Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties

Article abstract of DOI:10.1016/j.bmc.2016.06.052

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human

Full text of DOI:10.1016/j.bmc.2016.06.052

Accepted Manuscript
Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfo-
namides with subnanomolar carbonic anhydrase II and XII inhibitory properties
Murat Bozdag, Ahmed M. Alafeefy, Fabrizio Carta, Mariangela Ceruso, Abdul-
Malek S. Al-Tamimi, Abdulla A. Al-Kahtani, Fatmah A.S. Alasmary, Claudiu
T. Supuran
PII:
S0968-0896(16)30475-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.052
BMC 13108
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
1 June 2016
24 June 2016
25 June 2016
Please cite this article as: Bozdag, M., Alafeefy, A.M., Carta, F., Ceruso, M., Al-Tamimi, A.S., Al-Kahtani, A.A.,
Alasmary, F.A.S., Supuran, C.T., Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-
benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.052
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Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with
subnanomolar carbonic anhydrase II and XII inhibitory properties
a
b
a
a
Murat Bozdag, Ahmed M. Alafeefy, Fabrizio Carta, Mariangela Ceruso, Abdul-Malek S. Al-
c
c,d
d
a
Tamimi, Abdulla A. Al-Kahtani, Fatmah A.S. Alasmary, Claudiu T. Supuran*
a
Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via
Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
b
Department of Chemistry, Kulliyyah of Science, International Islamic University Malaysia, P.O.
Box 141, 25710 Kuantan, Pahang Darul Makmur, Malaysia.
c
Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz
University, P.O. Box 173, Alkharj, 11942, Saudi Arabia
d
Department of Chemistry, College of Science, King Saud University, Riyadh 11362, Saudi Arabia
Abstract. Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-
benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-
quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic
anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a
transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new
sulfonamides acted as medium potency inhibitors of hCA I (K s of 28.5 – 2954 nM), being highly
I
effective as hCA II (K s in the range of 0.62 – 12.4 nM) and XII (K s of 0.54 – 7.11 nM) inhibitors.
I
I
All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties,
in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA
II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in
pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II
and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated.
Keywords: carbonic anhydrase; sulfonamide; inhibitor; 2-mercapto-quinazolin-4-one; tail approach
Author of correspondence: CTS, Phone: +39-055-4573729; fax: +39-055-4573385; E-mail:
claudiu.supuran@unifi.it

1
. Introduction
1
In a previous work from our groups we reported a series of benzenesulfonamides obtained from
sulfanilamide, which was converted to the corresponding 4-isothiocyanato benzenesulfonamide,
and reacted with anthranilic acid derivatives, leading thus to 2-mercapto-3H-quinazolin-4-ones.
These sulfonamides possessed excellent inhibitory activity against the metalloenzyme carbonic
2
,3
anhydrase (CA, EC 4.2.1.1). Several CA isoforms (of the 15 presently known in humans) were
inhibited in the low nanomolar range with some of these compounds, among which the human (h)
2
,3
hCA II and XII (involved in glaucoma) as well as hCA IX and XII (transmembrane isoforms
4
-6
involved in tumorigenesis).
Although no crystal structures for adducts of 2-mercapto-3H-
quinazolin-4-one-benzenesulfonamides (such as A-C, Fig. 1) with any CA isozymes are available to
date, we hypothesized that the presence of the substituted-mercaptoquinazolinone tails in their
scaffolds exerted a beneficial effect on the interaction with the enzyme active site, and thus decided
to further investigate this type of tail for designing sulfonamide CA inhibitors (CAIs). Indeed,
7
8,9
pharmacological agents belonging to this class are clinically used as diuretics, antiglaucoma,
1
0,11
12
4-6
antiepileptic,
antiobesity, and more recently antitumor agents, with one such derivative,
1
3,14
SLC-0111 in Phase Ib clinical trials for the treatment of metastatic hypoxic cancers.
SO NH2
2
O
N
R
N
SH
A: R = 8-Cl, K (hCA II) = 0.31 nM; K (hCA XII) = 5.5 nM
I
I
B: R = 6-Me, K (hCA II) = 0.25 nM; K (hCA XII) = 5.2 nM
I
I
C: R = 7,8-(MeO) , K (hCA II) = 0.61 nM; K (hCA XII) = 0.67 nM
2
I
I
Fig. 1: Sulfonamides A-C incorporating 2-mercapto-3H-quinazolin-4-one functionalities and their
1
hCA II and XII inhibitory properties.
2
2
. Results and discussion
.1. Chemistry
The drug design strategy for obtaining novel sulfonamides used in this work is based on the tail
1
5
approach developed by one of our groups earlier. Substituted anthranilic acids 1a-o and 4-
isothiocyanatoethyl-benzenesulfonamide 2 have been employed for preparing the new sulfonamides
3
a-o (Scheme 1) by a chemistry already reported in the previous work, in which the derivatives

