Benzenesulfonamides incorporating bulky aromatic/heterocyclic tails with potent carbonic anhydrase inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2015.11.023

Three series of sulfonamides incorporating long, bulky tails were obtained by applying synthetic strategies in which substituted anthranilic acids, quinazolines and aromatic sulfonamides have been used as starting materials. They incorporate long, bulky diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl moieties in their molecules, and were investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides showed excellent inhibitory effects against the four isoforms, with K_Is of 7.6-322 nM against hCA I, of 0.06-85.4 nM against hCA II; of 6.7-152 nM against hCA IX and of 0.49-237 nM against hCA XII; respectively. However no relevant isoform-selective behavior has been observed for any of them, although hCA II and XII, isoforms involved in glaucoma-genesis were the most inhibited ones. The structure-activity relationship for inhibiting the four CAs with these derivatives is discussed in detail.

Full text of DOI:10.1016/j.bmc.2015.11.023

Bioorganic & Medicinal Chemistry 23 (2015) 7751–7764
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzenesulfonamides incorporating bulky aromatic/heterocyclic
tails with potent carbonic anhydrase inhibitory activity
Murat Bozdag a,y, Ahmed M. Alafeefy b,y, Daniela Vullo a, Fabrizio Carta a, Nurcan Dedeoglu a,
Abdul-Malek S. Al-Tamimi c, Nabila A. Al-Jaber d, Andrea Scozzafava a, Claudiu T. Supuran e,
⇑
a Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
b Chemistry Department, Kulliyyah of Science, International Islamic University, Kuantan, Malaysia
c Pharmaceutical Chemistry Dept., College of Pharmacy, Prince Sattam Bin Abdulaziz University, Saudi Arabia
d Chemistry Department, College of Science, King Saud University, Saudi Arabia
e Università degli Studi di Firenze, NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of sulfonamides incorporating long, bulky tails were obtained by applying synthetic strate-
gies in which substituted anthranilic acids, quinazolines and aromatic sulfonamides have been used as
starting materials. They incorporate long, bulky diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl
moieties in their molecules, and were investigated for the inhibition of four physiologically relevant car-
bonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, as well as the transmem-
brane hCA IX and XII. Most of the new sulfonamides showed excellent inhibitory effects against the four
isoforms, with KIs of 7.6–322 nM against hCA I, of 0.06–85.4 nM against hCA II; of 6.7–152 nM against
hCA IX and of 0.49–237 nM against hCA XII; respectively. However no relevant isoform-selective behav-
ior has been observed for any of them, although hCA II and XII, isoforms involved in glaucoma-genesis
were the most inhibited ones. The structure–activity relationship for inhibiting the four CAs with these
derivatives is discussed in detail.
Received 13 October 2015
Revised 8 November 2015
Accepted 20 November 2015
Available online 23 November 2015
Keywords:
Carbonic anhydrase
Sulfonamide
Quinazoline
Tail approach
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
ring approach2 which explored various aromatic/heterocyclic ring
systems on which the SO2NH2 moiety was bound, afforded CA inhi-
The sulfonamides continue to be one of the important families
of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC
4.2.1.1), with many representatives in clinical use as diuretics,
antiglaucoma, antiepileptic, antiobesity drugs, or agents useful
for the treatment of some neurological disorders, such as idio-
pathic intracranial hypertension.1–10 Recently, a sulfonamide CA
inhibitor (CAI) developed by one of our groups by applying the tail
approach, SLC-0111, entered Phase I clinical trials for the treatment
of patients with advanced solid, metastatic tumors overexpressing
CA IX/XII.1–4
bitors (CAIs) possessing both high affinity and desired pharmaco-
logic properties.4–10 This approach, based on an ‘extension’ of the
aromatic/heterocyclic scaffolds through the anchoring tails has
been thereafter explored for sulfamates, sulfamides, and dithiocar-
bamates (as alternative ZBGs to the sulfonamide) but also to CAIs
incorporating scaffolds belonging to the aliphatic or glycosidic
chemical species.10–12 The advantage of the tail approach over
other drug design strategies was thereafter explained at the molec-
ular level, after the report of many X-ray crystallographic struc-
tures of adducts of various CA isoforms with such inhibitors.
These studies demonstrated that the active site of most CA isoen-
zymes is a rather large conical cavity in which the Zn(II) ion is posi-
tioned at its bottom. The lining of the active site builds two
adjacent very diverse halves, one entirely hydrophilic, the oppos-
ing one completely hydrophobic,12 with the highest variability of
amino acid residues between the different isoforms on the edge/
entrance of the active site. This is exactly the region in which the
tails of the inhibitors are usually accomodated,12–16 explaining
why these specific interactions between the inhibitor tail and
amino acid residues at the entrance of the active site may lead to
The tail approach1 reported by one of these groups more than
15 years ago allowed for the facile preparation of a large number
of potent and isoform-selective classes of CA inhibitors (CAIs).1–4
Indeed, an initial drug design strategy based on appending tails
of different size, shape or nature to pharmacophores incorporating
sulfonamides as zinc-binding group (ZBG),4–10 as opposed to the
⇑
Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
These authors contributed equally to the work.
y
http://dx.doi.org/10.1016/j.bmc.2015.11.023
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

7752
M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
O
compounds showing selectivity for inhibiting isoforms with phar-
macological applications.1–4 In this way many sulfonamides/sulfa-
mates/sulfamides/dithiocarbamates with excellent CA IX/XII over
CA I and II selectivity were reported,16 whereas the coumarins,
which bind exclusively in this outward region of the active site,
showed the highest isoform selectivity among all CAI classes
reported to date.17–24
R1
O
O
OH
NH
TEA
R1
OH
NH2
Cl
+
DCM
R2
O
R2
2a-c
1a-b
3a-f
Here we report a new series of sulfonamide CAIs obtained by
applying the tail approach. Our interest was to obtain compounds
with even longer tails than the ones we reported earlier, in order to
investigate whether prolonging the scaffold towards the outside
part of the enzyme active cavity may have interesting conse-
quences for the inhibition profiles of such sulfonamides against
Ca isoforms with applications in pharmacology, such as CA I and
II (cytosolic isoforms) as well as CA IX and XII (trans-membrane,
tumor-associated isoforms).
Ac2O
reflux
SO2NH2
SO2NH2
O
R1
O
O
R1
n
N
N
H
+
nNH2
reflux
R2
NH
O
4a-f
R2
2. Results and discussion
2.1. Chemistry
R1 = H, NO2
R2 = H, Cl, F
n = 0, 2
6a-k
5a-b
Scheme 1. Preparation of 2-benzamido-N-(4-sulfamoylphenyl)benzamides 6a–k.
Aromatic benzene sulfonamides (sulfanilamide and 4-
aminoethylbenzenesulfonamide) have been used in the present
drug design as main scaffolds as they led to CA IX/XII-selective CAIs
in several tail-approach studies.15,24 Furthermore, the amino moi-
ety present in these two compounds has a good reactivity and it
is easily derivatizable by reaction with a variety of derivatives.
Several synthetic strategies have been thus employed for pro-
longing the scaffold of these aromatic sulfonamides in order to
obtain long-tailed CAIs, as shown in Schemes 1–3.
O
O
N
O
NH2
R1
R1
S
OH
NH2
+
O
O
reflux
H2N
R2
n
1a-c
7a-d
5a-b
reflux
The anthranilic acid derivatives 1 were chosen for the first
approach, due to the fact that the amino moiety in ortho to the
carboxylic acid has a good reactivity with acyl halides, can partic-
ipate in cyclization reactions and in addition, diamides with the
geometry shown in Scheme 1 were not investigated earlier. Thus,
2-amino-5-substituted benzoic acids 1a,b were reacted with
4-substituted-benzoyl chlorides 2a–c leading to intermediates
3a–f which were thereafter cyclized in the presence of acetic
anhydrides to the key intermediates 4a–f, which by reaction with
sulfanilamide (5a) or 4-aminoethylbenzenesulfonamide (5b) led
to a first group of long-tailed sulfonamides 6a–k incorporating
diamide functionalities (Scheme 1).
