3-Aminobenzenesulfonamides incorporating acylthiourea moieties selectively inhibit the tumor-associated carbonic anhydrase isoform IX over the off-target isoforms I, II and IV

Article abstract of DOI:10.1016/j.bioorg.2018.10.006

We describe the synthesis of a series of novel 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas (4a–k) acting as human carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors. Reaction of alkyl/aryl isothiocyanates with 3-aminobenzenesulfonamide afforded a series of the title compounds incorporating a variety of short as well as highly lipophilic long tails. The newly synthesized sulfonamides were evaluated against 4 physiologically relevant CA isoforms (hCA I, II, IV, and IX). Several compounds showed interesting inhibitory activity. The tumor-associated hCA IX was the most sensitive isoform to inhibition with these compounds, with K_Is in the range of 21.5–44.0 nM and selectivity ratios over the major cytosolic isoform hCA II in the range of 3.35–37.3. The sulfonamides incorporating the phenylacetylthioureido and pentadecanoylthioureido moieties were the most hCA IX-selective inhibitors detected in this work, making them of interest for further investigations.

Full text of DOI:10.1016/j.bioorg.2018.10.006

Accepted Manuscript
3
-Aminobenzenesulfonamides incorporating acylthiourea moieties selectively
inhibit the tumor-associated carbonic anhydrase isoform IX over the off-target
isoforms I, II and IV
Tanzeela Abdul Fattah, Silvia Bua, Aamer Saeed, Ghulam Shabir, Claudiu T.
Supuran
PII:
S0045-2068(18)30928-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.006
YBIOO 2540
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
23 August 2018
26 September 2018
4 October 2018
Please cite this article as: T. Abdul Fattah, S. Bua, A. Saeed, G. Shabir, C.T. Supuran, 3-Aminobenzenesulfonamides
incorporating acylthiourea moieties selectively inhibit the tumor-associated carbonic anhydrase isoform IX over the
off-target isoforms I, II and IV, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.006
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3
-Aminobenzenesulfonamides incorporating acylthiourea moieties selectively inhibit the
tumor-associated carbonic anhydrase isoform IX over the off-target isoforms I, II and IV
a
b
a
a
Tanzeela Abdul Fattah , Silvia Bua , Aamer Saeed , Ghulam Shabir, and Claudiu T.
b
Supuran *
a
Departmentof Chemistry Quaid-i-Azam University-45320, Islamabad, Pakistan.
b
University of Florence, NEUROFARBA Department, Pharmaceutical and Nutraceutical Sciences
Section, via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. We describe the synthesis of a series of novel 1-aroyl/acyl-3-(3-aminosulfonylphenyl)
thioureas (4a-k) acting as human carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors. Reaction of
alkyl/aryl isothiocyanates with 3-aminobenzenesulfonamide afforded a series of the title
compounds incorporating a variety of short as well as highly lipophilic long tails. The newly
synthesized sulfonamides were evaluated against 4 physiologically relevant CA isoforms (hCA I,
II, IV, and IX). Several compounds showed interesting inhibitory activity. The tumor-associated
hCA IX was the most sensitive isoform to inhibition with these compounds, with K
of 21.5 – 44.0 nM and selectivity ratios over the major cytosolic isoform hCA II in the range of
.35 37.3. The sulfonamides incorporating the phenylacetylthioureido and
I
s in the range
3
–
pentadecanoylthioureido moieties were the most hCA IX-selective inhibitors detected in this
work, making them of interest for further investigations.
Keywords:
carbonic
anhydrase;
inhibitors;
thioureas;
isothiocyanates;
3-
aminobenzenesulfonamide; acyl-thioureas.
_
___
*Corresponding authors. Fax: +92-51-9064-2241E-mail: asaeed@qau.edu.pk, aamersaeed@yahoo.com
(AS); claudiu.supuran@unifi.it (CTS).
1