1
,16
without the ethylene spacer, of types A-C, have been reported.
In fact we have prepared a large
number of thioureas incorporating benzenesulfonamide functionalities, by reaction of aromatic
sulfonamides incorporating isothiocyanato moieties with amines (aromatic, aliphatic or
1
7b
17a
17c
heterocyclic), amino acids, and oligopeptides. Many of the derivatives obtained by such
procedures (incorporating the NH-CS-NH-C H -SO NH functionality) showed excellent CA
6
4
2
2
1
7
inhibitory properties against isoforms such as hCA II, IV, IX and XII, involved in serious
2
,3
4,5
pathologies, such as glaucoma and cancer. Furthermore, some of these sulfonamides also
1
7
showed good degrees of isoform selectivity inhibitory profiles. Thus, in this work we extend our
1
,17
previous investigations
on the use of 4-isothiocyanatoethyl-benzenesulfonamide for obtaining
CAIs incorporating novel heterocyclic scaffolds (Scheme 1).
Scheme 1. General synthetic procedure for compounds 3a-o.
The reaction of the substituted anthranilic acid 1a-o with the isothiocyanate 2 initially led to an
ortho-carboxyphenyl-thiourea, which has not been isolated, as in the reaction conditions we used it
underwent cyclisation with the formation of mercapto-quinazolinones 3a-o (Scheme 1). The
chemical diversity of the obtained series was achieved by using variously substituted-anthranilic
acids which incorporated halogens (F, Cl, Br, I), methyl and methoxy moieties (one, two or three
1
such moieties) in their molecules, similar with the sulfonamides reported in the previous work
(Table 1).
The structure of the new compounds was confirmed by means of NMR, MS and elemental
1
analyses (see Experimental for details). The H-NMR spectra showed D O exchangeable singlet
2
peak corresponding to the SH group at around 13.0 ppm, D O exchangeable peaks due to the
2
SO NH protons at around 7.50 ppm. The aromatic protons were also detected at their expected
2
2
1
3
respected regions. The C-NMR spectra were also in agreement with the suggested structure of the
target compounds (see Experimental for details).The mass spectra (MS) showed the exact mass ion
for each compound and the corresponding isotopes when halogens were present in the new
compounds (see Experimental for details).

2
.2. Inhibition of CAs
We investigated the inhibition profile of sulfonamides 3a-3o against the isoforms hCA I and II
cytosolic, off-target isoforms when considering the anti-cancer applications of CAIs, but target
(
1
8
isoforms for glaucoma) and hCA XII (predominantly present in tumors but also overexpressed in
8
glaucomatous eyes). For comparison reasons we also discuss the inhibition profiles of the standard,
clinically used sulfonamide drug acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide,
AAZ) against the same isoforms (Table 1).
Table 1: Inhibition data of human CA isoforms hCA I, II and XII with sulfonamides 3a-o reported
here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO hydrase
2
1
8
assay.
K (nM)
I
Compound
R
hCAI
hCA II
hCA XII
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
H
31.5
212
0.62
12.4
9.1
0.59
0.62
0.64
0.54
0.66
1.45
0.76
5.53
0.61
0.73
0.54
0.47
5.74
0.65
6-F
7-F
39.9
153
6-Cl
6.5
8-Cl
41.4
104
0.78
0.91
0.71
56.2
1.2
7-Br
g
h
i
6-I
34.3
296
6-CH3
8-CH3
6,8-CH3
6-OCH3
7-OCH3
8-OCH3
6,7-OCH3
2954
1402
150
j
0.86
1.0
k
l
28.5
42.3
678
0.69
31.9
1.1
m
n

3
o
6,7,8-OCH3 268
- 250
10.7
12
7.11
5.7
AAZ
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 %
of the reported values).
The following structure-activity relationship (SAR) can be observed from data of Table 1:
(i) hCA I, the slow, off-target cytosolic isoform was inhibited by all sulfonamides 3a-3o with
inhibition constants ranging between 28.5 and 2954 nM. Some derivatives showed good inhibitory
properties, such as 3a, 3c, 3e, 3g, 3l and 3m, with K s ranging between 28.5 and 42.3 nM. They
I
incorporated either H at the quinazolinone ring (3a) or 7-F, 8-Cl, 6-I, 7-MeO and 8-MeO
substituents. Interestingly, small structural changes (such as the shift of the fluorine from the 7 to
the 6 position of the heterocyclic ring, e.g., in 3b), led to important differences of activity with 3b,
which resulted 5.3 fold weaker inhibitor of hCA I when compared to 3c (Table 1). The same is true
for the isomers 3d-3e, 3k-3l, etc. Some of these derivatives, such as 3b, 3d, 3f, 3h, 3k and 3o
showed medium potency hCA I inhibition, in the same range as acetazolamide (K = 250 nM), with
I
inhibition constants in the range of 104 – 268 nM. Three derivatives, 3i, 3j and 3n were even
weaker inhibitors, with K s ranging between 678 and 2954 nM. Thus, the main SAR conclusion is
I
that the type and, more importantly, the position of the R substituent in the quinazolinone ring have
great influence the hCA I inhibitory properties.
(ii) SAR was even simpler for the inhibition of the physiologically dominant cytosolic isoform hCA
II (Table 1). Thus, all the new sulfonamides 3a-3o were highly effective inhibitors with two of them
having K s in the range of 31.9 – 56.2 nM (compounds 3h and 3m) whereas the remaining ones
I
acting as very string inhibitors with K s in the range of 0.62 – 12.4 nM (as effective as AAZ or one
I
order of magnitude better inhibitors compared to this clinically used compound). Thus, basically all
substitution patterns present in these sulfonamides led to effective hCA II inhibitors except the 6-
Me and 8-MeO groups which led to slightly weaker inhibitors.
(iii) As hCA II, also the transmembrane isoform hCA XII was highly inhibited by all the new
sulfonamides reported here with K s in the range of 0.54 – 7.11 nM. The SAR is thus very flat and
I
all the substitution patterns from the new sulfonamides were highly beneficial for inducing high
affinity for hCA II (Table 1).
3
. Conclusions