O
N
R1
N
n
O
R2
S
NH2
O
8a-e
R1= H, NO2
R2= H, CH3
n = 0,2
, OCH3
Scheme 2. Preparation of 4-(4-oxoquinazolin-3(4H)-yl)benzenesulfonamides 8a–e.
For the second approach, again anthranilic acid derivatives 1
were considered, but this time in such a way as to obtain cyclic
compounds in which the amino and carboxylic moieties of the
starting material are involved. Again such derivatives were not
investigated earlier as CAIs. Condensation of 2-amino-5-substi-
tuted benzoic acids 1a–c with acetic anhydride or triethylorthofor-
mate afforded the cyclic intermediates 7a–d which were
subsequently reacted with the two sulfonamides mentioned above,
5a,b, leading to the second group of sulfonamides reported here
8a–e, incorporating 4-oxoquinazolin-3(4H)-yl tails in their mole-
cules (Scheme 2).
SO2NH2
Cl
N
SO2NH2
TEA
N
+
n
X
EtOH, reflux
XHn
N
X = NH, n = 0,2
X = O, n = 0
N
5a-d
9
10a-d
Scheme 3. Preparation of 4-substituted quinazolines 10a–d.
The last synthetic approach is shown in Scheme 3. In this case
we used as model reaction the one between cyanuryl chloride
and amino-sulfonamides,15b by which we have reported a large
series of highly isoform-selective CAIs. Indeed, the chlorine
atoms from cyanuryl chloride and the chlorine from 9 have a
good reactivity with various nucleophiles, leading thus to
The sulfonamides investigated here as CAIs were characterized
by physico-chemical and spectroscopic methods which confirmed
their structures (see Section 4).
chemical diversity by
a
facile synthetic approach. Thus,
2.2. Carbonic anhydrase inhibition
sulfanilamide, 4-aminoethyl-benzenesulfonamide or 4-hydroxy-
benzenesulfonamide were treated with 4-chloroquinazoline 9,
leading to the 4-substituted quinazolines 10a–d (Scheme 3).
Compounds 10a and 10b are also commercially available from
Aurora Screening library, but we prepared them by the procedure
illustrated above.
The sulfonamides 6a–6k, 8a–8e and 10–10d reported here were
investigated for their enzyme inhibitory action against four physi-
ologically relevant CA isoforms, the human (h) hCA I, II, IX and XII
(Table 1). Acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfon-
amide) was used as standard drug in the assay.25

M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
7753
Table 1
Inhibition of human (h) isoforms hCA I, II, IX and XII with the sulfonamides reported in the paper by a CO2 hydrase stopped-flow assay25
No.
R1
R2
n
KI* (nM)
hCA I
hCA II
hCA IX
hCA XII
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
H
NO2
NO2
NO2
H
H
F
H
Cl
F
H
Cl
F
H
Cl
F
CH3
CH3
CH3
CH3
H
0
0
0
0
0
2
2
2
2
2
2
0
2
2
2
2
9.7
7.6
2.6
0.41
9.5
18.2
9.3
21.3
20.1
9.1
6.7
7.2
0.85
7.0
7.2
3.3
0.49
26.3
6.5
5.4
18.2
42.3
25.8
183
8.5
237
34.7
31.5
48.1
62.6
86.7
85.2
261
67.1
64.3
322
7.2
8.0
7.1
H
H
75.5
32.3
85.4
65.5
7.3
0.84
0.76
4.3
25.0
79.1
79.5
41.4
8.5
35.9
8.0
79.3
88.9
NO2
NO2
NO2
NO2
H
NO2
OCH3
OCH3
6j
6k
8a
8b
8c
8d
8e
30.8
84.4
40.8
15.7
3.4
0.78
67.8
39.3
SO2NH2
HN
N
10a
—
9.5
0.06
8.1
1.3
2.2
N
SO2NH2
HN
10b
10c
—
—
9.1
6.9
8.4
N
N
HN
N
SO2NH2
55.5
0.08
152
0.96
N
N
SO2NH2
O
10d
AAZ
—
—
51.2
250
0.09
12.1
75.1
25.3
0.95
5.7
N
—
—
*
Mean from three different assays. Errors in the range of 5–10% of the reported values (data not shown).
The following structure–activity relationship (SAR) can be
drawn from the inhibition data shown in Table 1:
derivatives without this moiety (6a, 6b) and that most of
the time the sulfanilamide derivatives were more inhibitory
compared to the corresponding 4-aminoethylbenzenesul-
fonamides. All three main chemotypes investigated here, 6,
8 and 10, showed rather comparable biological activities in
inhibiting this isoform.
(i) The slow cytosolic isoform hCA I was inhibited by the new
sulfonamides 6, 8 and 10 reported here with KIs ranging
between 7.6 and 322 nM. Few compounds were low
nanomolar inhibitors, such as 6a, 6b, 10a and 10b (KIs of
7.6–9.5 nM). They usually incorporate sulfanilamide ‘heads’
(which led to better hCA I inhibitors compared to 4-
aminoethylbenzenesulfonamide), except 10b which has the
latter derivative in its molecule, as well as diamide tails
devoid of nitro moieties (in 6a and 6b) as well as quina-
zolin-4-yl moieties (in 10a and 10b). Two other derivatives,
6i and 8a, showed poor hCA I inhibitory power (similar to
AAZ), with inhibition constants of 261–322 nM. They do
not differ significantly from the remaining derivatives inves-
tigated here, which all of them showed a behavior of med-
ium potency hCA I inhibitor, with KIs ranging between 15.7
and 86.7 nM (Table 1), being more effective than the
standard drug acetazolamide against this isoform. The most
salient SAR features were that the nitro derivatives 6c–e
were less effective compared to the corresponding
(ii) The physiologically dominant isoform hCA II (cytosolic like
hCA I) was very effectively inhibited by most sulfonamides
investigated here, with KIs ranging between 0.06 and
85.4 nM. Four compounds were medium potency inhibitors,
that is, 6g–6j, with KIs ranging between 32.3 and 85.4 nM,
the remaining ones showing KIs <20 nM (Table 1). All the
less effective CAIs incorporate the diamide tail (with or
without the NO2 group) and are 4-aminoethylbenzenesul-
fonamide derivatives. It is interesting to note that the pres-
ence of the NO2 moiety did not influence markedly the CA
inhibitory properties of these derivatives, although we
expected that its acidifying effects may enhance CA inhibi-
tion. In fact many times the nitro-derivatives were less effec-
tive compared to the corresponding compounds without this
moiety (compare 6a and 6c, or 6b and 6e, etc). This is diffi-
cult to explain but probably the nitro moiety in that position

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M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
of the aromatic ring does not make good contacts with the
4. Experimental
4.1. Chemistry
enzyme active site. It should also be mentioned that several
subnanomolar hCA II inhibitors were detected here, such as
among others 6b, 8b, 8e, 10a, 10c and 10d. Especially the
last three compounds, which are quite simple and all of
them incorporate the quinazoline-4-yl moiety in their mole-
cules, are among the most effective CAIs ever reported, with
low picomolar activity against this isoform.
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, DEPT-135, DEPT-90, HSQC, HMBC) 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 pat-
terns are designated as follows: s, singlet; d, doublet; sept, septet;
t, triplet; q, quadruplet; m, multiplet; br s, broad singlet; dd, dou-
ble of doubles, appt, apparent triplet, appq, apparent quartet. The
assignment of exchangeable protons (OH and NH) was confirmed
by the addition of D2O. Analytical thin-layer chromatography
(TLC) was carried out on Merck silica gel F-254 plates. Flash chro-
matography 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 carried
out in open capillary tubes and are uncorrected.
