (m, 2H, CH2), 1.83–1.77
(m, 2H, CH2). 13C NMR (100 MHz, CDCl3) d 157.3 (C), 137.0 (C),
126.94 (C), 126.86 (CH), 121.1 (CH), 108.9 (CH), 55.6 (OCH3), 52.5
(CH–N), 30.1 (CH2), 22.9 (CH2), 18.6 (CH2). IR (CH2Cl2, cmꢀ1):
3669, 3271, 3064, 3031, 2952, 2913, 2845, 2297, 1721, 1611,
1457, 1358, 1306, 1228, 1160, 1088, 1052. Anal. Calcd for
(C11H16N2O3S): C, 51.54; H, 6.29; N, 10.93; S, 12.51. Found: C,
51.50; H, 6.31; N, 10.90; S, 12.48.
4.1.7. Benzyl N-(indane-2-yl)sulfamoylcarbamate (15)
White solid. Mp 144–146 °C. 1H NMR (400 MHz, CDCl3) d 7.7 (br
s, 1H, NH), 7.37 (br s, 5H, Ar-H), 7.18–7.12 (m, 4H, Ar-H), 5.57 (d,
1H, NH, J = 7.7 Hz), 5.18 (s, 2H, CH2), 4.24–4.15 (m, 1H, CH–N),
3.18 (A part of AB, dd, 2H, 2Ha of CH2, J = 7.3 and J = 15.6 Hz),
2.82 (B part of AB, dd, 2H, 2Hb of CH2, J = 6.42 and J = 15.6 Hz).
13C NMR (100 MHz, CDCl3) d 151.6 (CO), 139.9 (2C), 134.8 (C),
129.2 (CH), 129.0 (2CH), 128.9 (2CH), 127.3 (2CH), 124.8 (2CH),
68.8 (OCH2), 55.6 (CH–N), 39.8 (2CH2). IR (CH2Cl2, cmꢀ1): 3277,
3210, 3030, 2952, 2902, 2840, 1712, 1606, 1586, 1456, 1357,
1243, 1211, 1164, 1087, 1055. Anal. Calcd for (C17H18N2O4S): C,
58.94; H, 5.24; N, 8.09; S, 9.26. Found: C, 58.93; H, 5.20; N, 8.12;
S, 9.27.
4.1.11. N-(1,2,3,4-Tetrahydronaphthalene-2-yl)sulfamide (19)
Sulfamide 19 was synthesized from the hydrogenolysis reaction
of 13 according to the procedure given at 4.1.8 (80% yield). White
solid. Mp 136–138 °C. 1H NMR (400 MHz, CDCl3) d 7.14–7.04 (m,
4H, Ar-H), 4.80 (br s, 2H, NH2), 4.73 (d, 1H, NH, J = 6.2 Hz), 3.84–
3.77 (m, 1H, CH–N), 3.16 (A part of AB, dd, 1H, Ha of CH2, J = 4.9
and J = 16.3 Hz), 2.91–2.87 (m, 2H, CH2), 2.77 (B part of AB, dd,
1H, Hb of CH2, J = 8.2 and J = 16.3 Hz), 2.16–2.12 (m, 1H, CH2),
1.89–1.80 (m, 1H, CH2). 13C NMR (100 MHz, CDCl3) d 135.4 (C),
133.8 (C), 129.7 (CH), 129.1 (CH), 126.6 (CH), 126.3 (CH), 50.2
(CH–N), 36.6 (CH2), 29.6 (CH2), 27.2 (CH2). IR (CH2Cl2, cmꢀ1):
3619, 3271, 3017, 2924, 2845, 1552, 1494, 1452, 1437, 1320,
1255, 1239 1156, 1113, 1068. Anal. Calcd for (C10H14N2O2S): C,
53.08; H, 6.24; N, 12.38; S, 14.17. Found: C, 53.10; H, 6.22; N,
12.41; S, 14.13.
4.1.8. Standard procedure for the synthesis of sulfamides
(Hydrogenolysis of sulfamylcarbamates): N-(5,6-
dimethoxyindane-1-yl)sulfamide (16)
Into a 100-mL flask were placed Pd-C (50 mg) and sulfamoylc-
arbamate 10 (0.65 g, 1.60 mmol) in MeOH (50 mL). A balloon filled
with H2 gas (3 L) was fitted to the flask. The mixture was deoxy-
genated by flushing with H2 and then hydrogenated at rt for 4 h.
The catalyst was removed by filtration. Recrystallization of the res-
idue from EtOAc/hexane gave 16 (0.31 g, 71% yield). White solid.
Mp 175–177 °C. 1H NMR (400 MHz, CD3OD) d 7.06 (s,1H, Ar-H),
6.82 (s, 1H, Ar-H), 4.89 (br s, 3H, NH, NH2 and H of CD3OD),
4.84–4.80 (m, 1H, CH–N), 3.81 (s, 3H, OCH3), 3.80 (s, 3H, OCH3),
2.91 (A part of AB, ddd, 1H, Ha of CH2, J = 3.6, J = 8.8 and
J = 15.5 Hz), 2.78–2.70 (B part of AB, m, 1H, Hb of CH2), 2.59 (A part
of AB, dddd, 1H, Ha of CH2, J = 3.6, J = 7.8, J = 11.4 and J = 15.6 Hz),
2.00–1.95 (B part of AB, m, 1H, Hb of CH2). 13C NMR (100 MHz,
DMSO-d6) d 149.5 (C) 148.6 (C), 135.7 (C), 134.8 (C), 108.7 (CH),
4.1.12. N-(5,6-Dimethoxyindane-2-yl)sulfamide (20)
Hydrogenolysis of 14 gave compound 20 with a yield of 76%.