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to
series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These
sulfonamides were investigated as inhibitors of the following isoforms: hCA I and II (cytosolic
isozymes), as well as hCA XII (a trans-membrane, tumor-associated enzyme also involved in
glaucomagenesis). The new sulfonamides acted as medium potency inhibitors of hCA I (K s of 28.5
I
–
2954 nM), being highly effective as hCA II (K s in the range of 0.62 – 12.4 nM) and XII (K s of
I I
0
.54 – 7.11 nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens,
methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring)
led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as
preclinical candidates in pathologies in which the activity of these enzymes should be inhibited,
such as glaucoma (CA II and XII as targets) or some tumors in which the expression of isoforms
CA II and XII is dysregulated.
4
4
. Experimental protocols
.1. Chemistry. Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Alfa
Aesar and TCI. All reactions involving air- or moisture-sensitive compounds were performed under
a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear
1
13
magnetic resonance ( H-NMR, C-NMR) spectra were recorded using a Bruker Avance III 400
MHz spectrometer in DMSO-d . Chemical shifts are reported in parts per million (ppm) and the
6
coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s,
singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet; dd, double of doublets. The assignment
of exchangeable protons (OH and NH) was confirmed by the addition of D O. Analytical thin-layer
2
chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash chromatography
purifications were performed on Merck Silica gel 60 (230-400 mesh ASTM) as the stationary phase
and ethyl acetate/n-hexane were used as eluents. Melting points (mp) were measured in open
capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are uncorrected.
1
General procedure for the synthesis of benzenesulfonamides 3a-o.
The commercially available 2-aminobenzoic acid derivatives 1a-o (1.0 eq) and isothiocyanato-
benzenesulfonamide 2 (1.0 eq) were suspended in EtOH, and the reaction was refluxed until starting
materials were consumed (TLC monitoring). Then the mixture was cooled down to r.t. and the
precipitate formed was collected by filtration to give a residue that was purified by silica gel column

chromatography eluting with the appropriate ethyl acetate/n-hexane solution or by trituration from
diethyl ether to afford the desired compounds 3a-o.
2
Synthesis of 4-(2-isothiocyanato-ethyl)-benzenesulfonamide (2).
A suspension of ethylaminobenezene sulfonamide (1.0g, 1.0 eq) in dry pyridine (2.0 ml) was cooled
to -10 °C and then treated with CS (5.0 g, 13.2 eq) and DCC (1.0g, 1.0 eq). The reaction mixture
2
was stirred at r.t. under a nitrogen atmosphere until consumption of the starting material (TLC
monitoring). Then the solvent was removed under vacuo to give a pale yellow solid that was
crystallized from acetone. The solution was recovered, concentrated under vacuo and the residue
obtained was purified by silica gel column chromatography eluting with 5% MeOH/DCM to afford
the titled compound as a pale yellow solid.
4
5
-(2-Isothiocyanato-ethyl)-benzenesulfonamide 2: 52% yield; silica gel TLC R 0.17 (MeOH/DCM
f
2
% v/v); m.p. 142°C (lit. 146°C); δ (400 MHz, DMSO-d ) 3.08 (2H, t, J 6.8, N-CH -CH -), 4.00
H
6
2
2
(
2H, t, J 6.8, N-CH -CH -), 7.37 (2H, s, exchange with D O, SO NH ), 7.52 (2H, d, J 8.4, Ar-H),
2 2 2 2 2
7
1
.82 (1H, d, J 8.4, Ar-H); δ (100 MHz, DMSO-d ) 35.8, 46.4, 126.7, 128.8 (-N=C=S), 130.3,
C 6
42.7, 143.6; Elemental analysis: calc: C 44.61, H 4.16, N 11.56, S 26.47; found: C 44.65, H 4.15,
+
N 11.59, S 26.44; m/z (ESI positive) 243.02 [M+H] .
2
Experimental data are in agreement with reported data.
Synthesis of 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide (3a).
SO NH
2
2
O
N
N
SH
3a
2
-Aminobenzoic acid 1a (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-benzenesulfonamide 2
(0.17g, 1.0 eq) were treated in EtOH (10 ml) at reflux for 5h according to the general procedure

