Synthesis of new 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamides with strong inhibition properties against the tumor associated carbonic anhydrases IX and XII

Article abstract of DOI:10.1016/j.bmc.2017.03.054

We report a series of novel metanilamide-based derivatives 3a–q bearing the 2-mercapto-4-oxo-4H-quinazolin-3-yl moiety as tail. All compounds were synthesized by means of straightforward condensation procedures and were investigated in vitro for their inh

Full text of DOI:10.1016/j.bmc.2017.03.054

Accepted Manuscript
Synthesis of new 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfona-
mides with strong inhibition properties against the tumor associated carbonic
anhydrases IX and XII
Murat Bozdag, Ahmed Mahmoud Alafeefy, Abdul Malik Altamimi, Fabrizio
Carta, Claudiu T. Supuran, Daniela Vullo
PII:
S0968-0896(17)30444-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.054
BMC 13652
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 March 2017
21 March 2017
24 March 2017
Please cite this article as: Bozdag, M., Alafeefy, A.M., Altamimi, A.M., Carta, F., Supuran, C.T., Vullo, D., Synthesis
of new 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamides with strong inhibition properties against
the tumor associated carbonic anhydrases IX and XII, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.03.054
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Synthesis of new 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamides with strong
inhibition properties against the tumor associated carbonic anhydrases IX and XII
Murat Bozdag,1† Ahmed Mahmoud Alafeefy,2† Abdul Malik Altamimi,3 Fabrizio Carta,1
Claudiu T. Supuran1* and Daniela Vullo4*
1 Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche,
Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
2 Chemistry Department, Kulliyyah of Science, International Islamic University, Kuantan, Malaysia
3 Pharmaceutical Chemistry Dept., College of Pharmacy, Prince Sattam Bin Abdulaziz University,
Saudi Arabia.
4 Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica,
Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
Abstract: We report a series of novel metanilamide-based derivatives 3a-q bearing the 2-mercapto-
4-oxo-4H-quinazolin-3-yl moiety as tail. All compounds were synthesized by means of
straightforward condensation procedures and were investigated in vitro for their inhibition potency
against the human (h) carbonic anhydrase (CA; EC 4.2.1.1.1) isoforms I, II, IX and XII. Among all
compounds tested the 6-iodo 3g and the 7-fluoro 3i derivatives were the most potent inhibitors
against the tumor associated CA IX and XII isoform (KIs 1.5 and 2.7 nM respectively for the hCA
IX and KIs 0.57 and 1.9 nM respectively for the hCA XII).
The kinetic data reported here strongly support compounds of this type for their future
development as radiotracers in tumor pathologies which are strictly dependent on the enzymatic
activity of the hCA IX and XII isoforms.
Keywords: Carbonic anhydrase inhibitors (CAIs), Quinazolines, Tumors, Imaging.
_____
* Corresponding authors: daniela.vullo@unifi.it (DV) Phone: +39-055-4573005; Fax: +39-055-
4573385; claudiu.supuran@unifi.it. (CTS) Phone: +39-055-4573729; Fax: +39-055-4573385
† These author equally contributed to the work

1. Introduction
The carbonic anhydrase (CAs, EC 4.2.1.1) IX is a validated pharmacological target for the
treatment of hypoxic tumors. [1] To date the hCA IX monoclonal chimeric antibody Girentuximab
(Rencarex®) and its iodo radiolabeled derivative (Redectane®) are currently marketed for the
treatment and diagnosis of renal cell carcinomas (RCCs) respectively. Among the small molecule
drugs, the most advanced product in the pipeline is represented by the sulfanilamide derivative
SLC-0111 (figure 1) discovered by our group, which successfully ended its Phase-I trial program.
[2]
Figure 1. Chemical structure of the SLC-0111 carbonic anhydrase IX inhibitor.
Despite the availability of antibody-based drugs, the development of small molecules as
hCA IX selective inhibitors, such as the SLC-0111, still remains an attractive approach for two
main reasons: i) small molecule drugs do not suffer of the heavy drawbacks usually associated to
the antibodies such as immunogenicity, [3] long circulation times in the bloodstream, [4] and low
penetration into solid tumors; [5] ii) production costs of clinical grade antibodies are quite high
when compared to “classical” chemical drugs. [6]
Several classes of compound moieties have been investigated with the final intent to identify hCA
IX selective and druggable small molecules. The main contributions include phenols and
thiophenols, [7, 8] polyamines, [9] cyclic and/or fluorinated tertiary sulfonamides, [10]
dithio/monothiocarbamates and xanthates, [11, 12] as well as the coumarins and their thio
derivatives. [13-18] However, the primary sulfonamides (-SO2NH2) still represent the main class of
CA inhibitors (CAIs) investigated so far. They strongly coordinate the enzymatic metal ion (Zn2+ in
the humans), which is buried at the low edge of the conical active site, [19] whereas the tail of the
molecular scaffold is subjected to derivatizations with various chemical moieties. Such a strategy,
commonly referred as the tail approach, resulted particularly efficient in the obtainment of isoform
selective CAIs of the sulfonamide (-SO2NH2) type, [20] but also coumarin, [21] sulfocoumarin [22]
as well as dithiocarbamate derivatives [23] were obtained. From the molecular viewpoint the
isoform selectivity of small molecules through the tail approach resides in the modulation of the
number/type of chemical interactions interceding between the enzymatic rim of the targeted CA,