1
. Introduction
The zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) catalyzes the rapid hydration of
carbon dioxide into a proton and the bicarbonate ion utilizing a metal hydroxide nucleophilic
mechanism, this being a simple but vital reaction, involved in many physiological processes
including respiration, pH homeostasis, electrolyte secretion, biosynthesis of important molecules
(
urea, glucose and lipids etc.) and as shown recently, tumorigenicity [1-2]. These processes are
crucial for most organisms, and as a result, during the evolution of life on Earth, seven
genetically diverse families of this enzyme (classified as α-, the β-, γ-, δ-, ζ-, η- and -CAs)
evolved [3-5]. There are 15 CA isoforms known in humans (hCAs) and they belong to the α-CA
class [1-3]. These enzymes differ in their catalytic/inhibition activity, subcellular localization
(
mitochondria, cytosol, anchored to the cell membrane, transmembrane or secreted isoforms),
tissue distribution and affinity for the different classes of inhibitors [6-7]. CA II is the most
investigated and physiologically dominant isoform, being expressed in many tissues and organs
in mammals [1-3]. Its presence in the endothelium of neo-vessels in several cancer tissues,
including esophageal, melanoma, renal and lung cancers [8] made it of interest also as an
antitumor target. However, CA this cytosolic isoform is crucial for the antiglaucoma effects of
the CA inhibitors (CAIs) of the sulfonamide type, which are clinically used agents for decades
[
1,3]. hCA I and hCA II inhibitors are utilized for the management of glaucoma, cerebral edema
and altitude sickness among others [9-12]. CAIs are in clinical use for more than 70 years as
antiglaucoma drugs and diuretics. Glaucoma is a multi-factorial ocular disorder characterized by
optic nerve deterioration usually associated with high intraocular pressure (IOP) which may lead
to blindness [13]. Sulfonamide CAIs such as brinzolamide (BRZ) are effective in reducing IOP
after topical administration. Examples of other clinically employed CAIs are Acetazolamide
AAZ, Ethoxzolamide EZA, Brinzolamide BRZ, and Methazolamide MZA, and some of the
structurally related sulfonamides found in the literature are shown in Fig. 1. Due to larger
number of hCA isoforms, there is a need to improve the selectivity profile and inhibition of such
clinically used CAIs, in order to avoid the side effects [14-17].
Dysregulation of CAs expression has been linked to many diseases apart glaucoma,
including cancer and epilepsy [10-11]. CA II is one of the most effective catalyst for CO
2
hydration [12].
2

The 4-(3,4-dichloro-phenylureido)thioureido-benzene sulfonamide A having thiourea moiety
was shown to be an efficient in vitro inhibitor of Plasmodium falciparum CA (PfaCA) [18].
Various anti-plasmodial 7-chloro-4-aminoquinolyl- derived sulfonamides, as well as
sulfonamides of type B (Fig. 1) have been prepared and tested against chloroquine-resistant and
chloroquine susceptible P. falciparum [19]. Previously, some of us reported sulfanilamide
thiourea hybrids acting as antimicrobial agents and urease inhibitors but those compounds have
not yet been assayed for their CA inhibitory properties against all physiologically relevant
isoforms [20]. In fact, aminobenzenesulfonamide-thiourea conjugates were assayed only as
bovine CA II inhibitors [21]. In this paper we report the synthesis of 3-
aminobenzenesulfonamide thioureas with aliphatic acid chlorides and determined their inhibition
activity against hCA I, II, IV and IX.
Fig. 1: Pharmacologically used CAIs, AAZ and BRZ. Compounds A and B were also reported to
possess interesting pharmacological effects [8].
3

SOCl2
DMF
O
O
R
OH
a-k
R
Cl
a-k
reflux for 3 h
2
1
-
SO , HCl
2
SO NH2
2
SO NH2
2
O
O
KSCN
Dry acetone
reflux 1.5 h
Dry acetone
reflux 7 h
S
O
R
Cl
a-k
R
N C S
N
H
N
R
NH2
2
H
3
(4a-k)
2
SO NH2
SO2NH2
SO NH2
2
S
O
S
O
S
O
N
H
N
N
H
N
N
N
13
H
H
H
H
4c
4
b
4a
SO NH2
2
SO NH2
SO NH2
2
2
S
O
S
O
S
O
Cl
N
H
N
H
N
H
N
N
N
H
1
6
H
H
4
f
4d
4e
Cl
SO NH2
SO2NH2
SO NH2
2
2
S
O
S
O
S
O
N
H
N
H
N
H
N
H
N
N
H
1
4
H
4h
4i
4g
SO NH2
2
SO NH2
2
S
O
S
O
N
H
N
H
N
H
N
H
4k
4
j
Scheme 1: Synthesis of 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas (4a-k)
2
2
.
Experimental
.1. Methods and materials
4