previously described. Then the solvent was removed under vacuo to give a residue that was
triturated from diethyl ether to afford the title compound 3a as a white solid
4
-[2-(2-Mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3a: 74% yield; silica gel
TLC R 0.21 (Ethyl acetate/n-hexane 40 % v/v); m.p. 274°C sub.; δ (400 MHz, DMSO-d ) 3.10
f
H
6
(2H, m, N-CH -CH -), 4.65 (2H, m, N-CH -CH -), 7.33 (2H, s, exchange with D O, SO NH ), 7.39
2 2 2 2 2 2 2
(2H, m, Ar-H), 7.53 (2H, d, J 8.4, Ar-H), 7.78 (3H, m, Ar-H), 8.02 (1H, d, J 8.4, Ar-H), 13.05 (1H,
s, exchange with D O, -SH); δ (100 MHz, DMSO-d ) 33.0, 47.5, 116.4, 116.6, 125.5, 126.9, 128.2,
2
C
6
1
30.0, 136.4, 140.0, 143.3, 143.6, 160.1, 175.8; Elemental analysis: calc: C 53.17, H 4.18, N 11.63,
+
S 17.7; found: C 53.20, H 4.15, N 11.62, S 17.9; m/z (ESI positive) 361.06 [M+H] .
Synthesis of 4-[2-(6-fluoro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide (3b)
2
-Amino-5-fluorobenzoic acid 1b (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-benzenesulfonamide
2
(0.16g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the general procedure
previously described. The precipitate formed was collected by filtration and triturated from diethyl
ether to afford the title compound 3b as a white solid.
4
-[2-(6-Fluoro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3b: 80% yield;
silica gel TLC R 0.19 (Ethyl acetate/n-hexane 50 % v/v); m.p. >310°C; δ (400 MHz, DMSO-d )
f
H
6
3
.10 (2H, t, J 5.6, N-CH -CH -), 4.64 (2H, t, J 5.6, N-CH -CH -), 7.35 (2H, s, exchange with D O,
2 2 2 2 2
SO NH ), 7.49 (1H, m, Ar-H), 7.52 (2H, d, J 8.4, Ar-H), 7.70 (1H, m, Ar-H), 7.72 (1H, d, J 8.1, Ar-
2
2
H), 7.82 (2H, d, J 8.4, Ar-H), 13.13 (1H, s, exchange with D O, -SH); δ (376 MHz, DMSO-d ) -
2
F
6
2
3
1
16.43 (1F, s); δ (100 MHz, DMSO-d ) 32.9, 47.6, 113.1 (d, J 24), 117.7 (d, J 8), 119.3 (d,
C 6 C-F C-F
3
2
1
4
JC-F 8), 124.7 (d, J 24), 126.9, 130.0, 136.9, 143.3, 143.5, 159.3 (d, J 241), 159.5 (d, J 3),
C-F
C-F
C-F
1
1
75.3; Elemental analysis: calc: C 50.65, H 3.72, N 11.07, S 16.90; found: C 50.66, H 3.70, N
+
1.10, S 16.78; m/z (ESI positive) 380.06 [M+H] .
Synthesis of 4-(2-(7-fluoro-2-mercapto-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (3c)

2
-Amino-4-fluorobenzoic acid 1c (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-benzenesulfonamide
2
(0.16g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the general procedure
previously described. The precipitate formed was collected by filtration and triturated from diethyl
ether to afford the title compound 3c as a white solid.
4
-[2-(7-Fluoro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3c: 78% yield;
silica gel TLC R 0.17 (Ethyl acetate/n-hexane 40 % v/v); m.p. 246°C; δ (400 MHz, DMSO-d )
f
H
6
3
7
.09 (2H, t, J 5.6, N-CH -CH -), 4.62 (2H, t, J 5.6, N-CH -CH -), 7.14 (1H, dd, J 8.1, 2.2, Ar-H),
2 2 2 2
.23 (1H, m, Ar-H), 7.35 (2H, s, exchange with D O, SO NH ), 7.52 (2H, d, J 8.4, Ar-H), 7.83 (2H,
2
2
2
d, J 8.4, Ar-H), 8.08 (1H, m, Ar-H), 13.09 (1H, s, exchange with D O, -SH); δ (376 MHz, DMSO-
2
F
d ) -101.87 (1F, s); δ (100 MHz, DMSO-d ) 33.0, 47.5, 102.7 (d, J 27), 113.7 (d, JC-F 12), 113.9,
6
C
6
C-F
1
5
3
27.0, 130.1, 131.9, 141.8 (d, JC-F 13), 143.5 (d, JC-F 23), 159.5, 176.4; Elemental analysis: calc: C
0.65, H 3.72, N 11.07, S 16.90; found: C 50.68, H 3.69, N 11.11, S 16.85; m/z (ESI positive)
+
80.06 [M+H] .
Synthesis of 4-[2-(6-chloro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide (3d)
2
-Amino-5-chlorobenzoic acid 1d (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.14g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the
general procedure previously described. The precipitate formed was collected by filtration and
triturated from diethyl ether to afford the title compound 3d as a light-brown solid.