highly variable, and the tail end of the inhibitor. [19c, 20] Thus we considered that the 2-mercapto-
4-oxo-4H-quinazolin-3-yl scaffold is of particular relevance in this context, since it offers multiple
sites for hydrogen bonding interactions, and the additional substitutions within the scaffolds may
serve for further enhancing the ligand-enzyme complex (figure 1).
Figure 1. Interaction sites offered by the 2-mercapto-4-oxo-4H-quinazolin-3-yl scaffold.
Herein, as extension of our recent previous reports [24-26] and with the aim to identify
selective hCA IX drug leads, we report a series of sulfonamide compounds possessing the 3-(2-
mercapto-4-oxo-4H-quinazolin-3-yl)-moiety and we explored their inhibitory effects against the
four physiological relevant hCA isoforms such as the cytosolic and abundantly expressed I, II and
the tumor associated IX and XII.
2. Results and Discussion
2.1. Chemistry
All compounds reported here , 3a-q, were obtained by using the same synthetic strategy developed
from our groups for the obtainment of 2-mercapto-4-oxo-4H-quinazolin-3-yl scaffolds substituted at
the 3 position with ethylaminobenzene sulfonamides [24] or sulfanilamides. [25] Specifically for
this work we coupled the commercially available anthranilic acids 1a-q with the freshly synthesized
3-isothiocyanato-benzenesulfonamide 2 in alcohol at reflux (scheme 1).

Scheme 1. General synthetic scheme for the obtainment of compounds 3a-q.
All new compounds reported here were properly characterized by means of NMR, MS and
elemental analyses.
2.2. Carbonic anhydrase inhibition
All synthesized compounds 3a-q were investigated for their inhibition potencies against the four
physiological relevant hCAs I, II, IX and XII, by means of the stopped flow CO2 hydrase assay,
[27] and their data compared to the standard CAI acetazolamide (AAZ).
Table 1: Inhibition data of human CA isoforms hCA I, II, IX and XII with compounds 3a-q and the
standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay. [27]
SO NH
2
2
O
N
N
R
SH
3 a-q
KI (nM)*
hCA IX
Compound
R
H
hCAI
hCA II
hCA XII
3a
189
14.6
76.1
10.5

3b
3c
6-Cl
276
164
348
296
143
387
135
169
563
719
1650
3200
7650
965
19.8
0.98
10.7
1.1
45.1
15.4
9.5
12.0
5.8
7-Cl
3d
3e
8-Cl
3.5
7-Br
16.8
4.0
2.4
3f
8-Br
0.82
27.6
7.0
2.6
3g
3h
3i
6-I
1.5
0.57
3.2
6-F
4.4
7-F
6.6
2.7
1.9
3j
6-Me
8-Me
6,7-(Me)2
6-MeO
7-MeO
8-MeO
18.6
65.4
210
98.4
104
77.8
126
76.5
12.0
58.9
25.2
34.5
13.0
25.0
15.6
19.8
20.1
25.0
45.2
7.6
3k
3l
9.8
3m
3n
3o
3p
3q
AAZ
10.5
23.4
23.9
4.7
6,7-(MeO)2 3570
6,7,8-(MeO)3 4600
6.0
-
250
5.7
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5-10 %
of the reported values).
Overall the compounds 3a-q reported showed interesting inhibitory activities. A structure-activity-
relationship (SAR) is below reported.
i)
The cytosolic and abundantly expressed hCA I was the least inhibited isoform among those
considered in this study with KI values, reported in table 1, spanning from 135 to 7650 nM.
In general 3a and its halogen substituted derivatives 3b-i showed higher inhibition potencies
when compared to the alkyl and alkoxy containing counterparts 3j-q and in the same order
of magnitude of the AAZ (table 1). Among the first series, the nature of the halide as well as
its position within the 2-mercapto-4-oxo-4H-quinazolin scaffold strongly affected the hCA I
inhibition values. For instance the 7-chloro derivative 3c was 1.7 and 2.1 times stronger than
its 6- and 8- chloro-substituted regioisomers respectively. The 8-bromo derivative 3f was
more potent than its 7-bromo derivative 3e (2.1 fold). Conversely when the fluorine atom
was considered, the 6-substituted regioisomer 3h showed higher inhibition potency when
compared to its 7-fluoro counterpart 3i (KIs of 135 and 169 nM respectively). The bulky 6-