The chemicals, 3-aminobenzenesulfonamide, potassium thiocyanate,thionyl chloride, substituted
acid chlorides, all synthetic starting materials, reagents and solvents were of analytical reagent
grade or of the highest quality commercially available and were purchased from Sigma Aldrich
Merck (Milan, Italy) and were used without further purification. Acetone was freshlydried over
KMnO
4
and distilled.R -values were determined using pre-coated silica gel aluminum plates
f
6
0F254 from Merck (Germany). All Melting points were determined in open glass capillaries
TM
using Stuart SMP3(UK) melting point apparatus and are uncorrected.Infrared spectra (IR) of
the compounds were recorded on a Bio-Rad-Excalibur Series Model No. FTS 300 MX
1
13
spectrophotometeras pure compounds. H and C-NMR spectra were obtained on a Bruker
00MHz and 75.5 MHz NMR spectrometerusing teteramethylsilane (TMS) as internal reference
standard. Elemental analyses were conducted using a LECO-183 CHNS analyser.
.2. Synthesis of 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas (4a-k)
A solution of suitable carboxylic acids 1a-k (1.0 mmol) and DMF (0.05 ml) in thionyl chloride
1.2 mmol) were refluxed for 3.5 hr. After cooling at room temperature the mixture was
3
2
(
concentrated to afford the acid chlorides 2a-k. A solution of acid chlorides 2a-k (1.0 mmol) in
dry distilled acetone (20 ml) was added drop wise to a suspension of potassium thiocyanate (1.0
o
mmol) in dry acetone and refluxed for 1.5 hr at 50 C to afford the corresponding isothiocyanates.
After cooling on room temperature, a solution of 3-aminobenzenesulfonamide 3 (1.0 mmol) in
dry acetone was added in it and the reaction mixture was refluxed for 9 hr. After completion
checked by TLC (n-Hexane: Ethyl acetate 1: 1) the reaction mixture was poured onto crushed
ice and the resulting precipitates were collected via simple filtration, washed, dried and
recrystallized from ethanol to obtain the 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas4a-k
in excellent yields (scheme 1).
2
.2.1. 1-Butanoyl-3-(3-aminosulfonylphenyl)thiourea (4a)
o
-1
Off white solid; yield: 74%; m.p: 137 C; R
f
: 0.52 (n-Hexane: Ethyl acetate 1:1);IR (pure, cm ):
3
1
254(N-H), 1660 (C=O), 1595 (C=C), 1530 (thioamide I), 1331 (C-S), 1240 (thioamide II),
1
150, 1092 (thioamide III), 742 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.04 (1H, brs), 7.50 (1H, t, J = 7.5 Hz), 7.60
1H, d, J = 7.4 Hz), 7.70 (1H, d, J = 7.6 Hz), 2.17 (t, 2H, CH ), 1.60 (m, 2H, CH ), 0.91 (s, 3H,
6
): δ (ppm); 12.98
(
s, 1H, N-H), 11.90 (s, 1H, N-H), 7.42 (SO
2
2
(
2
2
1
3
CH
3
); C NMR (75.5 MHz, DMSO-d
6
); 184.2 (C=S), 178.9 (C=O), 148.1, 140.4, 137.1, 132.2,
5