4
-[2-(6-Chloro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3d: 68% yield;
silica gel TLC R 0.21 (Ethyl acetate/n-hexane 50 % v/v); m.p. >310°C; δ (400 MHz, DMSO-d )
f
H
6
3
.09 (2H, t, J 5.6, N-CH -CH -), 4.63 (2H, t, J 5.6, N-CH -CH -), 7.35 (2H, s, exchange with D O,
2 2 2 2 2
SO NH ), 7.45 (1H, d, J 8.1, Ar-H), 7.52 (2H, d, J 8.4, Ar-H), 7.83 (2H, d, J 8.4, Ar-H), 7.84 (1H,
2
2
d, J 8.1, Ar-H), 7.95 (1H, s, Ar-H), 13.16 (1H, s, exchange with D O, -SH); δ (100 MHz, DMSO-
2
C
d6) 32.9, 47.7, 117.9, 119.0, 127.0, 127.2, 129.4, 130.1, 136.5, 138.9, 143.4,. 143.6, 159.3, 175.8;
Elemental analysis: calc: C 48.54, H 3.56, N 10.61, S 16.20; found: C 48.56, H 3.58, N 10.57, S
+
1
6.17; m/z (ESI positive) 396.02 [M+H] .
Synthesis of 4-[2-(7-chloro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide (3e)
2
-Amino-4-chlorobenzoic acid 1e (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-benzenesulfonamide
2
(0.14g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the general procedure
previously described. The precipitate formed was collected by filtration and triturated from diethyl
ether to afford the title compound 3e as a white solid.
4
-[2-(7-Chloro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3e: 65% yield;
silica gel TLC R 0.25 (Ethyl acetate/n-hexane 50 % v/v); m.p. >310°C; δ (400 MHz, DMSO-d )
f
H
6
3
.09 (2H, t, J 5.6, N-CH -CH -), 4.63 (2H, t, J 5.6, N-CH -CH -), 7.35 (2H, s, exchange with D O,
2 2 2 2 2
SO NH ), 7.42 (2H, m, Ar-H), 7.52 (2H, d, J 8.4, Ar-H), 7.80 (2H, d, J 8.4, Ar-H), 8.00 (1H, s, Ar-
2
2
H), 13.08 (1H, s, exchange with D O, -SH); δ (100 MHz, DMSO-d ) 33.6, 48.2, 116.1, 116.6,
2
C
6
1
4
3
26.4, 127.6, 130.7, 131.2, 141.6, 141.6, 144.0, 144.3, 160.3, 177.0; Elemental analysis: calc: C
8.54, H 3.56, N 10.61, S 16.20; found: C 48.59, H 3.60, N 10.58, S 16.16; m/z (ESI positive)
+
96.02 [M+H] .
Synthesis of 4-[2-(7-Bromo-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide (3f)

2
-Amino-4-bromobenzoic acid 1f (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-benzenesulfonamide
2
(0.11g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the general procedure
previously described. The precipitate formed was collected by filtration and triturated from diethyl
ether to afford the title compound 3f as a white solid.
4
-[2-(7-Bromo-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3f: 55% yield;
silica gel TLC R 0.40 (Ethyl acetate/n-hexane 50 % v/v); m.p. 307°C; δ (400 MHz, DMSO-d )
f
H
6
3
.07 (2H, t, J 5.6, N-CH -CH -), 4.62 (2H, t, J 5.6, N-CH -CH -), 7.33 (2H, s, exchange with D O,
2 2 2 2 2
SO NH ), 7.54 (3H, m, Ar-H), 7.60 (1H, s, Ar-H), 7.79 (2H, d, J 8.4, Ar-H), 7.90 (1H, d, J 8.1, Ar-
2
2
H), 13.07 (1H, s, exchange with D O, -SH); δ (100 MHz, DMSO-d ) 34.7, 49.5, 119.0, 125.0,
2
C
6
1
4
26.4, 128.0, 128.3, 130.0, 131.0, 141.0, 143.0, 149.1, 159.3, 177.1. Elemental analysis: calc: C
3.64, H 3.20, N 9.54, S 14.56; found: C 43.66, H 3.25, N 9.50, S 14.52; m/z (ESI positive) 441.34
+
[
M+H] .
Synthesis of 4-[2-(6-iodo-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide (3g)
2
-Amino-5-iodobenzoic acid 1g (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-benzenesulfonamide
2
(0.09g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the general procedure
previously described. The precipitate formed was collected by filtration and triturated from diethyl
ether to afford the title compound 3g as a white solid.
4
-[2-(6-Iodo-2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3g: 68% yield;
silica gel TLC R 0.12 (Ethyl acetate/n-hexane 40 % v/v); m.p. 230°C with dec; δ (400 MHz,
f
H