(iii) The transmembrane, tumor-associated isoform hCA IX was
also effectively inhibited by most sulfonamides investigated
here, which showed KIs ranging between 6.7 and 152 nM
(Table 1). Several low nanomolar hCA IX inhibitors were
detected, such as 6c, 6g, 8a, 8c, 10a, and 10b (KIs ranging
between 6.7 and 9.1 nM), but unlike other classes of sulfon-
amides investigated earlier15 these derivatives did not show
any selectivity for inhibiting hCA IX over hCA II (or hCA I).
(iv) The second transmembrane, tumor-associated isoform hCA
XII, was very effectively inhibited by sulfonamides 6, 8 and
10 investigated here, with KIs ranging between 0.49 and
237 nM. Few compounds (8a, 8c and 8d) showed weak inhi-
bitory action, with KIs ranging between 67.8 and 237 nM,
whereas the vast majority of the remaining ones were highly
effective inhibitors. Several subnanomolar hCA XII inhibitors
were thus discovered, such as 6a, 6e, 10c and 10d. Thus, the
simple scaffolds present in 10c and 10d as well as the least
elaborated long tails from 6a and 6e may lead to effective,
subnanomolar inhibitors of this isoform involved both in
cancerogenesis as well as glaucoma.3,19,20
(v) No compounds with relevant isoform selectivity were
detected in the study, probably because the tails were too
long and they may adopt quite variable conformations when
bound within the enzyme active site. For example in the case
of 10c and 10d, where the position sulfonamide moieties (in
meta and respectively para to the bulky scaffold) should
force the molecules to adopt very different orientation, the
activity of these compounds is rather similar against the var-
ious isoforms. This is probably due to the fact that, as shown
by X-ray crystallography,11–14 due to the fact that the active
site is rather voluminous, the tail of the inhibitors may bind
either in the hydrophobic or hydrophilic half of the active
site with similar efficacy. The consequence is that the inhibi-
tors may show similar potency although their binding
modes are quite diverse.11–14 Some of the compounds
reported here were low nanomolar/subnanomolar inhibitors
of two isoforms involved in glaucoma (hCA II and XII) and
may be thus interesting candidates for such studies.
(a) General procedure for the synthesis of 2-benzamidoben-
zoic acids 3a–f: Anthranilic acid 1a,b (1.0 equiv) was treated
with benzoyl chloride 2a–c (1.1 equiv) in DCM followed by
drop-wise addition of TEA (1.5 equiv) at 0 °C. The mixture
was warmed-up to rt and stirred until the consumption of
starting materials (TLC monitoring). The reaction was
quenched with 1.0 M aqueous HCl solution, obtained precip-
itate was filtered, washed with n-hexane (3 ꢀ 10 ml) and
dried under vacuum to obtain desired compounds 3a–f.
(b) General procedure for the synthesis of benzoxazin-4-ones
4a–f: 2-Benzoylamino-benzoic acid 3a–f (1.0 equiv) was
refluxed in acetic anhydride (15.0 equiv) until the consump-
tion of starting material (TLC monitoring). Excess of solvents
were removed under vacuum and n-hexane (20 ml) was
added to mixture and stirred vigorously for 30 min, filtered
and washed with n-hexane (3 ꢀ 10 ml) and dried under vac-
uum to obtain desired compound 4a–f.
(c) General procedure for 2-benzamido-N-(4-sulfamoylphen-
yl)benzamides 6a–k: Sulfonamide 5a,b (1.0 equiv) was
treated withbenzoxazin-4-ones 4a–f (1.0 equiv) in a proper
solvent and refluxed until the consumption of starting mate-
rials (TLC monitoring). The reaction was treated accordingly
to obtain desired products 6a–k.
(d) General procedure for 2-methyl-4H-1,3-benzoxazin-4-
ones 7a–c: Anthranilic acid 1a–c (1.0 equiv) was refluxed
in Ac2O (10 equiv) until the consumption of starting material
(TLC monitoring). The reaction treated accordingly to give
the title compounds 7a–c.
(e) General procedure for 4-(2-methyl-4-oxoquinazolin-3
(4H)-yl)benzenesulfonamides 8a–e: Compound 5a–b was
treated with 2-methyl-4H-1,3-benzoxazin-4-one 7a–c in a
proper dry solvent and the stirring mixture was heated
(120–160 °C) until the consumption of starting materials
(TLC monitoring). The reaction treated accordingly to obtain
desired products 8a–e.
(f) General procedure for the synthesis of 4-substituted
quinazolines 10a–d: Sulfonamide 5a–d (1.0 equiv) was
treated with 4-chloroquinazoline 9 (1.0 equiv) in EtOH and
followed by addition of TEA (1.2 equiv).The reaction refluxed
until the consumption of starting materials (TLC monitor-
ing). Reaction quenched with slush and acidified with 1 M
3. Conclusions
Three series of sulfonamides incorporating long, bulky tails
were obtained by applying synthetic strategies in which substi-
tuted anthranilic acids, quinazolines and aromatic sulfonamides
have been used as starting materials. They incorporate long, bulky
diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl moieties in
their molecules, and were investigated for the inhibition of four
physiologically relevant CA isoforms, the cytosolic hCA I and II, as
well as the transmembrane hCA IX and XII. Most of the new sulfon-
amides reported here showed excellent inhibitory effects against
the four isoforms, with KIs of 7.6–322 nM against hCA I, of 0.06–
85.4 nM against hCA II; of 6.7–152 nM against hCA IX and of
0.49–237 nM against hCA XII; respectively. However no relevant
isoform-selective behavior has been observed for any of them,
although hCA II and XII, isoforms involved in glaucoma-genesis
were the most inhibited ones.

M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
7755
aqueous HCl solution to obtain a precipitate which was
filtered and washed with Et2O (3 ꢀ 10 ml) and dried under
vacuum to obtain title product 10a–d.
Anthranilic acid 1a (1.5 g, 1.0 equiv) was treated with 4-fluo-
robenzoyl chloride 2b (1.1 equiv) in DCM (25 ml) followed by
drop-wise addition of TEA (1.5 equiv) at 0 °C. The reaction mixture
was treated according to general procedure a, previously reported,
to give the title compound 3c as a white solid.
4.1.1. Synthesis of 2-benzamidobenzoic acid 3a
2-(4-Fluorobenzamido)benzoic acid 3c: yield 78%; silica gel TLC
Rf = 0.7 (Ethyl acetate/n-hexane 40% v/v); mp 183–184 °C; dH
(400 MHz, DMSO-d6) 7.26 (1H, t, J 8.0), 7.48 (2H, m), 7.71 (1H, t,
J 8.0), 8.07 (3H, m), 8.70 (1H, d, J 8.0), 12.17 (1H, s, exchange with
D2O, NH), 13.83 (1H, br s, exchange with D2O, OH); dC (100 MHz,
O
O
OH
NH
O
TEA
2
3
OH
NH2
+
DMSO-d6) 116.9 (d, JC–F 22), 117.6, 120.9, 123.9, 130.7 (d, JC–F
1
DCM
Cl
9), 132.0, 132.2, 135.2, 142.0, 164.5, 165.3 (d, JC–F 248), 171.0; dF
O
(376 MHz, DMSO-d6) ꢁ107.9 (1F, s).
1a
2a
3a
4.1.4. Synthesis of 2-benzamido-5-nitrobenzoic acid 3d
Anthranilic acid 1a (2.0 g, 1.0 equiv) was treated with benzoyl
chloride 2a (1.1 equiv) in DCM (30 ml) followed by drop-wise addi-
tion of TEA (1.5 equiv) at 0 °C. The reaction mixture was treated
according to general procedure a, previously reported, to give the
title compound 3a as a white solid.