1
2
28.4, 125.4, 38.9, 20.1, 14.1; Anal. Calcd. for C11
1.28 found: C,43.81; H, 5.00;N,13.91; S, 21.24.
H
15
N
3
O
3
2
S : C, 43.84; H, 5.02; N, 13.94; S,
2
.2.2. 1-Pentadecanoyl-3-(3-aminosulfonylphenyl)thiourea (4b)
o
-1
Black solid; yield: 79%; m.p: 129 C; R
f
: 0.58 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ):
3
1
250 (N-H), 1669 (C=O), 1589 (C=C), 1529 (thioamide I), 1338 (C-S), 1246 (thioamide II),
1
153, 1099 (thioamide III), 743 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.06 (1H, brs), 7.59 (1H, t, J = 7.4 Hz), 7.62
1H, d, J = 7.5 Hz), 7.71 (1H, d, J = 7.4 Hz), 2.19 ( t, 2H, J = 6.8 Hz), 1.59 (quin, 2H, CH CH ),
); C NMR
); 188.2 (C=S), 180.9 (C=O), 147.1, 141.4, 136.2, 132.9, 128.9, 126.4,
6
): δ (ppm); 12.90
(
s, 1H, N-H), 11.99 (s, 1H, N-H), 7.44 (SO
2
2
(
2
2
1
3
1
.28 (quin, 20H, CH
2
CH
2
), 1.34 (m, 2H, CH
2
CH
3
), 0.92 (t, 3H, J = 7.1 Hz, CH
3
(
75.5 MHz, DMSO-d
6
3
5
8.4, 26.5, 28.9, 29.3, 29.3, 29.1 (6C), 31.8, 22.7, 14.3; Anal. Calcd. for C22
7.99; H, 8.18; N, 9.22; S, 14.07found: C, 57.96; H, 8.15; N, 9.20; S, 14.05.
H
37
N
3
O
3
S
2
: C,
2
.2.3. 1-Octanoyl-3-(3-aminosulfonylphenyl)thiourea (4c)
o
-
Light brown solid; yield: 85%; m.p: 138 C; R
f
: 0.89 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm
1
)
: 3257 (N-H), 1672 (C=O), 1579 (C=C), 1535 (thioamide I), 1336 (C-S), 1252 (thioamide II),
1
1
188, 1100 (thioamide III), 750 (thioamide IV); H NMR (300 MHz, DMSO-d
s, 1H, N-H), 11.01 (s, 1H, N-H), 7.42 (SO NH ), 8.06 (1H, brs), 7.62 (1H, t, J = 7.3 Hz), 7.59
1H, d, J = 7.6 Hz), 7.68 (1H, d, J = 7.3 Hz), 2.17 ( t, 2H, J = 6.9 Hz), 1.58 (quin, 2H, CH CH ),
); C NMR (75.5
); 189.1 (C=S), 176.2 (C=O), 149.2, 144.2, 134.4, 133.4, 128.7, 126.6, 37.4,
6.1, 28.8 (2C), 31.9, 22.5, 14.3; Anal. Calcd. for C15 : C, 50.40; H, 6.48; N, 11.75; S,
7.94found: C, 50.37; H, 6.45; N, 11.72; S, 17.91.
6
): δ (ppm); 12.99
(
2
2
(
2
2
1
3
1
.28 (quin, 6H, CH
2
CH
2
), 1.38 (m, 2H, CH
2
CH
3
), 0.99 (t, 3H, J = 7.2 Hz, CH
3
MHz, DMSO-d
6
2
1
H
23
N
3
O S
3 2
2
.2.4. 1-(4-Chorobenzoyl)-3-(3-aminosulfonylphenyl)thiourea (4d)
o
-1
Brown; yield: 78%; m.p: 219 C; R
f
: 0.65 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ): 3278
(
N-H), 1674 (C=O), 1593 (C=C), 1529 (thioamide I), 1316 (C-S), 1250 (thioamide II), 1150,
1
1
001 (thioamide III), 749 (thioamide IV); H NMR (300 MHz, DMSO-d
6
): δ (ppm); 12.51 (s, 1H,
N-H), 11.73 (s, 1H, N-H), 7.80 (d, 2H, H-Ar), 7.47 (SO
2
NH ), 7.30 (d, 2H, J = 5.7 Hz, H-Ar),
2
1
3
6
.39 (d, 1H, J = 4.6 Hz, H-Ar), 7.16 (d, 1H, H-Ar); C NMR (75.5 MHz, DMSO-d ); 181.7
6
6

(
C=S), 170.9 (C=O), 139.5 (C-S), 138.8 (C-Cl), 136.1 (C-Ar), 135.1 (C-CO), 129.7 (2C-Ar),
29.1 (C-Ar), 128.6 (2C-Ar), 128.1 (C-Ar), 124.7 (C-Ar), 123.9 (C-Ar); Anal. Calcd. for
: C, 45.46; H, 3.27; Cl, 9.59; N, 11.36; S, 17.34 found: C, 45.43; H, 3.25; Cl,
1
C
14
H12ClN
3
O S
3 2
9
.61; N,11.30; S, 17.29.
2
.2.5. 1-Octadecanoyl-3-(3-aminosulfonylphenyl)thiourea (4e)
o
-1
Black solid; yield: 89%; m.p: 161 C; R
f
: 0.70 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ):
3
1
251 (N-H), 1688 (C=O), 1560 (C=C), 1555 (thioamide I), 1341 (C-S), 1247 (thioamide II),
1
178, 1091 (thioamide III), 743 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.10 (1H, brs), 7.71 (1H, t, J = 7.4 Hz), 7.62
1H, d, J = 7.4), 7.69 (1H, d, J = 7.4 Hz), 2.17 ( t, 2H, J = 6.9 Hz), 1.56 (quin, 2H, CH CH ),
); C NMR
); 185.2 (C=S), 175.3 (C=O), 148.3, 141.2, 136.4, 132.4, 128.4,
6
): δ (ppm); 13.01
(
s, 1H, N-H), 11.88 (s, 1H, N-H), 7.43 (SO
2
2
(
2
2
1
3
1
.28 (quin, 26H, CH
2
CH
2
), 1.35 (m, 2H, CH
2
CH
3
), 0.91 (t, 3H, J = 7.1 Hz, CH
3
(
75.5 MHz, DMSO-d
6
1
6
26.3,36.1, 26.2, 28.7, 29.1, 29.6 (9C), 29.3, 31.6, 22.1, 14.9; Anal. Calcd. for C25
H
43
N
3
O
3
S
2
: C,
0.32; H, 8.71; N, 8.44; S, 12.88found: C, 60.30; H, 8.69; N, 8.41; S, 12.65.
2
.2.6. 1-(2-chloroacetyl)-3-(3-aminosulfonylphenyl)thiourea (4f)
o
-1
Dark brown; yield: 67%; m.p: 137 C; R
f
: 0.62 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ):
3
1
256 (N-H), 1675 (C=O), 1534 (C=C), 1545 (thioamide I), 1333 (C-S), 1246 (thioamide II),
1
159, 1076 (thioamide III), 742 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.13 (1H, brs), 7.57 (1H, t, J = 7.4 Hz), 7.62
1H, d, J = 7.3), 7.76 (1H, d, J = 7.3 Hz),4.34 (s, 1H, CH
88.2 (C=S), 179.2 (C=O), 149.4, 143.1, 136.2, 131.2, 128.1, 126.3, 44.1 (CH
for C : C, 35.12; H, 3.27; N, 13.65; S, 15.60found: C, 35.10; H, 3.25; N, 13.61; S,
6
): δ (ppm); 12.81
(
(
s, 1H, N-H), 11.88 (s, 1H, N-H), 7.42 (SO
2
2
1
3
2
-Cl); C NMR (75.5 MHz, DMSO-d
6
);
1
2
-Cl); Anal. Calcd.
9
H10ClN
3
O S
3 2
1
5.58.
2
.2.7. 1-Pentanoyl-3-(3-aminosulfonylphenyl)thiourea (4g)
o
-1
White solid; yield: 79%; m.p: 146 C; R
f
: 0.51 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ):
3
250 (N-H), 1678 (C=O), 1559 (C=C), 1560 (thioamide I), 1330 (C-S), 1242 (thioamide II),
7