DMSO-d ) 3.10 (2H, t, J 5.6, N-CH -CH -), 4.63 (2H, t, J 5.6, N-CH -CH -), 7.40 (5H, m,
6
2
2
2
2
SO NH , Ar-H), 7.79 (2H, d, J 8.4, Ar-H), 8.10 (1H, brs, Ar-H), 8.25 (1H, m, Ar-H), 13.11 (1H, s,
2
2
exchange with D O, -SH); δ (100 MHz, DMSO-d ) 33.0, 47.5, 98.4, 120.0, 126.4, 127.1, 129.0,
2
C
6
1
1
30.2, 136.1, 137.2, 140.7, 143.6, 160.1, 177.0. Elemental analysis: calc: C 39.43, H 2.90, N 8.62, S
+
3.16; found: C 39.40, H 2.87, N 8.59, S 13.12; m/z (ESI positive) 487.94 [M+H] .
Synthesis of 4-[2-(2-mercapto-6-methyl-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide
3h)
(
2
-Amino-5-methylbenzoic acid 1h (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.16g, 1.0 eq) were treated in EtOH (10 ml) at reflux for 5h according to the
general procedure previously described. The precipitate formed was collected by filtration and was
triturated from diethyl ether to afford the title compound 3h as a light-brown solid
4
-[2-(2-Mercapto-6-methyl-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3h: 32% yield;
silica gel TLC R 0.25 (Ethyl acetate/n-hexane 40 % v/v); m.p. >310°C; δ (400 MHz, DMSO-d )
f
H
6
2
.41 (3H, s, CH ), 3.10 (2H, t, J 5.6, N-CH -CH -), 4.66 (2H, t, J 5.6, N-CH -CH -), 7.35 (2H, s,
3 2 2 2 2
exchange with D O, SO NH ), 7.37 (1H, d, J 8.1, Ar-H), 7.52 (2H, d, J 8.4, Ar-H), 7.63 (1H, dd, J
2
2
2
8
.1, 2.2, Ar-H), 7.82 (2H, d, J 8.4, Ar-H), 7.83 (1H, d, J 2.2, Ar-H), 13.12 (1H, s, exchange with
D O, -SH); δ (100 MHz, DMSO-d ) 22.0, 33.4, 48.0, 121.0, 126.8, 127.1, 129.0, 130.2, 136.2,
2
C
6
1
1
37.0, 137.1, 140.7, 143.6, 160.1, 176.4. Elemental analysis: calc: C 54.38, H 4.56, N 11.19, S
+
7.08; found: C 54.40, H 4.60, N 11.17, S 17.12; m/z (ESI positive) 376.09 [M+H] .
Synthesis of 4-[2-(2-mercapto-8-methyl-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide (3i)

2
-Amino-3-methylbenzoic acid 1i (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.16g, 1.0 eq) were treated in EtOH (10 ml) at reflux for 5h according to the
general procedure previously described. The precipitate formed was collected by filtration and was
triturated from diethyl ether to afford the title compound 3i as a light-brown solid.
4
-[2-(2-Mercapto-8-methyl-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3i: 20% yield;
silica gel TLC R 0.22 (Ethyl acetate/n-hexane 40 % v/v); m.p. 286 °C; δ (400 MHz, DMSO-d )
f
H
6
2
7
8
.53 (3H, s, CH ), 3.10 (2H, t, J 5.6, N-CH -CH -), 4.66 (2H, t, J 5.6, N-CH -CH -), 7.31 (1H, t, J
3 2 2 2 2
.6, Ar-H), 7.35 (2H, s, exchange with D O, SO NH ), 7.54 (2H, d, J 8.4, Ar-H), 7.63 (1H, dd, J
2
2
2
.1, 2.2, Ar-H), 7.82 (2H, d, J 8.4, Ar-H), 7.89 (1H, d, J 7.6, Ar-H), 13.00 (1H, s, exchange with
D O, -SH); δ (100 MHz, DMSO-d ) 18.2, 33.0, 47.8, 116.8, 125.5, 125.5, 126.2, 127.0, 130.1,
2
C
6
1
1
37.8, 138.5, 143.4, 143.7, 160.3, 176.2. Elemental analysis: calc: C 54.38, H 4.56, N 11.19, S
+
7.08; found: C 54.42, H 4.58, N 11.20, S 17.15; m/z (ESI positive) 376.09 [M+H] .
Synthesis of 4-[2-(2-mercapto-6,8-dimethyl-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide
3j)
(
2
-Amino-3,5-dimethylbenzoic acid 1j (0.1 g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.15g, 1.0 eq) were treated in EtOH (10 ml) at reflux for 5h according to the
general procedure previously described. The precipitate formed was collected by filtration and was
triturated from diethyl ether to afford the title compound 3j as a light-brown solid.
4
-[2-(2-Mercapto-6,8-dimethyl-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3j: 22%
yield; silica gel TLC R 0.30 (Ethyl acetate/n-hexane 40 % v/v); m.p. 275°C; δ (400 MHz, DMSO-
f
H
d ) 2.37 (3H, s, CH ), 2.49 (3H, s, CH ), 3.11 (2H, t, J 5.6, N-CH -CH -), 4.68 (2H, t, J 5.6, N-CH -
6
3
3
2
2
2
CH -), 7.35 (2H, s, exchange with D O, SO NH ), 7.47 (1H, s, Ar-H), 7.53 (2H, d, J 8.4, Ar-H),
2
2
2
2
7
.70 (1H, s, Ar-H), 7.82 (2H, d, J 8.4, Ar-H), 11.80 (1H, s, exchange with D O, -SH); δ (100 MHz,
2 C
DMSO-d ) 18.4, 20.0, 33.4, 48.1, 116.9, 124.4, 125.5, 126.0, 126.8, 130.2, 136.6, 138.5, 143.4,
6