iodo-2-mercapto-4-oxo-4H-quinazolin derivative 3g was the least potent among the halogen
containing compounds in inhibiting the hCA I isoform (KI 387 nM).
As above stated the introduction in 3a of alkyl moieties resulted detrimental for the
inhibition potency. The 6-methyl derivative 3j was the most potent in this series in
inhibiting the hCA I isoform (KI 563 nM), followed by the 8-methyl regioisomer 3k which
resulted 1.3 times less potent. All remaining compounds showed remarkably higher KI
values which were all in the µM range (table 1).
ii)
In analogy to the hCA I, also the cytosolic CA II isoform was efficiently inhibited from the
unsubstituted 3a (KI 14.6 nM) and from its halogen derivatives 3b-i (KIs between 27.6 and
0.82 nM). Among the halogen containing series a similar enzymatic profile to the hCA I is
also shown. Thus the 7-chlorosubstituted 2-mercapto-4-oxo-4H-quinazolin scaffold 3c (KI
0.98 nM) was by far more potent in inhibiting the hCA II than its 6- and 8-regioisomers (KIs
19.8 and 10.7 nM respectively), and again the 8-bromo derivative 3f was slightly more
potent when compared to its 7-substituted regioisomer (KIs 0.82 and 1.1 nM respectively).
The bulky iodo derivative 3g was confirmed as the weaker inhibitor among the halogen
containing compounds and even less potent when compared to the standard CAI AAZ (table
1). In the case of the fluoro substituted derivatives 3h and 3i it is worth noticing that their
inhibition profiles against the hCA II isoform was opposite when compared to the hCA I. In
fact the 7-fluoroderivative was slightly more potent (0.9 fold) than its 6-substituted
regioisomer.
Finally as for the hCA I, the alkyl containing derivatives showed weak KI data, with the 6-
and 8-methyl substituted compounds 3j and 3k being the strongest in the series (KIs of 18.6
and 65.4 nM respectively).
iii)
An interesting enzymatic profile was shown for the transmembrane and tumor associated
hCA IX. The unsubstituted 3a was the least potent within all the series reported (KI 76.1
nM) and far less active when compared to the standard CAI AAZ (KI 25 nM). The
introduction of a chloro moiety within the 2-mercapto-4-oxo-4H-quinazolin scaffold to
afford the regioisomers 3b-d, determined a remarkable increase of the inhibition potency.
As shown in table 1, the 8-substituted derivative 3d was the most potent among the series
with a KI value of 9.5 nM and thus 4.7 and 1.6 times more effective than 3b and 3c
respectively. In analogy to the cytosolic hCA I and II, also for the IX isoform the in vitro
inhibition profiles of the 7- and 8-bromosubstituted derivatives 3e and 3f were similar (KIs
16.8 and 4.0 nM respectively). It is noteworthy that in this case their selectivity index (KI
3f/KI 3e) for the hCA IX isoform is the highest reported, including the hCA XII discussed