1
1
179, 1099 (thioamide III), 742 (thioamide IV); H NMR (300 MHz, DMSO-d
s, 1H, N-H), 11.60 (s, 1H, N-H), 7.48 (SO NH ), 8.00 (1H, brs), 7.76 (1H, t, J = 7.3 Hz), 7.63
1H, d, J = 7.3 Hz), 7.70 (1H, d, J = 7.3 Hz), 2.19 ( t, 2H, J = 6.8 Hz), 1.59 (quin, 2H, CH ), 1.31
6
): δ (ppm); 12.32
(
(
(
2
2
2
1
3
m, 2H, CH
2
), 0.97 (t, 3H, J = 7.2 Hz, CH
3
); C NMR (75.5 MHz, DMSO-d
6
); 188.1 (C=S),
1
77.3 (C=O), 148.4, 142.1, 135.4, 132.9, 128.5, 126.6, 36.1, 28.9, 21.6, 13.4; Anal. Calcd. for
: C, 45.70; H, 5.43; N, 13.32; S, 15.22 found: C, 45.68; H, 4.47; N, 13.30; S,
C
12
H
17
N
3
O S
3 2
1
5.20.
2
.2.8. 1-Heptanoyl-3-(3-aminosulfonylphenyl)thiourea (4h)
o
-1
Brown solid; yield: 81%; m.p: 141 C; R
f
: 0.60 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ):
3
1
250 (N-H), 1670 (C=O), 1569 (C=C), 1542 (thioamide I), 1339 (C-S), 1243 (thioamide II),
1
178, 1107 (thioamide III), 748 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.12 (1H, brs), 7.58 (1H, t, J = 7.3 Hz), 7.62
1H, d, J = 7.6 Hz), 7.69 (1H, d, J = 7.3 Hz), 2.18 ( t, 2H, J = 6.9 Hz), 1.59 (quin, 2H, CH CH ),
); C NMR (75.5
); 181.1 (C=S), 174.2 (C=O), 149.3, 144.8, 134.3, 133.2, 128.7, 126.2, 36.1,
6.0, 28.3, 31.4, 22.9, 14.5; Anal. Calcd. for C14 : C, 48.96; H, 6.16; N, 12.23; S,
8.67 found: C, 48.91; H, 6.14; N, 12.20; S, 18.65.
6
): δ (ppm); 12.43
(
s, 1H, N-H), 11.54 (s, 1H, N-H), 7.42 (SO
2
2
(
2
2
1
3
1
.27 (quin, 4H, CH
2
CH
2
), 1.37 (m, 2H, CH
2
CH
3
), 0.94 (t, 3H, J = 7.3 Hz, CH
3
MHz, DMSO-d
6
2
1
H
21
N
3
O S
3 2
2
.2.9. 1-Hexadecanoyl-3-(3-aminosulfonylphenyl)thiourea (4i)
o
-1
Blackish solid; yield: 88%; m.p: 143 C; R
f
: 0.62 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ):
3
1
245(N-H), 1659 (C=O), 1594 (C=C), 1538 (thioamide I), 1345 (C-S), 1251 (thioamide II),
1
165, 1123 (thioamide III), 744 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.10 (1H, brs), 7.65 (1H, t, J = 7.3 Hz), 7.77
1H, d, J = 7.4 Hz), 7.72 (1H, d, J = 7.3 Hz), 2.17 ( t, 2H, J = 6.9 Hz), 1.58 (quin, 2H, CH CH ),
); C NMR
); 183.2 (C=S), 179.9 (C=O), 147.4, 145.4, 139.2, 1324, 128.1, 126.1,
6
): δ (ppm); 12.78
(
s, 1H, N-H), 11.65 (s, 1H, N-H), 7.47 (SO
2
2
(
2
2
1
3
1
.29 (quin, 22H, CH
2
CH
2
), 1.31 (m, 2H, CH
2
CH
3
), 0.97 (t, 3H, J = 7.2 Hz, CH
3
(
75.5 MHz, DMSO-d
6
3
5
8.4, 26.1, 28.8, 29.0, 29.7 (7C), 29.5, 31.6, 22.7, 14.4; Anal. Calcd. for C23
8.81; H, 8.37; N, 8.95; S, 13.65found: C, 58.78; H, 8.35; N, 8.93; S, 13.63.
H
39
N
3
O
3
2
S : C,
2
.2.10. 1-(2-phenylacetyl)-3-(3-aminosulfonylphenyl)thiourea (4j)
8