O
O2N
O
OH
NH
O
TEA
O2N
OH
NH2
+
DCM
Cl
O
2-Benzamidobenzoic acid 3a: yield 95%; silica gel TLC Rf = 0.55
(Ethyl acetate/n-hexane 50% v/v); mp 176–177 °C; dH (400 MHz,
DMSO-d6) 7.26 (1H, t, J 8.0), 7.62–7.74 (4H, m), 8.01 (2H, d, J
8.0), 8.10 (1H, d, J 8.0), 8.75 (1H, d, J 8.0), 12.21 (1H, br s, exchange
with D2O, NH); dC (100 MHz, DMSO-d6) 117.5, 120.8, 123.9, 127.9,
129.9, 132.2, 133.1, 135.2, 135.5, 142.0, 165.6, 170.9. Experimental
in agreement with reported data.26
1b
2a
3d
5-Nitroanthranilic acid 1b (1.5 g, 1.0 equiv) was treated with
benzoyl chloride 2a (1.1 equiv) in DCM (25 ml) followed by drop-
wise addition of TEA (1.5 equiv) at 0 °C. The reaction mixture
was treated according to general procedure a, previously reported,
to give the title compound 3d as a pale yellow solid.
4.1.2. Synthesis of 2-(4-chlorobenzamido)benzoic acid 3b
2-Benzamido-5-nitrobenzoic acid 3d: yield 85%; silica gel TLC
Rf = 0.45 (Ethyl acetate/n-hexane 50% v/v); mp 175–176 °C; dH
(400 MHz, DMSO-d6) 7.66 (2H, t, J 7.2), 7.73 (1H, t, J 7.2), 8.02
(2H, d, J 7.2), 8.55 (1H, dd, J 2.8, 9.2), 8.20 (1H, d, J 2.8), 8.97 (1H,
d, J 9.2), 12.54 (1H, s, exchange with D2O, NH); dC (100 MHz,
DMSO-d6) 117.6, 121.0, 127.6, 128.2, 130.1, 133.8, 134.6, 142.3,
147.3, 147.3, 166.1, 169.5. Experimental in agreement with
reported data.28
O
O
OH
NH
O
TEA
OH
NH2
+
Cl
DCM
Cl
O
Cl
1a
2b
3b
4.1.5. Synthesis of 2-(4-chlorobenzamido)-5-nitrobenzoic
acid 3e
Anthranilic acid 1a (1.5 g, 1.0 equiv) was treated with 4-
chlorobenzoyl chloride 2b (1.1 equiv) in DCM (25 ml) followed
by drop-wise addition of TEA (1.5 equiv) at 0 °C. The reaction mix-
ture was treated according to general procedure a, previously
reported, to give the title compound 3b as a white solid.
O
O2N
O
OH
NH
O
TEA
O2N
OH
NH2
+
Cl
2-(4-Chlorobenzamido)benzoic acid 3b: yield 95%; silica gel TLC
Rf = 0.57 (Ethyl acetate/n-hexane 20% v/v); mp 202–203 °C; dH
(400 MHz, DMSO-d6) 7.27 (1H, t, J 8.0), 7.70 (3H, m), 8.00 (2H, d,
J 8.8), 8.10 (1H, d, J 8.0), 8.69 (1H, d, J 8.0), 12.19 (1H, s, exchange
with D2O, NH); dC (100 MHz, DMSO-d6) 117.7, 120.9, 124.0, 129.7,
129.9, 132.1, 134.2, 135.0, 137.9, 141.7, 164.5, 170.7.
DCM
Cl
O
Cl
1b
3e
2b
Experimental in agreement with reported data.27
5-Nitroanthranilic acid 1b (1.5 g, 1.0 equiv) was treated with 4-
chlorobenzoyl chloride 2b (1.1 equiv) in DCM (25 ml) followed by
drop-wise addition of TEA (1.5 equiv) at 0 °C. The reaction mixture
was treated according to general procedure a, previously reported,
to give the title compound 3e as a pale brown solid.
2-(4-Chlorobenzamido)-5-nitrobenzoic acid 3e: yield 80%; sil-
ica gel TLC Rf = 0.60 (Ethyl acetate/n-hexane 50% v/v); mp 192–
193 °C; dH (400 MHz, DMSO-d6) 7.75 (2H, d, J 8.8), 8.03 (2H, d, J
8.8), 8.56 (1H, dd, J 2.8, 9.2), 8.83 (1H, d, J 2.8), 8.94 (1H, d, J 9.2),
12.68 (1H, s, exchange with D2O, NH); dC (100 MHz, DMSO-d6)
121.3, 127.7, 130.1, 130.3, 130.3, 133.7, 138.7, 142.6, 147.2,
165.3, 169.5.
4.1.3. Synthesis of 2-(4-fluorobenzamido)benzoic acid 3c
O
O
OH
NH
O
TEA
OH
NH2
+
F
DCM
Cl
O
F
1a
2c
3c

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M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
4.1.6. Synthesis of 2-(4-fluorobenzamido)benzoic acid 3f
A suspension of 2-(4-chlorobenzamido)benzoic acid 3b (2.5 g,
1.0 equiv) was refluxed in Ac2O (15.0 equiv) and treated accord-
ingly to general procedure b, previously reported, to obtain title
compound 4b as a white solid.
O
O2N
2-(4-Chlorophenyl)-4H-benzo[d][1,3]oxazin-4-one 4b: yield
60%; silica gel TLC Rf = 0.80 (Ethyl acetate/n-hexane 50% v/v); mp
189–190 °C; dH (400 MHz, DMSO-d6) 7.70 (3H, m), 7.77 (1H, d, J
8.0), 8.00 (1H, t, J 8.0), 8.20 (1H, d, J 8.0), 8.23 (2H, d, J 8.8); dC
(100 MHz, DMSO-d6) 117.6, 127.7, 128.8, 129.5, 129.7, 129.9,
130.3, 137.6, 138.4, 146.9, 156.4, 159.3. Experimental in agreement
with reported data.29
O
OH
NH
O
TEA
O2N
OH
NH2
+
F
DCM
Cl
O
F
1b
2c
3f
4.1.9. Synthesis of 2-(4-fluorophenyl)-4H-benzo[d][1,3]oxazin-
4-one 4c
5-Nitroanthranilic acid 1b (1.5 g, 1.0 equiv) was treated with
4-fluorobenzoyl chloride 2c (1.1 equiv) in DCM (25 ml) followed
by drop-wise addition of TEA (1.5 equiv) at 0 °C. The reaction
mixture was treated according to general procedure a,
previously reported, to give the title compound 3f as a pale
yellow solid.
O
O
OH
Ac2O
reflux
O
2-(4-Fluorobenzamido)benzoic acid 3f: yield 83%; silica gel
TLC Rf = 0.59 (Ethyl acetate/n-hexane 50% v/v); mp 198–199 °C;
dH (400 MHz, DMSO-d6) 7.50 (2H, m), 8.06 (2H, m), 8.53 (1H,
dd, J 2.8, 9.2), 8.79 (1H, d, J 2.8), 8.91 (1H, d, J 9.2),12.49 (1H, s,
NH
N
O
F
F
3c
4c
2
exchange with D2O, NH); dC (100 MHz, DMSO-d6) 117.1 (d, JC–F
3
22), 117.4, 120.8, 127.5, 129.9, 130.9, 131.0 (d, JC–F10), 142.1,
1
147.2, 164.8, 165.7 (d, JC–F250), 169.5; dF (376 MHz, DMSO-d6)
ꢁ106.8 (1F, s).
A suspension of 2-(4-fluorobenzamido)benzoic acid 3c (2.75 g,
1.0 equiv) was refluxed in Ac2O (15.0) and treated accordingly to
general procedure b, previously reported, to obtain title compound
4c as a white solid.