later. The fluoro containing derivatives 3h and 3i showed low nanomolar inhibition
potencies against the hCA IX and no particular differences between the two regioisomers
were reported (KIs 4.4 and 2.7 nM respectively). Interestingly the iodo derivative 3g showed
a remarkable inhibition potency (KI 1.5 nM), which makes it as the most potent inhibitor
against the IX isoform among all the compound series here reported.
As for the alkyl and alkoxy substituted derivatives 3j-q, the 6-methyl regioisomer 3j was the
least effective in inhibiting the hCA IX (KI 58.9 nM), followed by its doubling 3l (KI 34.5
nM) and by its 8-substituted derivative 3k which showed a KI value almost identical to the
standard CAI AAZ (25.2 nM and 25.0 nM respectively). Better results were obtained when
the methoxy moieties were introduced as in compounds 3m-q. Among the monosubstituted
methoxy derivatives, the 6- and the 8-regioisomers were the most effective (KIs 13.0 and
15.6 nM respectively), whereas the 7-methoxy 3n was the weakest (25.0 nM). Further
introduction of methoxy moieties to afford the di- or tri-substituted compounds 3p and 3q
didn’t result in any significant effect on the kinetic data (KI 19.8 and 20.1 nM respectively).
The second tumor associated hCA, the XII isoform, resulted strongly inhibited from all the
compounds reported. The only exception was represented from the 6-methylsubstituted
derivative 3j which showed a KI value of 45.2 nM (table 1). The introduction within 3a (KI
10.5 nM) of the chloro moiety at 6-, 7- and 8-position to afford 3b-d, resulted in a
progressive increase of the inhibition potency (KIs 12.0, 5.8 and 3.5 nM). The bromo
regioisomers 3e and 3f resulted nearly equal for their ability to inhibit the hCA IX isoform
(KIs 2.4 and 2.6 nM respectively), whereas among the fluoro derivatives, the 7-substituted
isomer 3i showed a preferential inhibition over its 6-fluoro regioisomer 3h (KIs 1.9 and 3.2
nM respectively). As for the hCA IX, also the XII isoform resulted strongly inhibited from
the iodo derivative 3g, which was the strongest among all the compound series reported (KI
0.57 nM).
iv)
As above stated, the 6-methyl compound 3j was the weakest, among all compounds
reported, in inhibiting the hCA XII (KI 45.2 nM). It is interesting to note that the 8-methyl
regioisomer 3k (KI 7.6 nM) and the 6,7-dimethyl derivative 3l (KI 9.8 nM) resulted 5.9 and
4.6 times respectively more potent against this isoform. A significant influence of the
regioselectivity on the hCA XII inhibition data was also evident for the methoxy substituted
derivatives 3m-o. In this case the 7- and the 8-methoxy compounds 3n and 3o were 2.2. and
2.3 times respectively less potent when compared to the 6-methoxy isomer 3m (KI 10.5
nM). Finally the multiple introduction of methoxy groups within the 2-mercapto-4-oxo-4H-
quinazolin-3-yl scaffold, as for 3p and 3q, resulted beneficial for the inhibition potency

against the hCA XII, as they showed KI values comparable to the standard CAI AAZ (see
table 1).
3. Conclusions
We reported a series of new metanilamides bearing the 2-mercapto-4-oxo-4H-quinazolin-3-yl as
tails. All compounds were obtained by means of condensation of the freshly prepared 3-
isothiocyanatobenzene sulfonamide 2 with commercially available anthranilic acids. All compounds
were investigated in vitro for their ability to inhibit the most physiological relevant hCA isoforms
such as the cytosolic hCA I, II and the tumor associated IX and XII isoforms. In general all halogen
containing compounds 3b-i showed higher inhibition potencies against all the hCA isoforms
considered when compared to the unsubstituted 3a as well as to the alkyl bearing derivatives 3j-q.
The hCA I was the least inhibited isoform, whereas the hCA II, which is deeply involved in
glaucoma, is effectively inhibited from the 7-chloro 3c, 7- and 8-bromo derivatives 3e and 3f
respectively (KIs 0.98, 1.1 and 0.82 nM). As for the tumor associated hCA IX isoform, the 6-iodo
3g and the 7-fluoro 3i derivatives resulted the most potent inhibitors among all compounds tested
(KIs 1.5 and 2.7 nM respectively). Such result is of particular interest, since the presence of fluorine
and iodine makes them attractive leads for their possible development as radiotracers for diagnostic
purposes in hypoxic tumors positively expressing the hCA IX isoform. In analogy to the hCA IX,
also the XII isoform resulted potently inhibited from 3g and 3i (KIs 0.57 and 1.9 nM respectively).
In addition the alkyl poly-substituted derivatives 3p and 3q resulted also quite effective in
inhibiting this isoform with KIs of 4.7 and 6.0 nM and thus comparable to the halogen derivatives
3b-i.
We conclude that some of these compounds are of particular relevance for their future development
of new leads with diagnostic applications in pathologies expressing the hCAs IX and XII such as
the hypoxic tumors.
4. Experimental protocols
4.1. 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 magnetic
resonance (1H-NMR, 13C-NMR) spectra were recorded using a Bruker Avance III 400 MHz