1
4
43.6, 160.2, 176.2. Elemental analysis: calc: C 55.51, H 4.92, N 10.79, S 16.47; found: C 54.53, H
+
.89, N 10.82, S 16.50; m/z (ESI positive) 390.50 [M+H] .
Synthesis of 4-[2-(2-mercapto-6-methoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide
3k)
(
2
-Amino-5-methoxy-benzoic acid 1k (0.2g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.29g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the
general procedure previously described. The precipitate formed was collected by filtration and was
triturated from diethyl ether to afford the title compound 3k as a light-brown solid.
4
-[2-(2-Mercapto-6-methoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3k: 28% yield;
silica gel TLC R 0.11 (MeOH/CDM 5 % v/v); m.p. 273°C; δ (400 MHz, DMSO-d ) 3.09 (2H, t, J
f
H
6
5
.6, N-CH -CH -), 3.87 (3H, s, OCH ), 4.66 (2H, t, J 5.6, N-CH -CH -), 7.35 (2H, s, exchange with
2 2 3 2 2
D O, SO NH ), 7.42 (3H, m, Ar-H), 7.52 (2H, d, J 8.4, Ar-H), 7.82 (2H, d, J 8.4, Ar-H), 13.05 (1H,
2
2
2
s, exchange with D O, -SH); δ (100 MHz, DMSO-d ) 33.6, 48.2, 57.3, 109.4, 117.8, 119,2, 126.4,
2
C
6
1
1
27.5, 130.8, 135.0, 143.5, 144.5, 157.8, 161.0, 174.9; Elemental analysis: calc: C 52.16, H 4.38, N
+
0.73, S 16.38; found: C 52.20, H 4.35, N 10.75, S 16.40; m/z (ESI positive) 392.09 [M+H] .
Synthesis of 4 4-[2-(2-mercapto-7-methoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide
3l)
(
2
-Amino-4-methoxybenzoic acid 1l (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.14g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the

general procedure previously described. The solvent was removed under vacuo to give a residue
that was triturated from diethyl ether and then purified by silica gel column chromatography eluting
with 50% ethyl acetate/n-hexane to afford the title compound 3l as a light-brown solid.
4
-[2-(2-Mercapto-7-methoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3l: 21% yield;
silica gel TLC R 0.10 (MeOH/DCM 5 % v/v); m.p. 235°C; δ (400 MHz, DMSO-d ) 3.07 (2H, t, J
f
H
6
5
.6, N-CH -CH -), 3.90 (3H, s, OCH ), 4.70 (2H, t, J 5.6, N-CH -CH -), 7.34 (2H, s, exchange with
2 2 3 2 2
D O, SO NH ), 7.48 (3H, m, Ar-H), 7.50 (2H, d, J 8.4, Ar-H), 7.90 (2H, d, J 8.4, Ar-H), 12.90 (1H,
2
2
2
s, exchange with D O, -SH); δ (100 MHz, DMSO-d ) 34.0, 48.1, 56.1, 118.9, 119.4, 126.2, 128.0,
2
C
6
1
1
31.1, 135.4, 143.3, 144.0, 153.0, 156.1, 161.1, 177.6. Elemental analysis: calc: C 52.16, H 4.38, N
+
0.73, S 16.38; found: C 52.18, H 4.36, N 10.76, S 16.35; m/z (ESI positive) 392.09 [M+H] .
Synthesis of 4-[2-(2-mercapto-8-methoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide
3m)
(
2
-Amino-3-methoxybenzoic acid 1m (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.14g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the
general procedure previously described. The solvent was removed under vacuo to give a residue
that was triturated from diethyl ether to afford the title compound 3m as a light-brown solid.
4
-[2-(2-Mercapto-8-methoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3m: 33%
yield; silica gel TLC R 0.15 (MeOH/DCM 5 % v/v); m.p. 295°C; δ (400 MHz, DMSO-d ) 3.10
f
H
6
(
2H, t, J 5.6, N-CH -CH -), 4.00 (3H, s, OCH ), 4.67 (2H, t, J 5.6, N-CH -CH -), 7.36 (3H, m,
2 2 3 2 2
exchange with D O, SO NH , Ar-H), 7.44 (1H, m, Ar-H), 7.62 (2H, d, J 8.4, Ar-H), 7.68 (1H, m,
2
2
2
Ar-H), 7.75 (2H, d, J 8.4, Ar-H), 11.80 (1H, brs, exchange with D O, -SH); δ (100 MHz, DMSO-
2
C
d6) 33.6, 48.2, 56.0, 116.0, 118.8, 119.1, 126.0, 127.6, 130.7, 135.1, 143.5, 144.4, 152.9, 161.1,
1
1
76.9. Elemental analysis: calc: C 52.16, H 4.38, N 10.73, S 16.38; found: C 52.22, H 4.39, N
+
0.75, S 16.40; m/z (ESI positive) 392.09 [M+H] .