4.1.7. Synthesis of 2-phenyl-4H-benzo[d][1,3]oxazin-4-one 4a
2-(4-Fluorophenyl)-4H-benzo[d][1,3]oxazin-4-one 4c: yield
83%; silica gel TLC Rf = 0.7 (Ethyl acetate/n-hexane 50% v/v); mp
174–175 °C; dH (400 MHz, DMSO-d6) 7.48 (2H, m), 7.67 (1H, t, J
8.0), 7.76 (1H, d, J 8.0), 8.00 (1H, t, J 8.0), 8.20 (1H, d; J 8.0),8.30
O
O
OH
Ac2O
reflux
O
NH
2
(2H, m); dC (100 MHz, DMSO-d6) 117.1 (d, JC–F 22), 117.8, 127.5
N
4
3
O
(d, JC–F 3), 127.8, 129.0, 129.5, 131.4 (d, JC–F 9), 137.8, 147.1,
1
156.5, 159.7, 165.7 (d, JC–F 250); dF (376 MHz, DMSO-d6) ꢁ106.7
3a
4a
(1F, s).
Experimental in agreement with reported data.30
4.1.10. Synthesis of 6-nitro-2-phenyl-4H-benzo[d][1,3]oxazin-4-
one 4d
A suspension of 2-benzamidobenzoic acid 3a (0.32 g, 1.0 equiv)
was refluxed in Ac2O (15.0 equiv) and treated accordingly to gen-
eral procedure b, previously reported, to obtain title compound
4a as a white solid.
2-Phenyl-4H-benzo[d][1,3]oxazin-4-one 4a: yield 40%; silica
gel TLC Rf = 0.80 (Ethyl acetate/n-hexane 50% v/v); mp 124–
125 °C; dH (400 MHz, DMSO-d6) 7.70 (4H, m), 7.78 (1H, d, J 8.0),
8.00 (1H, t, J 8.0), 8.21 (1H, d, J 8.0), 8.25 (2H, d, J 8.0); dC
(100 MHz, DMSO-d6) 117.8, 127.8, 128.7, 129.0, 129.5, 129.9,
130.9, 133.6, 137.7, 147.2, 157.3, 159.8. Experimental in agree-
ment with reported data.26
O
O
N
O2N
OH
NH
O2N
Ac2O
reflux
O
O
3d
4d
4.1.8. Synthesis of 2-(4-chlorophenyl)-4H-benzo[d][1,3]oxazin-
4-one 4b
A suspension of 2-benzamido-5-nitrobenzoic acid 3d (1.6 g,
1 equiv) was refluxed in Ac2O (15.0 equiv) and treated accordingly
to general procedure b, previously reported, to obtain title com-
pound 4d as a pale yellow solid.
6-Nitro-2-phenyl-4H-benzo[d][1,3]oxazin-4-one 4d: yield 83%;
silica gel TLC Rf = 0.72 (Ethyl acetate/n-hexane 50% v/v); mp 167–
168 °C; dH (400 MHz, DMSO-d6) 7.68 (2H, t, J 8.0), 7.77 (1H, t, J
8.0), 7.96 (1H, d, J 8.8), 8.29 (2H, d, J 8.0), 8.70 (1H, dd, J 2.8, 8.8),
8.81 (1H, d, J 2.8); dC (100 MHz, DMSO-d6) 118.8, 124.3, 129.3,
129.5, 130.1, 130.3, 131.6, 134.6, 146.8, 151.6, 158.7, 160.0.
Experimental in agreement with reported data.31
O
O
OH
Ac2O
reflux
O
NH
N
O
Cl
Cl
3b
4b

M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
7757
4.1.11. Synthesis of 2-(4-chlorophenyl)-6-nitro-4H-benzo[d]
[1,3]oxazin-4-one 4e
7.60 (1H, t, J 7.6), 8.00 (1H, d, J 7.6), 8.49 (1H, d, J 7.6); dC
(100 MHz, DMSO-d6) 25.7, 117.4, 120.8, 123.3, 131.8, 134.7,
141.7, 169.2, 170.2.
O
4.1.14. Synthesis of 2-methyl-6-nitro-4H-benzo[d][1,3]oxazin-
4-one 7b
O
O2N
OH
O2N
Ac2O
reflux
O
NH
N
O
O
O
N
Cl
Ac2O
reflux
Cl
O2N
O2N
OH
NH2
O
3e
4e
1b
7b
A suspension of 2-(4-chlorobenzamido)-5-nitrobenzoic acid 3e
(1.0 g, 1.0 equiv) was refluxed in Ac2O (15.0 equiv) and treated
accordingly to general procedure b, previously reported, to obtain
title compound 4e as pale yellow solid.
2-(4-Chlorophenyl)-4H-benzo[d][1,3]oxazin-4-one 4e: yield
35%; silica gel TLC Rf = 0.75 (Ethyl acetate/n-hexane 50% v/v); mp
239–240 °C; dH (400 MHz, DMSO-d6) 7.76 (2H, d, J 8.4), 7.97 (1H,
d, J 8.8), 8.29 (2H, d, J 8.4), 8.72 (1H, dd, J 2.8, 8.8), 8.82 (1H, d, J
2.8); dC (100 MHz, DMSO-d6) 118.9, 124.4, 129.4, 129.5, 130.3,
131.1, 131.7, 139.5, 147.0, 151.5, 158.6, 159.2.
2-Amino-5-nitrobenzoic acid 1b (5.0 g, 1.0 equiv) was refluxed
in Ac2O (10.0 equiv) until the consumption of starting materials
(TLC monitoring). The excess of solvents were evaporated under
vacuum to obtain the residue that washed with n-hexane
(3 ꢀ 10 ml), filtered and dried under vacuum to obtain title com-
pound 7b as a brown solid.
2-Methyl-6-nitro-4H-benzo[d][1,3]oxazin-4-one 7b: yield 85%;
silica gel TLC Rf = 0.55 (Ethyl acetate/n-hexane 50% v/v); mp 220–
221 °C; dH (400 MHz, DMSO-d6) 2.51 (3H, s), 7.82 (1H, d, J 8.8),
8.66 (1H, dd, J 2.8, 8.8), 8.77 (1H, d, J 2.8); dC (100 MHz, DMSO-
d6) 22.2, 118.3, 124.2, 128.8, 131.6, 146.8, 151.4, 159.0, 164.5;
m/z (ESI negative) 205.08 [MꢁH]ꢁ. Experimental in agreement
with reported data.33
4.1.12. Synthesis of 2-(4-fluorophenyl)-6-nitro-4H-benzo[d]
[1,3]oxazin-4-one 4f
O
O
O2N
4.1.15. Synthesis of 6-methoxy-2-methyl-4H-benzo[d][1,3]
oxazin-4-one 7c
OH
NH
O2N
Ac2O
reflux
O
N
O
O
O
N
F
Ac2O
reflux
O
O
F
OH
NH2
O
3f
4f
1c
7c
A suspension of 2-(4-fluorobenzamido)-5-nitrobenzoic acid 3f
(1.9 g, 1.0 equiv) was refluxed in Ac2O (15.0 equiv) and treated
accordingly to general procedure b, previously reported, to obtain
title compound 4f as a pale yellow solid.
5-Methoxyanthranilic acid 1c (1.5 g, 1.0 equiv) was refluxed in
acetic anhydrate (10.0 equiv) until the consumption of starting
materials (TLC monitoring). The excess of solvents were evapo-
rated under vacuum to obtain the residue that washed with n-hex-
ane, filtered and dried under vacuum to obtain title compound 7c
as a pale yellow solid.