spectrometer in DMSO-d6. Chemical shifts are reported in parts per million (ppm) and the coupling
constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd, double of doublets. The
assignment of exchangeable protons was confirmed by the addition of D2O. Analytical thin-layer
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 (m.p.) were carried out in open
capillary tubes and are uncorrected.
Synthesis of 3-isothiocyanato-benzenesulfonamide (2). [28]
Metanilamide (1.0 eq) was dissolved in a 1.0 M hydrochloric acid aqueous solution and then treated
at r.t. with thiophosgene (1.0 eq). A precipitate was readily formed and the reaction mixture was
stirred overnight The precipitate formed was collected by filtration, washed with H2O and dried
under vacuum to afford the titled compounds as white solids, which were used without further
purification.
3-Isothiocyanato-benzenesulfonamide 2: 81% yield; silica gel TLC Rf 0.46 (MeOH/DCM 10 %
v/v); mp 128 °C (lit. [28] 130°C); δH (400 MHz, DMSO-d6) 7.48 (2H, s, exchange with D2O,
SO2NH2), 7.63 (1H, d, J 8.4, Ar-H), 7.68 (1H, dd, J 8.4, Ar-H), 7.89 (1H, d, J 8.4, Ar-H), 7.92 (1H,
s, Ar-H); δC (100 MHz, DMSO-d6) 123.0, 124.2, 129.7, 129.9, 130.1 (-N=C=S), 132.0, 142.0;
Elemental analysis: calc: C 39.24, H 2.82, N 13.07, S 29.93; found: C 39.26, H 2.84, N 13.09, S
29.91; m/z (ESI positive) 215.00 [M+H]+.
General procedure for the synthesis
of 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-
benzenesulfonamides 3a-q. [24, 25]

The commercially available 2-aminobenzoic acid derivatives 1a-q (1.0 eq) and 2-isothiocyanato-
benzenesulfonamide 2 (1.0 eq) were suspended in EtOH and the reaction mixture 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 trituration
from diethyl ether to afford the desired compounds 3a-q.
Synthesis of 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamide (3a). The titled
compound 3a was obtained according to the general procedure previously reported using 2-
aminobenzoic acid 1a and 2-isothiocyanato-benzenesulfonamide 2. 90% yield, mp > 300 °C; δH
(400 MHz, DMSO-d6) 7.50 (2H, s, exchange with D2O, SO2NH2), 7.55 (4H, m), 7.67 (2H, m), 7.93
(1H, d, J 7.2), 8.33 (1H, s), 13.16 (1H, s, exchange with D2O, SH); δC (100 MHz, DMSO-d6) 119.7,
120.6, 122.3, 122.9, 125.1, 127.3, 129.2, 129.4, 134.1, 134.7, 140.2, 147.5, 160.2, 176.9; m/z (ESI
negative) 332.0 [M-H]-.
Synthesis of 3-(6-chloro-2-mercapto-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3b). The
titled compound 3b was obtained according to the general procedure previously reported using 2-
amino-5-chlorobenzoic acid 1b and 2-isothiocyanato-benzenesulfonamide 2. 88% yield, mp > 300
°C; δH (400 MHz, DMSO-d6) 7.42 (2H, s, exchange with D2O, SO2NH2), 7.58 (3H, m), 7.94 (2H,
m), 8.11 (1H, s), 8.38 (1H, s), 13.17 (1H, s, exchange with D2O, SH); δC (100 MHz, DMSO-d6)
119.6, 121.1, 125.2, 126.3, 127.2, 128.2, 129.3, 130.4, 133.6, 136.5, 140.4, 140.5, 160.6, 176.9; m/z
(ESI negative) 366.0 [M-H]-.
Synthesis of 3-(7-chloro-2-mercapto-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3c) The titled
compound 3c was obtained according to the general procedure previously reported using 2-amino-
4-chlorobenzoic acid 1c and 2-isothiocyanato-benzenesulfonamide 2. 69% yield; mp 191 °C; δH
(400 MHz, DMSO-d6) 7.43 (2H, s, exchange with D2O, SO2NH2), 7.50 (7H, m), 11.34 (1H, s,
exchange with D2O, SH); δC (100 MHz, DMSO-d6) 119.0, 119.5, 122.8, 123.0, 126.8, 127.3, 132.2,
133.5, 133.6, 138.4, 140.0, 148.8, 160.0, 177.5; m/z (ESI positive) 368.00 [M+H]+.