White solid. Mp 178–180 °C. 1H NMR (400 MHz, CD3OD) d 6.81
(s, 2H, Ar-H), 4.84 (br s, 3H, NH and NH2 and H of CD3OD), 4.19–
4.15 (m, 1H, CH–N), 3.78 (s, 6H, 2OCH3), 3.21 (A part of AB, dd,
2Ha of CH2, J = 7.6 and J = 15.5), 2.85 (B part of AB, dd, 2Hb of
CH2, J = 7.2 and J = 15.5). 13C NMR (100 MHz, CD3OD) d 148.6
(2C), 132.9 (2C), 108.5 (2CH), 55.5 (2OCH3), 55.1 (CH–N), 39.7
(2CH2). IR (CH2Cl2, cmꢀ1): 3322, 3249, 3142, 2957, 2929, 2840,

1384
A. Akıncıog˘lu et al. / Bioorg. Med. Chem. 21 (2013) 1379–1385
1605, 1560, 1504, 1443, 1404, 1311, 1247, 1219, 1188, 1169, 1152,
1096. Anal. Calcd for (C11H16N2O4S): C, 48.52; H, 5.92; N, 10.29; S,
11.77. Found: C, 48.48; H, 5.96; N, 10.33; S, 11.72.
novel sulfonamides and 4-nitrophenylacetate as the substrate at
five different concentrations to construct Lineweaver–Burk
curves53 described previously.54–56
4.5. Protein determination
4.1.13. N-(Indane-2-yl)sulfamide (21)
Compound 21 was synthesized from 15 (82%). White solid. Mp
153–155 °C. 1H NMR (400 MHz, CD3OD) d 7.18–7.10 (m, 4H, Ar-H),
4.85 (br s, 3H, NH, NH2 and H of CD3OD), 4.21–4.13 (m, 1H, CH–N),
3.26 (A part of AB, dd, 2H, 2Ha of CH2, J = 7.5 and J = 15.6 Hz), 2.91
(B part of AB, dd, 2H, 2Hb of CH2, J = 7.3 and J = 15.6 Hz). 13C NMR
(100 MHz, CD3OD) d 140.9 (2C), 126.5 (2CH), 124.2 (2CH), 54.8
(CH–N), 39.7 (2CH2). IR (KBr, cmꢀ1): 3347, 3279, 3064, 3025,
2958, 2891, 2846, 2504, 2450, 1567, 1459, 1384, 1348, 1320,
1305, 1225, 1107, 1021. Anal. Calcd for (C9H12N2O2S): C, 50.92;
H, 5.70; N, 13.20; S, 15.11. Found: C, 50.90; H, 5.75; N, 13.20; S,
15.14.
The yield of protein during the purification steps was deter-
mined spectrophotometrically at 595 nm according to the Bradford
method,57 explained previously58 using bovine serum albumin as
the standard protein.59
4.6. SDS–polyacrylamide gel electrophoresis
SDS–polyacrylamide gel electrophoresis (SDS–PAGE) by the
method of Laemmli60 was performed for determination of isoen-
zymes purity as described previously.61,62 The running and stack-
ing gels contained acrylamide (10% and 3%), and SDS (0.1%),
respectively. A 20 mg sample was applied to the electrophoresis
medium. Gels were stained for 1.5 h in Coomassie Brilliant Blue
R-250 (0.1%) in methanol (50%) and acetic acid (10%), then dis-
tained with several changes of the same solvent without the dye.63
4.2. Purification of hCA I and hCA II by affinity chromatography
Erythrocytes were isolated from fresh human blood, which was
obtained from the University Hospital Blood Centre following low-
speed centrifugation at 3000ꢂg for 15 min (HERMLE Z 383 K) by
removal of plasma and buffy coat. The red cells were washed twice
with NaCl (w/v 0.9%) and haemolysed with 1.5 volumes of ice-cold
water. Ghost and intact cells were then removed by high-speed
centrifugation at 20,000ꢂg for 30 min at 4 °C and the pH of the
haemolysate adjusted to 8.7 with solid Tris. The pH-adjusted
haemolysate was then subjected to affinity chromatography using
Acknowledgment
This study was financed by Ataturk University (BAP2012/152)
for (A.A., Y.A. and S.G.), and by a FP7 EU project (Metoxia) for
(C.T.S.).
Supplementary data
a column packed with Sepharose-4B-
L-Tyrosine-sulfanilamide re-
sin.31 CA isoenzymes were purified via a simple one-step method
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.019.
using Sepharose-4B-L-Tyrosine-sulfanilamide affinity gel chroma-
tography; described previously.50 For this purpose, pH value of
affinity column was adjusted to 8.7 with solid Tris. Sepharose-
References and notes
4B-L-Tyrosine-sulfanylamide affinity column equilibrated with
Tris–HCl (25 mM)/Na2SO4 (0.1 M, pH 8.7). The affinity gel was
washed with Tris–HCl (25 mM)/Na2SO4 (22 mM, pH 8.7). hCA I
and hCA II isoenzymes were eluted with NaCl (1.0 M)/sodium
phosphate (0.25 M; pH 6.3) and sodium acetate (0.1 M)/NaClO4
(0.5 M, pH 5.6), respectively. Column flow rate was 20 mL/h and
was 4 mL fractions were collected. All procedures were performed
at 4 °C.31
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