2-(4-Fluorophenyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one 4f:
yield 57%; silica gel TLC Rf = 0.70 (Ethyl acetate/n-hexane 50% v/
v); mp 181–182 °C; dH (400 MHz, DMSO-d6) 7.52 (2H, m), 7.95
(1H, d, J 8.8), 8.35 (2H, m), 8.71 (1H, dd, J 2.8, 8.8), 8.81 (1H, d, J
6-Methoxy-2-methyl-4H-benzo[d][1,3]oxazin-4-one 7c: yield
91%; silica gel TLC Rf = 0.25 (Ethyl acetate/n-hexane 20% v/v); mp
157–158 °C; dH (400 MHz, DMSO-d6) 2.41 (3H, s), 3.92 (3H, s),
7.55 (3H, m); dC (100 MHz, DMSO-d6) 21.6, 56.8, 109.5, 118.1,
126.1, 128.7, 141.1, 158.8, 159.4, 160.2.
2
2.8); dC (100 MHz, DMSO-d6) 117.3 (d, JC–F 22), 118.6, 124.3,
3
126.8, 129.5, 131.7, 132.1 (d, JC–F 10), 146.8, 151.6, 158.5, 159.1,
1
166.1 (d, JC–F 251); dF (376 MHz, DMSO-d6) ꢁ105.0 (1F, s).
4.1.13. Synthesis of 2-methyl-4H-benzo[d][1,3]oxazin-4-one
Experimental in agreement with reported data.33
7a32
4.1.16. Synthesis of 6-methoxy-4H-benzo[d][1,3]oxazin-4-one
7d
O
O
N
Ac2O
reflux
OH
NH2
O
O
O
N
O
O
O
O
OH
NH2
+
O
1a
7a
O
reflux
1c
7d
Anthranilic acid 1a (3.75 g, 1.0 equiv) was refluxed in Ac2O
(10.0 equiv) until the consumption of starting material (TLC mon-
itoring). The solvent was evaporated under vacuum to obtain the
residue that was crystallized with EtOAc/n-hexane mixture to
obtain title compound 7a as a white solid.
2-Methyl-4H-benzo[d][1,3]oxazin-4-one 7a: yield 60%; silica
gel TLC Rf = 0.16 (Ethyl acetate/n-hexane 80% v/v); mp 120–
121 °C; dH (400 MHz, DMSO-d6) 2.16 (3H, s), 7.16 (1H, t, J 7.6),
5-Methoxyanthranilic acid 1c (3.0 g, 1.0 equiv) was refluxed in
triethylorthoformate (5.0 equiv) and the mixture was stirred until
the consumption of starting materials (TLC monitoring). The excess
of solvents were evaporated under vacuum to obtain the residue
that was washed with n-hexane, filtered and dried under vacuum
to obtain title compound 7d as a pale yellow solid.

7758
M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
3
4
6-Methoxy-4H-benzo[d][1,3]oxazin-4-one 7d: yield 89%; silica
123.0, 124.6, 125.1, 127.4, 130.0, 130.8 (d, JC–F 9), 131.9 (d, JC–F
1
gel TLC Rf = 0.63 (Ethyl acetate/n-hexane 50% v/v); mp 180–
181 °C (decomp.); dH (400 MHz, DMSO-d6) 3.93 (3H, s), 7.55 (2H,
m), 7.64 (1H, m), 8.14 (1H, s); dC (100 MHz, DMSO-d6) 56.9,
109.7, 120.4, 126.0, 129.3, 140.0, 149.7, 159.4, 160.1.
3), 133.2, 139.0, 140.0, 142.6, 164.7, 165.2 (d, JC–F 248), 168.5; dF
(376 MHz, DMSO-d6) ꢁ108.1 (1F, s); m/z (ESI negative) 412.1
[MꢁH]ꢁ.
4.1.19. Synthesis of 2-benzamido-5-nitro-N-(4-sulfamoyl-
4.1.17. Synthesis of 2-benzamido-N-(4-sulfamoylphenyl)
phenyl)benzamide 6c
benzamide 6a
O
N
SO2NH2
O2N
SO2NH2
O
+
O
SO2NH2
HN
NH2
O
O
+
AcOH,reflux
N
5a
NH
4d
NH2
O
SO2NH2
O
O2N
5a
4a
6a
N
H
NH
AcOH, reflux
O
Sulfanilamide 5a (0.15 g, 1.0 equiv) was treated with 4a
(1.0 equiv) in glacial acetic acid (3.0 ml) according to the general
procedure, previously reported. The reaction was quenched with
slush, obtained precipitate was filtered, washed with Et2O
(3 ꢀ 10 ml) and dried under vacuum to obtain the title compound
6a as a white solid.
6c
Sulfanilamide 5a (0.1 g, 1.0 equiv) was treated with 4d
(1.0 equiv) in glacial acetic acid (3.0 ml) according to the general
procedure c, previously reported. The reaction was quenched with
slush and obtained precipitate was filtered, washed with Et2O
(3 ꢀ 10 ml) and dried under vacuum to obtain the title compound
6c as a white solid.
2-Benzamido-N-(4-sulfamoylphenyl)benzamide 6a: yield 45%;
silica gel TLC Rf = 0.16 (Ethyl acetate/n-hexane 50% v/v); mp 270–
271 °C; dH (400 MHz, DMSO-d6) 7.33 (2H, s, exchange with D2O,
SO2NH2), 7.36 (1H, t, J 8.0), 7.58–7.70 (4H, m), 7.84 (2H, d, J 8.9),
7.93 (5H, m), 8.40 (1H, d, J 8.0), 10.84 (1H, s, exchange with D2O,
NH), 11.45 (1H, s, exchange with D2O, NH); dC (100 MHz, DMSO-
d6) 121.4, 122.8, 124.4, 124.6, 127.4, 128.0, 129.8, 130.0, 132.9,
133.3, 135.4, 139.3, 140.1, 142.6, 165.7, 168.5; m/z (ESI negative)
394.1 [MꢁH]ꢁ.
2-Benzamido-5-nitro-N-(4-sulfamoylphenyl)benzamide
6c:
yield 52%; silica gel TLC Rf = 0.25 (Ethyl acetate/n-hexane 50% v/
v); mp >300 °C; dH (400 MHz, DMSO-d6) 7.35 (2H, s, exchange with
D2O, SO2NH2),7.64 (2H, t, J 7.2), 7.70 (1H, t, J 7.2), 7.90 (4H, m), 7.98
(2H, d, J 7.2), 8.55 (1H, dd, J 2.4, 9.2), 8.71 (1H, d, J 9.2), 8.38 (1H, d, J
2.4), 11.16 (1H, s, exchange with D2O, NH), 11.89 (1H, s, exchange
with D2O, NH); dC (100 MHz, DMSO-d6) 121.8, 122.5, 123.7, 125.8,
127.4, 128.3, 128.5, 129.9, 133.5, 134.7, 140.6, 142.1, 142.7, 145.2,
166.1, 166.9; m/z (ESI negative) 439.0 [MꢁH]ꢁ.
Experimental in agreement with reported data.34a
4.1.18. Synthesis of 2-(4-fluorobenzamido)-N-(4-sulfamoyl-
phenyl)benzamide 6b
4.1.20. Synthesis of 2-(4-chlorobenzamido)-5-nitro-N-(4-sulfa-
moylphenyl)benzamide 6d
SO2NH2
O
N
SO2NH2
HN
O
N
SO2NH2
O
O
+
O2N
O
AcOH, reflux
NH
+
NH2
F
O
NH2
Cl
F
5a
4c
6b
5a
4e
SO2NH2
O
Sulfanilamide 5a (0.18 g, 1.0 equiv) was treated with 4c
(1.0 equiv) in glacial acetic acid (3.0 ml) according to the general
procedure c, previously reported. The reaction was quenched with
slush and obtained precipitate was filtered, washed with Et2O
(3 ꢀ 10 ml) and dried under vacuum to obtain the title compound
6b as a white solid.