Synthesis of 3-(8-chloro-2-mercapto-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3d). The
titled compound 3d was obtained according to the general procedure previously reported using 2-
amino-3-chlorobenzoic acid 1d and 2-isothiocyanato-benzenesulfonamide 2. 69% yield, mp > 300
°C; δH (400 MHz, DMSO-d6) 7.50 (2H, s, exchange with D2O, SO2NH2), 7.62 (3H, m), 7.93 (3H,
m), 8.03 (1H, s), 13.01 (1H, s, exchange with D2O, SH); δC (100 MHz, DMSO-d6) 119.4, 121.8,
125.7, 126.5, 127.2, 127.5, 130.6, 133.5, 136.5, 137.7, 140.6, 146.0, 160.1, 177.4; m/z (ESI
negative) 366.0 [M-H]-.
Synthesis of 3-(7-bromo-2-mercapto-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3e). The titled
compound 3e was obtained according to the general procedure previously reported using 2-amino-
4-bromobenzoic acid 1e and 2-isothiocyanato-benzenesulfonamide 2. 62% yield, mp > 300 °C; δH
(400 MHz, DMSO-d6) 7.48 (2H, s, exchange with D2O, SO2NH2), 7.56 (4H, m), 7.87 (1H, d, J 7.1),
7.93 (2H, m), 13.16 (1H, s, exchange with D2O, SH); δC (100 MHz, DMSO-d6) 111.5, 117.3, 124.9,
127.1, 128.6, 129.7, 130.1, 130.9, 135.5, 140.8, 142.6, 143.9, 159.4, 177.1; m/z (ESI negative)
409.9 [M-H]-
Synthesis of 3-(8-bromo-2-mercapto-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3f). The titled
compound 3f was obtained according to the general procedure previously reported using 2-amino-3-
bromobenzoic acid 1f and 2-isothiocyanato-benzenesulfonamide 2. 62% yield; mp 285 °C; δH (400
MHz, DMSO-d6) 7.43 (2H, s, exchange with D2O, SO2NH2), 7.60 (7H, m), 13.17 (1H, s, exchange
with D2O, SH); δC (100 MHz, DMSO-d6) 110.0, 120.1, 122.5, 122.7, 124.2, 127.3, 127.8, 130.8,
132.5, 134.4, 140.4, 149.9, 159.9, 177.4; m/z (ESI positive) 411.9 [M+H]+.
Synthesis of 3-(6-iodo-2-mercapto-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3g). The titled
compound 3g was obtained according to the general procedure previously reported using 2-amino-
5-iodobenzoic acid 1g and 2-isothiocyanato-benzenesulfonamide 2. 82% yield, mp > 300 °C; δH
(400 MHz, DMSO-d6) 7.19 (2H, m), 7.42 (1H, d, J 7.3), 7.46 (1H, d, J 7.2), 7.51 (1H, m), 7.55 (2H,
s, exchange with D2O, SO2NH2), 8.15 (1H, d, J 7.3), 8.32 (1H, d, J 7.2), 13.25 (1H, s, exchange
with D2O, SH); δC (100 MHz, DMSO-d6) 92.8, 119.6, 122.5, 123.8, 126.2, 128.1, 129.4, 134.1,
137.8, 141.3, 143.6, 147.3, 160.4, 177.2; m/z (ESI negative) 457.9 [M-H]-.
Synthesis of 3-(6-fluoro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamide (3h). The
titled compound 3h was obtained according to the general procedure previously reported using 2-
amino-5-fluorobenzoic acid 1h and 2-isothiocyanato-benzenesulfonamide 2. 53% yield, mp > 300