Synthesis
of
4-[2-(2-mercapto-6,7-dimethoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-
benzenesulfonamide (3n)
2
-Amino-4,5-dimethoxybenzoic acid 1n (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.12g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the
general procedure previously described. The precipitate formed was collected by filtration and
triturated from diethyl ether to afford the title compound 3n as a white solid.
4
-[2-(2-Mercapto-6,7-dimethoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3n: 80%
yield; silica gel TLC R 0.20 (MeOH/DCM 5 % v/v); m.p. 302°C; δ (400 MHz, DMSO-d ) 3.09
f
H
6
(
2H, t, J 5.6, N-CH -CH -), 3.88 (3H, s, OCH ), 3.91 (3H, s, OCH ), 4.66 (2H, t, J 5.6, N-CH -
2 2 3 3 2
CH -), 6.98 (1H, s, Ar-H), 7.35 (3H, s, exchange with D O, SO NH , Ar-H), 7.52 (2H, d, J 8.4, Ar-
2
2
2
2
H), 7.82 (2H, d, J 8.4, Ar-H), 12.86 (1H, brs, exchange with D O, -SH); δ (100 MHz, DMSO-d )
2
C
6
3
1
3.2, 47.4, 56.8, 57.0, 107.8, 109.1, 126.9, 130.1, 130.2, 136.1, 143.3, 143.7, 147.8, 156.4, 159.7,
74.7; Elemental analysis: calc: C 51.29, H 4.54, N 9.97, S 15.22; found: C 51.30, H 4.56, N 10.01,
+
S 15.25; m/z (ESI positive) 422.09 [M+H] .
Synthesis
of
4-[2-(2-mercapto-6,7,8-trimethoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-
benzenesulfonamide (3o)
2
-Amino-3,4,5-trimethoxybenzoic acid 1o (0.1g, 1.0 eq) and 4-(2-isothiocyanato-ethyl)-
benzenesulfonamide 2 (0.11g, 1.0 eq) were treated in EtOH (10 ml) at reflux O.N. according to the

general procedure previously described. The precipitate formed was collected by filtration and
triturated from diethyl ether to afford the title compound 3o as a white solid.
4
-[2-(2-Mercapto-6,7,8-trimethoxy-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamide 3o: 75%
yield; silica gel TLC R 0.27 (Ethyl acetate/n-hexane 50 % v/v); m.p. 260°C; δ (400 MHz, DMSO-
f
H
d ) 3.09 (2H, t, J 5.6, N-CH -CH -), 3.92 (3H, s, OCH ), 3.93 (3H, s, OCH ), 3.94 (3H, s, OCH ),
6
2
2
3
3
3
4
7
.67 (2H, t, J 5.6, N-CH -CH -), 7.27 (1H, s, Ar-H), 7.35 (2H, s, exchange with D O, SO NH ),
2 2 2 2 2
.53 (2H, d, J 8.4, Ar-H), 7.82 (2H, d, J 8.4, Ar-H), 12.18 (1H, s, exchange with D O, -SH); δ (100
2
C
MHz, DMSO-d ) 33.0, 48.2, 56.8, 56.9, 59.2, 117.2, 119.1, 127.0, 131.0, 131.2, 136.2, 143.0,
6
1
46.0, 148.1, 156.4, 160.4, 170.0. Elemental analysis: calc: C 50.54, H 4.69, N 9.31, S 14.20;
+
found: C 50.57, H 4.71, N 9.27, S 14.16; m/z (ESI positive) 453.00 [M+H] .
4
.2. CA inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA-catalyzed
1
8
CO hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
2
working at the absorbance maximum of 557 nm, with 20 mM Hepes buffer (pH 7.5) and 20 mM
NaClO for maintaining constant ionic strength, following the initial rates of the CA-catalyzed CO
4
2
hydration reaction for a period of 10-100 s, at 20 °C. The CO concentrations ranged from 1.7 to 17
2
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 to determine the initial velocity.
The uncatalyzed rates were measured in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (1 mM) were prepared in distilled-deionized water and dilutions
down to 0.01 nM were made thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated 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
1
9-21
methods using PRISM 3 and the Cheng-Prusoff equation as reported earlier,
and represent the
mean from at least three different determinations. All CA isoforms were recombinant ones obtained
1
9-21
in huse as reported earlier,
and their concentrations in the assay system were: 12.3 nM for hCA
I, 8.5 nM for hCA II and 10.2 nM for hCA XII, respectively.
Acknowledgements
This project was supported by the Deanship of Scientific Research at Prince Sattam bin Abdulaziz
University, Saudi Arabia under the research project number ‘‘2014/03/2048, and by two European
Union FP7 projects, Metoxia and Dynano.

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Graphical abstract
K (hCA I) = 28.5 – 2954 nM
I
K (hCA II) = 0.625 – 12.4 nM
I
K (hCA XII) = 0.54 – 7.11 nM
I