o
-1
White solid; yield: 76%; m.p: 154 C; R
f
: 0.76 (n-Hexane : Ethyl acetate 1:1); IR (pure, cm ):
3
1
267 (N-H), 1674 (C=O), 1539 (C=C), 1540 (thioamide I), 1338 (C-S), 1252 (thioamide II),
1
176, 1096 (thioamide III), 744 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.14 (1H, brs), 7.55 (1H, t, J = 7.3 Hz), 7.63
1H, d, J = 7.2), 7.78 (1H, d, J = 7.2 Hz), 3.49 (s, 2H, CH -Ph), 7.09 (d, 2H, Ar-H), 7.16 (dd, 2H,
); 187.2 (C=S), 177.2 (C=O), 149.8,
43.2, 136.5, 131.4, 128.7, 126.7, 138.5, 129.8 (2C-Ar), 129.2 (2C-Ar), 129.7 (1C-Ar), 40.9
CH -Ph); Anal. Calcd. for C15 : C, 51.56; H, 4.33; N, 12.03; S, 18.35found: C, 51.54;
H, 4.30; N, 12.00; S, 18.33.
6
): δ (ppm); 12.76
(
(
s, 1H, N-H), 11.54 (s, 1H, N-H), 7.43 (SO
2
2
2
1
3
Ar-H), 7.07 (t, 1H, Ar-H); C NMR (75.5 MHz, DMSO-d
6
1
(
2
H
15
N
3
O S
3 2
2
.2.11. 1-acetyl-3-(3-aminosulfonylphenyl)thiourea (4k)
o
-1
Light brown; yield: 81%; m.p: 121 C; R
f
: 0.72 (n-Hexane: Ethyl acetate 1:1);IR (pure, cm ):
3
1
250 (N-H), 1664 (C=O), 1591 (C=C), 1532 (thioamide I), 1329 (C-S), 1241 (thioamide II),
1
152, 1082 (thioamide III), 748 (thioamide IV); H NMR (300 MHz, DMSO-d
NH ), 8.12 (1H, brs), 7.54 (1H, t, J = 7.6), 7.64 (1H,
d, J = 7.3), 7.79 (1H, d, J = 7.4), 2.05 (s, 3H, CH
C=S), 181.9 (C=O),149.1, 143.4, 135.1, 133.2, 127.4, 126.4, 23.2; Anal. Calcd. for
: C, 39.55; H, 4.06; N, 15.37; S, 23.46found: C,39.51; H, 4.04;N,15.30; S, 23.43.
6
): δ (ppm); 12.91
(
s, 1H, N-H), 11.92 (s, 1H, N-H), 7.44 (SO
2
2
1
3
3
); C NMR (75.5 MHz, DMSO-d ); 185.2
6
(
C
9
H
11
N
3
O S
3 2
2
.3.
Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA-catalyzed
CO hydration activity [22]. Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 20 mMHepes (pH 7.5) as buffer,
and 20 mM Na SO (for maintaining constant the ionic strength), following the initial rates of the
CA-catalyzed CO hydration reaction for a period of 10–100 s. The CO concentrations ranged
2
2
4
2
2
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
determining the initial velocity. The un catalyzed 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.
9