°C; δH (400 MHz, DMSO-d6) 7.38 (3H, m), 7.54 (2H, s, exchange with D2O, SO2NH2), 7.96 (2H,
m), 8.17 (1H, s), 8.33 (1H, s), 13.19 (1H, s, exchange with D2O, SH); δF (376 MHz, DMSO-d6) -
2
116.35 (1F, s); δC (100 MHz, DMSO-d6) 113.5 (d, JC-F 24), 117.9 (d, 3JC-F 8), 119.6 (d, 3JC-F 8),
2
1
124.5 (d, JC-F 24), 125.3, 129.8, 133.6, 138.2 ,140.4, 143.5, 159.4 (d, JC-F 273), 159.8 (d, 4JC-F 3),
161.5, 177.1; m/z (ESI negative) 350.0 [M-H]-.
Synthesis of 3-(7-fluoro-2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamide (3i). The titled
compound 3i was obtained according to the general procedure previously reported using 2-amino-4-
fluorobenzoic acid 1i and 2-isothiocyanato-benzenesulfonamide 2. 58% yield, mp > 300 °C; δH
(400 MHz, DMSO-d6) 7.23 (2H, m), 7.55 (2H, s, exchange with D2O, SO2NH2), 7.96-8.01 (4H, m),
8.27 (1H, s), 13.18 (1H, s, exchange with D2O, SH); δF (376 MHz, DMSO-d6) -101.78 (1F, s); δC
2
2
(100 MHz, DMSO-d6) 107.3 (d, JC-F 26), 114.7 (d, JC-F 13), 116.5, 119.4, 123.1, 125.0, 129.4,
3
2
1
130.3, 133.9, 142.3 (d, JC-F 13), 145.5 (d, JC-F 26), 159.6 (d, JC-F 273), 162.1, 176.7; m/z (ESI
negative) 350.0 [M-H]-.
Synthesis of 3-(2-mercapto-6-methyl-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamide (3j). The
titled compound 3j was obtained according to the general procedure previously reported using 2-
amino-5-methylbenzoic acid 1j and 2-isothiocyanato-benzenesulfonamide 2. 62% yield, mp > 300
°C; δH (400 MHz, DMSO-d6) 2.44 (3H, s), 7.23 (2H, m), 7.55 (2H, s, exchange with D2O,
SO2NH2), 7.87 (3H, m), 7.94 (2H, m), 13.13 (1H, s, exchange with D2O, SH); δC (100 MHz,
DMSO-d6) 22.9, 119.5, 120.7, 122.2, 123.1, 125.0, 129.3, 129.8, 133.5, 134.3, 137.5, 140.4, 144.6,
159.3, 177.1; m/z (ESI negative) 346.0 [M-H]-.
Synthesis of 3-(2-mercapto-8-methyl-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3k). The
titled compound 3k was obtained according to the general procedure previously reported using 2-
amino-3-methylbenzoic acid 1k and 2-isothiocyanato-benzenesulfonamide 2. 63% yield, mp > 300
°C; δH (400 MHz, DMSO-d6) 2.42 (3H, s), 7.25 (2H, m), 7.55 (2H, s, D2O exchangeable, SO2NH2),
7.78 (3H, m), 7.97 (2H, m), 13.03 (1H, s, exchange with D2O, SH); δC (100 MHz, DMSO-d6) 23.0,
118.9, 121.1, 123.4, 125.3, 126.3, 127.8, 129.5, 130.9, 132.3, 133.0, 139.1, 140.7, 159.2, 176.5; m/z
(ESI negative) 346.0 [M-H]-.
Synthesis of 3-(2-mercapto-6,7-dimethyl-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3l). The
titled compound 3l was obtained according to the general procedure previously reported using 2-
amino-4,5-dimethylbenzoic acid 1l and 2-isothiocyanato-benzenesulfonamide 2. 68% yield, mp >

300 °C; δH (400 MHz, DMSO-d6) 2.85 (6H, s), 6.84 (1H, s), 7.23 (1H, s), 7.56 (2H, s, exchange
with D2O, SO2NH2), 7.91 (4H, m), 13.17 (1H, s, exchange with D2O, SH); δC (100 MHz, DMSO-
d6) 23.7, 27.5, 118.2, 119.8, 122.5, 123.3, 124.2, 127.5, 128.1, 131.9, 134.2, 138.6, 140.8, 143.4,
159.6, 166.5; m/z (ESI negative) 360.0 [M-H]-.
Synthesis of 3-(2-mercapto-6-methoxy-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3m). The
titled compound 3m was obtained according to the general procedure previously reported using 2-
amino-5-methoxybenzoic acid 1m and 2-isothiocyanato-benzenesulfonamide 2. 71% yield; mp >
300 °C; δH (400 MHz, DMSO-d6) 3.87 (3H, s), 7.40 (1H, s), 7.48 (2H, s, exchange with D2O,
SO2NH2), 7.54 (2H, s), 7.59 (1H, m), 7.73 (1H, m), 7.78 (1H, s), 7.91 (1H, d, J 2.0), 13.12 (1H, s,
exchange with D2O, SH); δC (100 MHz, DMSO-d6) 57.8, 105.0, 118.7, 120.0, 120.2, 123.0, 127.2,
128.3, 131.6, 131.8, 138.7, 140.0 ;158.8, 160.6, 177.8; m/z (ESI negative) 362.0 [M-H]-.
Synthesis of 3-(2-mercapto-7-methoxy-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3n). The
titled compound 3n was obtained according to the general procedure previously reported using 2-
amino-4-methoxybenzoic acid 1n and 2-isothiocyanato-benzenesulfonamide 2. 71% yield; mp 297
°C; δH (400 MHz, DMSO-d6) 3.90 (3H, s), 6.95 (1H, s) 7.20 (1H, t, J 7.2), 7.42 (2H, s, exchange
with D2O, OH), 7.65-7-75 (3H, m), 8.05 (2H, m), 13.10 (1H, s, exchange with D2O, SH); δC (100
MHz, DMSO-d6) 57.8, 106.8, 114.2, 115.3, 120.1, 123.1, 125.8, 130.3, 133.2, 133.3, 140.0, 146.6,
160.0, 163.2, 172.5; m/z (ESI positive) 364.0 [M+H]+.
Synthesis of 3-(2-mercapto-8-methoxy-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3o). The
titled compound 3o was obtained according to the general procedure previously reported using 2-
amino-3-methoxybenzoic acid 1o and 2-isothiocyanato-benzenesulfonamide 2. 62% yield, mp >
300 °C; δH (400 MHz, DMSO-d6) 3.94 (3H, s), 7.00 (2H, m), 7.54 (2H, s, exchange with D2O,
SO2NH2), 7.77 (3H, m), 7.94 (2H, m), 12.54 (1H, s, exchange with D2O, SH); δC (100 MHz,
DMSO-d6) 57.1, 119.2, 119.9, 121.1, 121.7, 122.9, 124.3, 125.7, 127.5, 128.4, 132.2, 139.0, 151.2,
159.7, 176.7; m/z (ESI negative) 362.0 [M-H]-.
Synthesis of 3-(2-mercapto-6,7-dimethoxy-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3p).
The titled compound 3p was obtained according to the general procedure previously reported using
2-amino-4,5-dimethoxybenzoic acid 1p and 2-isothiocyanato-benzenesulfonamide 2. 80% yield, mp
> 300 °C; δH (400 MHz, DMSO-d6) 3.86 (3H, s), 3.92 (3H, s), 6.85 (1H, s), 7.33 (1H, s), 7.55 (2H,
s, exchange with D2O, SO2NH2), 7.97 (4H, m), 13.02 (1H, s, exchange with D2O, SH); δC (100