O2N
N
H
NH
AcOH, reflux
O
Cl
6d
2-(4-Fluorobenzamido)-N-(4-sulfamoylphenyl)benzamide 6b:
yield 50%; silica gel TLC Rf = 0.17 (Ethyl acetate/n-hexane 50% v/
v); mp 272–273 °C; dH (400 MHz, DMSO-d6) 7.32 (2H, s, exchange
with D2O, SO2NH2), 7.36 (1H, t, J 8.0), 7.44 (2H, m), 7.67 (1H, t, J
8.0), 7.84 (2H, d, J 8.8), 7.92 (3H, m), 8.01 (2H, m), 8.33 (1H, d, J
8.0), 10.82 (1H, s, exchange with D2O, NH), 11.38 (1H, s, exchange
Sulfanilamide 5a (0.1 g, 1.0 equiv) was treated with 4e
(1.0 equiv) in glacial acetic acid (3 ml) according to the general
procedure c, previously reported. Reaction was quenched with
slush and obtained precipitate was filtered and washed with
Et2O (3 ꢀ 10 ml), and dried under vacuum to obtain the title com-
pound 6d as a white solid.
2
with D2O, NH); dC (100 MHz, DMSO-d6) 116.8 (d, JC–F 22), 121.3,

M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
7759
2-(4-Chlorobenzamido)-5-nitro-N-(4-sulfamoylphenyl)benza-
mide 6d: yield 60%; silica gel TLC Rf = 0.35 (Ethyl acetate/n-hexane
50% v/v); mp 299–300 °C (decomp.); dH (400 MHz, DMSO-d6) 7.36
(2H, s, exchange with D2O, SO2NH2), 7.70 (2H, d, J 8.4), 7.90 (4H,
m), 7.97 (2H, d, J 8.4), 8.53 (1H, dd, J 2.8, 9.2), 8.63 (1H, d, J 9.2),
8.81 (1H, d, J 2.8),11.15 (1H, s, exchange with D2O, NH), 11.85
(1H, s, exchange with D2O, NH); dC (100 MHz, DMSO-d6) 121.8,
122.8, 124.3, 125.8, 127.5, 128.4, 130.0, 130.2, 133.5, 138.4,
140.5, 142.1, 142.9, 144.9, 165.2, 166.8; m/z (ESI negative) 473.0
[MꢁH]ꢁ.
2-Benzamido-N-(4-sulfamoylphenethyl)benzamide 6f: yield
47%, silica gel TLC Rf = 0.11 (Ethyl acetate/n-hexane 50% v/v); mp
258–259 °C; dH (400 MHz, DMSO-d6) 3.00 (2H, t, J 6.8), 3.62 (2H,
q, J 6.8), 7.23 (1H, t, J 8.0), 7.33 (2H, s, exchange with D2O, SO2NH2),
7.49 (2H, d, J 8.4), 7.59–7.70 (4H, m), 7.78 (3H, m),7.97 (2H, m),
8.67 (1H, d, J 8.0), 9.02 (1H, t, J 6.8, exchange with D2O, NH),
12.55 (1H, s, exchange with D2O, NH); dC (100 MHz, DMSO-d6)
35.3, 41.3, 121.2, 121.2, 123.8, 126.6, 127.9, 129.0, 129.9, 130.1,
133.0, 133.2, 135.5, 140.3, 143.1, 144.5, 165.3, 169.6; m/z (ESI
negative)422.17 [MꢁH]ꢁ.
4.1.21. Synthesis of 2-(4-fluorobenzamido)-5-nitro-N-(4-sulfa-
4.1.23. Synthesis of 2-(4-chlorobenzamido)-N-(4-sulfamoyl-
moylphenyl)benzamide 6e
phenethyl)benzamide 6g
SO2NH2
O
N
SO2NH2
SO2NH2
O
O
O2N
O
O
N
+
+
H
DMA, 160 o
C
N
NH
NH2
F
Cl
O
NH2
Cl
5a
4f
5b
4b
6g
SO2NH2
O
O2N
4(2-Aminoethyl)benzenesulfonamide 5b (0.2 g, 1.0 equiv) was
treated with 4b (1.0 equiv) in dry DMA (10 ml) according to the
general procedure c, previously reported. Reaction was quenched
with slush and obtained precipitate was filtered and washed with
Et2O (3 ꢀ 10 ml), and dried under vacuum to obtain the title com-
pound 6g as a white solid.
N
H
NH
AcOH, reflux
O
F
6e
2-(4-Chlorobenzamido)-N-(4-sulfamoylphenethyl)benzamide
6g: yield 75%, silica gel TLC Rf = 0.63 (Ethyl acetate/n-hexane 80% v/v);
mp 272–273 °C (decomp.); dH (400 MHz, DMSO-d6) 3.00 (2H, t, J
6.8), 3.61 (2H, q, J 6.8), 7.25 (1H, t, J 8.0), 7.32 (2H, s, exchange with
D2O, SO2NH2), 7.49 (2H, d, J 8.4), 7.61 (1H, t, J 8.0), 7.72 (2H, d, J
8.4), 7.80 (3H, m), 7.97 (2H, d, J 8.4), 8.64 (1H, d, J 8.0), 9.04 (1H,
t, J 6.8, exchange with D2O, NH), 12.59 (1H, s, exchange with
D2O, NH); dC (100 MHz, DMSO-d6) 35.3, 41.3, 121.3, 121.3, 124.0,
126.6, 129.0, 129.8, 130.0, 130.1, 133.2, 134.2, 137.8, 140.1,
143.1, 144.5, 164.3, 169.5; m/z (ESI negative) 456.17 [MꢁH]ꢁ.
Sulfanilamide 5a (0.1 g, 1.0 equiv) was treated with 4f
(1.0 equiv) in glacial acetic acid (3 ml) according to the general
procedure c, previously reported. Reaction was quenched with
slush and obtained precipitate was filtered and washed with
Et2O (3 ꢀ 10 ml), and dried under vacuum to obtain the title com-
pound 6e as a white solid.
2-(4-Fluorobenzamido)-5-nitro-N-(4-sulfamoylphenyl)benza-
mide 6e: yield 73%; silica gel TLC Rf = 0.35 (Ethyl acetate/n-hexane
50% v/v); mp >300 °C; dH (400 MHz, DMSO-d6) 7.36 (2H, s,
exchange with D2O, SO2NH2), 7.48 (2H, m), 7.89 (4H, m), 8.05
(2H, m), 8.55 (1H, dd, J 2.4, 9.2), 8.63 (1H, d, J 9.2), 8.81 (1H, d, J
2.4),11.15 (1H, s, exchange with D2O, NH), 11.82 (1H, s, exchange
4.1.24. Synthesis of 2-(4-fluorobenzamido)-N-(4-sulfamoyl-
phenethyl)benzamide 6h
2
with D2O, NH); dC (100 MHz, DMSO-d6) 116.9 (d, JC–F 22), 121.8,
3
122.6, 124.0, 125.8, 127.4, 128.4, 131.1 (d, JC–F 9), 140.6, 142.1,
1
142.8, 145.1, 165.1, 165.5 (d, JC–F 248), 166.8; dF (376 MHz,
SO2NH2
DMSO-d6) ꢁ107.0 (1F, s); m/z (ESI negative) 457.0 [MꢁH]ꢁ.
SO2NH2
O
O
O
4.1.22. Synthesis of 2-benzamido-N-(4-sulfamoylphenethyl)
benzamide 6f
N
+
H
N
DMA,160 o
C
NH
F
O
NH2
F
SO2NH2
5b
4c
6h
SO2NH2
O
O
O
4(2-Aminoethyl)benzenesulfonamide 5b (0.4 g, 1.0 equiv) was
treated with 4c (1.0 equiv) in dry DMA (20 ml) according to the
general procedure c, previously reported. Reaction was quenched
with slush and extracted with ethyl acetate (3 ꢀ 20 ml) and the
combined organic layers were washed with H2O (3 ꢀ 20 ml), dried
over Na2SO4, filtered and concentrated in vacuum to give the title
compound 6h as a white solid.