Inhibitor and enzyme solutions were pre-incubated together for 15 min at room temperature prior
to assay, in order to allow for the formation of the E-I complex. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation,
as reported earlier[23] and represent the mean from at least three different determinations. All
CA isofoms were recombinant ones obtained in-house as reported earlier [23, 24].
3
3
.
Results and discussion
.1. Chemistry
A variety of novel 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas (4a-k) were synthesized in
situ by reaction of corresponding acid chlorides with an equimolar quantity of potassium
thiocyanate in dry acetone to afford the isothiocyanates. Treatment of isothiocyanates with 3-
aminobenzenesulfonamide in dry acetone in 1:1 M ratio afforded the 1-aroyl/acyl-3-(3-
aminosulfonylphenyl) thioureas(4a-k) in good yields as shown in scheme 1.
-1
FT-IR spectrum showed peak at around 3250 cm due to N-H stretching vibrations and a
-1
strong peak at 1664 cm corresponding to C=O (amide). The aromatic stretching frequency
-1
1
bands appear at 1591 cm . In H-NMR, the characteristic singlets for N
thiourea were found in a comparatively wide range of 11.11-12.99 ppm respectively. The rest of
aromatic proton appears in their respective aromatic region. The –SO NH protons appear at
1
and N
3
protons in the
2
2
1
3
around 7.44 ppm. C-NMR showed C=S and C=O signal at 185.2 and 181.9 ppm, while the rest
of the aromatic and aliphatic carbons appear at their respective regions in the spectrum.
3
.2. Carbonic anhydrase inhibitory activity
The inhibitory potential of the synthesized compounds (4a-k) on human hCA isoforms (hCAI,
hCA II, hCA IV and hCA IX) was evaluated using a stopped-flow CO hydrase assay. CA
2
isoforms, the ubiquitous cytosolic hCA I and hCA II, the membrane-anchored hCA IV, and the
tumor-associated trans-membrane hCA IX were selected for the biological testing. Results are
summarized in Table 1, the standard inhibitor acetazolamide (AAZ) containing sulfonamide
moiety was used as a reference compound. All the compounds were able to inhibit the four hCA
isoforms. The following structure–activity relationship (SAR) can be drawn from the data of
Table 1.
10

Table 1:
reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow
CO hydrase assay.
Inhibition data of human CA isoforms hCA I, II, IV and IX with compounds 4a-k
2
a
compd
K
I
(nM)
Selectivity ratio
b
hCA I
hCA II
hCA IV
hCAIX
hCA II/IX
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
293.1
435.1
438.4
316.6
582.5
81.6
140.1
968.1
123.0
283.2
919.6
50.9
6343.7
8594.8
7315.0
5087.5
8934.5
7406.2
7019.0
7981.4
8030.7
6986.5
5650.0
74
44.0
238.1
46.2
83.9
274.2
23.3
30.9
21.5
26.4
36.3
40.2
25
3.18
4.06
2.66
3.37
3.35
2.18
5.27
5.93
37.3
7.25
3.68
0.48
g
h
i
334.7
486.4
826.3
394.2
712.5
250
163.1
127.5
986.2
263.3
148.1
12
j
k
AAZ
a
Mean values from three different assays. Errors were within ±5-10 % of the reported values
b
(
data not shown). Catalytic domain.
(
i) Most thiourea derivatives (4a-k) investigated here showed moderate inhibitory properties
against the cytosolic isoform hCA I. Compound 4i exhibited the lowest inhibition of this
isoform, with a K value of 826.3 nM. It is interesting to note that the slight shortening of the
aliphatic chain, as in compound 4b (K = 435.1 nM) respect to 4i, causes a considerable increase
of the inhibitory activity against hCA I.
I
I
The introduction of 6 and 7 carbons aliphatic chains determines a further improvement of the
inhibitory activity, as shown by 4h and 4c, with K s 486.4 and 438.4 nM, respectively. Also, the
I
11