MHz, DMSO-d6) 56.8, 56.9, 99.0, 108.0, 109.5, 126.2, 127.3, 130.4, 133.6, 136.5, 144.5, 145.9,
147.6, 156.4, 160.2, 175.6; m/z (ESI negative) 392.0 [M-H]-.
Synthesis of 3-(2-mercapto-6,7,8-trimethoxy-4-oxoquinazolin-3(4H)-yl)benzenesulfonamide (3q).
The titled compound 3q was obtained according to the general procedure previously reported using
2-amino-3,4,5-trimethoxybenzoic acid 1q and 2-isothiocyanato-benzenesulfonamide 2. 75% yield,
mp > 300 °C; δH (400 MHz, DMSO-d6) 3.91 (3H, s), 3.95 (3H, s), 3.96 (3H, s), 7.25 (1H, s), 7.55
(3H, m, 2H exchange with D2O SO2NH2), 7.73 (2H, m), 7.90 (1H, d, J 8), 12.36 (1H, s, exchange
with D2O, SH); δC (100 MHz, DMSO-d6) 57.1, 61.8, 62.6, 104.2, 112.5, 126.3, 127.2, 130.0, 130.6,
133.6, 140.6, 140.9, 145.9, 148.6, 151.3, 160.1, 175.9; m/z (ESI negative) 422.0 [M-H]-.
4.3. CA inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity. [27] Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM
Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed
CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged 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
uncatalyzed rates were determined in the same manner and subtracted from the total observed rates.
Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to
0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min – 6 h at room temperature prior to assay, in order to allow for the
formation of the E-I complex. Data from Table 1 were obtained after 15 minutes incubation of
enzyme and inhibitor. The inhibition constants were obtained by non-linear least-squares methods
using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, [30] and represent the mean
from at least three different determinations. All CA isoforms were recombinant ones obtained in-
house as reported earlier. [30]
Acknowledgments:
The authors are grateful to Dr. Mariangela Ceruso of the Università degli Studi di Firenze for
helpful discussion and execution of the in vitro enzymatic experiments. This research was financed
by two EU grants of the seventh framework program (Metoxia and Dynano projects to C.T.S.). This

project was supported by the National Plan of Science, Technology and Innovation (Grant No. 12-
MED2980-54), Prince Sattam bin Abdulaziz University, Alkharj, PO Box 173, 11942 to A.M.A and
F.C.

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Synthesis of new 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamides with strong
inhibition properties against the tumor associated carbonic anhydrases IX and XII
Murat Bozdag,1† Ahmed Mahmoud Alafeefy,2† Abdul Malik Altamimi,3 Fabrizio Carta,1
Claudiu T. Supuran1* and Daniela Vullo4*