introduction of a phenyl group has a positive effect on the inhibitory activity, as in the case of the
compounds 4d and 4j (K s= 316.6 and 394.2 nM, respectively), which only differ for a chlorine
atom in the aromatic group.
I
Finally, compound 4f showed the best activity against hCA I (K
I
= 81.6 nM) and is three times
more potent than the reference drug AAZ K 250 nM, and this result is presumably associated to
I
the presence of halogens, the absence of a long aliphatic chain and likelythe absence of bulky
groups.
(
ii) The physiologically dominant hCA II was moderately inhibited by most thiourea derivatives
reported with K s values spanning between 123.0 and 986.2 nM. The general tendencies
described above are also applicable for this isoform, in fact compound 4f stood out again as the
best hCA II inhibitor with K value of 50.9 nM. The presence of aliphatic chains of 13, 14 and 16
carbon atoms determined a reduction of the inhibitory activity of compounds 4b, 4i and 4e (K s =
68.1, 986.2, 919.6, respectively). Unlike the trend showed against hCA IX discussed below, the
I
I
I
9
compounds 4d and 4j showed very similar K s (283.2 and 263.3 nM, respectively), confirming
I
that the addition of a halogen atom on the phenyl ring has not substantial impact on the
inhibitory activity against hCA II. Small variations in the length of the aliphatic chain in this
particular type of scaffold correspond to minimal variations of the inhibitory activity as in the
cases of the compounds 4a and 4k (K
I
s = 140.1 and 148.1 nM, respectively) and of compounds
4
c and 4h (K s = 123.0 and 127.5 nM, respectively). These evidences are nevertheless important
I
to confirm that the compounds with a medium-length aliphatic chain are the best inhibitors
against hCA II.
(
iii) All derivatives herein studied show low inhibitory activityagainst the membrane-bound
isoform hCA IV (with K s in the range between 5087.5 and 8934.5 nM) and exhibit rather flat
SAR.
I
(
iv) The tumor associated hCA IX was the most inhibited isoform among those considered in this
study, with K s in the range between 21.5 and 274.2 nM. Compounds bearing a medium-length
alkyl chain showed the best K s as in the case of compounds 4a, 4g, 4h, and 4k (K s = 44.0, 30.9,
1.5 and 40.2 nM, respectively). Conversely, 4b and 4e that possess long alkyl chains, displayed
a medium nanomolar (K s = 238.1 and 274.2 nM, respectively) inhibitory action against hCA IX.
It is important to emphasize that compound 4j, holding an un-substituted phenyl, is an effective
I
I
I
2
I
12

inhibitor, with a K
determines a considerable reduction of inhibitory potency of compound 4d with a K
3.9 nM.
I
value of 36.3, whereas the presence of chlorine atom on the phenyl ring
I
value of
8
(
v) We can conclude that all derivatives bearing medium-length aliphatic chain tails have
reported the best CA inhibitory activities particularly against the tumor associated hCA IX. In
fact, as seen from the selectivity ratios (Table 1), some of the derivatives possess good such
indices for the inhibition of hCA IX over hCA II, in the range of 3.35 – 37.3. The most hCA IX –
selective CAIs designed here were the sulfonamides incorporating the phenylacetylthioureido
(
4j) and pentadecanoylthioureido (4i) moieties, that shown selectivity ratios of 7.25 – 37.3.
Following these evidences, compounds 4j and 4i have been selected as a guide for the synthesis
of new derivatives.
4
.
Conclusions
We have reported the synthesis of a novel series of 1-aroyl/acyl-3-(3-
aminosulfonylphenyl) thioureas (4a-k) in good yields. The new compounds were evaluated for
their human carbonic anhydrase inhibition potential. Several compounds displayed interesting
activity, with K s in the range of 21.5 – 44.0 nM and selectivity ratios over the major cytosolic
I
isoform hCA II in the range of 3.35 – 37.3. The sulfonamides incorporating the
phenylacetylthioureido and pentadecanoylthioureido moieties were the most hCA IX-selective
inhibitors detected in this work, making them of interest for further investigations.
Acknowledgments
We are thankful to Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan for
providing encouraging environment and facilities for research work.
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17

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18

3
-Aminobenzenesulfonamides incorporating acylthiourea moieties selectively inhibit the
tumor-associated carbonic anhydrase isoform IX over the off-target isoforms I, II and IV
a
b
a
a
Tanzeela Abdul Fattah , Silvia Bua , Aamer Saeed , Ghulam Shabir Claudiu T.
b
Supuran *
R = alkyl, aryl; K
I
(hCA IX) = 20-80 nM
Selectivity ratio hCA IX/hCA II = 7.25 – 37.3
Highlights




1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas were synthesized
these sulfonamide act as human carbonic anhydrase inhibitors
human isoforms I, II, IV and IX were investigated
potent CA IX Inhibitors were detected
19