2-(4-Fluorobenzamido)-N-(4-sulfamoylphenethyl)benzamide
6h: yield 94%; silica gel TLC Rf = 0.37 (Ethyl acetate/n-hexane 50%
v/v); mp 242–243 °C; dH (400 MHz, DMSO-d6) 3.00 (2H, t, J 6.8),
3.61 (2H, q, J 6.8), 7.24 (1H, t, J 7.8), 7.32 (2H, s, exchange with
D2O, SO2NH2), 7.48 (4H, m), 7.60 (1H, d, J 7.8), 7.78 (3H, m), 8.00
N
+
H
DMA, 160 o
C
N
NH
O
NH2
5b
4a
6f
4(2-Aminoethyl)benzenesulfonamide 5b (0.2 g, 1.0 equiv) was
treated with 4a (1.0 equiv)in dry DMA (10 ml) according to the
general procedure c, previously reported. Reaction was quenched
with slush and obtained precipitate was filtered and washed with
Et2O (3 ꢀ 10 ml) and dried under vacuum to obtain the title com-
pound 6f as a white solid.

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M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
(2H, m), 8.64 (1H, d, J 7.8), 9.03 (1H, t, J 6.8, exchange with D2O,
NH), 12.56 (1H, s, exchange with D2O, NH); dC (100 MHz, DMSO-
quenched with slush and obtained precipitate was filtered and
washed with Et2O (3 ꢀ 10 ml), and dried under vacuum to obtain
the title compound 6j as a white solid.
2
d6) 35.2, 41.2, 116.8 (d, JC–F 22), 121.2, 123.7, 126.5, 128.9,
3
4
129.9, 130.4 (d, JC–F 9), 131.9 (d, JC–F 3), 132.0 133.0, 140.2,
2-(4-Chlorobenzamido)-5-nitro-N-(4-sulfamoylphenethyl)ben-
zamide 6j: yield 65%; silica gel TLC Rf = 0.60 (Ethyl acetate/n-hex-
ane 50% v/v); mp >300 °C; dH (400 MHz, DMSO-d6) 3.03 (2H, t, J
6.8), 3.65 (2H, q, J 6.8), 7.33 (2H, s, exchange with D2O, SO2NH2),
7.51 (2H, d, J 8.4), 7.76 (4H, m), 8.01 (2H, d, J 8.4), 8.50 (1H, dd, J
2.8, 9.2), 8.78 (1H, d, J 2.8), 8.91 (1H, d, J 9.2), 9.5 (1H, t, J 6.8,
exchange with D2O, NH), 13.03 (1H, s, exchange with D2O, NH);
dC (100 MHz, DMSO-d6) 120.7, 121.3, 124.9, 126.7, 128.5, 130.0,
130.1, 130.2, 133.5, 138.5, 142.5, 143.1, 144.3, 145.9, 164.9,
167.9; m/z (ESI negative) 501.0 [MꢁH]ꢁ.
1
143.0, 144.4, 164.2 165.1 (d, JC–F 248), 169.5; dF (376 MHz,
DMSO-d6) ꢁ108.1 (1F, s); m/z (ESI negative) 440.2 [MꢁH]ꢁ.
4.1.25. Synthesis of 2-benzamido-5-nitro-N-(4-sulfamoyl-
phenethyl)benzamide 6i
SO2NH2
O
O2N
O
+
N
4.1.27. Synthesis of 2-(4-fluorobenzamido)-5-nitro-N-(4-sulfa-
moylphenethyl)benzamide 6k
NH2
5b
4d
SO2NH2
SO2NH2
O
O
O2N
O2N
O
N
H
NH
+
N
AcOH,reflux
F
NH2
O
5b
4f
6i
SO2NH2
O
O2N
N
H
NH
4-(2-Aminoethyl)benzenesulfonamide 5b (0.1 g, 1.0 equiv) was
treated with 4d (1.0 equiv) in glacial acetic acid (3 ml) according to
the general procedure c, previously reported. Reaction was
quenched with slush and obtained precipitate was filtered and
washed with Et2O (3 ꢀ 10 ml), and dried under vacuum to obtain
the title compound 6i as a white solid.
AcOH, reflux
O
F
6k
2-Benzamido-5-nitro-N-(4-sulfamoylphenethyl)benzamide 6i:
yield 60%; silica gel TLC Rf = 0.23 (Ethyl acetate/n-hexane 50% v/
v); mp >300 °C; dH (400 MHz, DMSO-d6) 3.04 (2H, t, J 7.2), 3.65
(2H, q, J 7.2), 7.33 (2H, s, exchange with D2O, SO2NH2), 7.51 (2H,
d, J 8.4), 7.70 (3H, m), 7.80 (2H, d, J 8.4), 8.00 (2H, d, J 8.4), 8.50
(1H, dd, J 2.4, 9.2), 8.78 (1H, d, J 2.4), 8.95 (1H, d, J 9.2), 9.49 (1H,
t, J 7.2), 13.01 (1H, s, exchange with D2O, NH); dC (100 MHz,
DMSO-d6) 35.2, 41.6, 120.6, 121.3, 124.9, 126.7, 128.1, 128.5,
130.1, 130.1, 133.6, 134.7, 142.4, 143.1, 144.4, 146.2, 165.9,
168.0; m/z (ESI negative) 467.0 [MꢁH]ꢁ.
4-(2-Aminoethyl)benzenesulfonamide 5b (0.15 g, 1.0 equiv)
was treated with 4f (1.0 equiv) in glacial acetic acid (3 ml) accord-
ing to the general procedure c, previously reported. Reaction was
quenched with slush and obtained precipitate was filtered and
washed with Et2O (3 ꢀ 10 ml), and dried under vacuum to obtain
the title compound 6k as a white solid.
2-(4-Fluorobenzamido)-5-nitro-N-(4-sulfamoylphenethyl)ben-
zamide 6k: yield 69%; silica gel TLC Rf = 0.24 (Ethyl acetate/n-hex-
ane 50% v/v); mp >300 °C; dH (400 MHz, DMSO-d6) 3.03 (2H, t, J
6.8), 3.65 (2H, q, J 6.8), 7.33 (2H, s, exchange with D2O, SO2NH2),
7.52 (4H, m), 7.80 (2H, d, J 8.4), 8.06 (2H, m), 8.50 (1H, dd, J 2.8,
9.2), 8.77 (1H, d, J 2.8), 8.91 (1H, d, J 9.2), 9.49 (1H, t, J 6.8, exchange
with D2O, NH), 13.00 (1H, s, exchange with D2O, NH); dC (100 MHz,
4.1.26. Synthesis of 2-(4-chlorobenzamido)-5-nitro-N-(4-sulfa-
moylphenethyl)benzamide 6j
2
DMSO-d6) 117.1 (d, JC–F 22), 120.7, 121.3, 124.9, 126.6, 126.7,
3
SO2NH2
128.5, 130.0, 130.1, 130.9 (d, JC–F 9), 131.2, 142.5, 143.1, 144.3,
O
1
146.1, 164.9, 165.6 (d, JC–F 249), 168.0; dF (376 MHz, DMSO-d6)
O2N
O
ꢁ106.9 (1F, s); m/z (ESI negative) 485.0 [MꢁH]ꢁ.
+
N
Cl
4.1.28. Synthesis of 4-(2-methyl-6-nitro-4-oxoquinazolin-3
NH2
(4H)-yl)benzenesulfonamide 8a
5b
4e
SO2NH2
O
SO2NH2
SO2NH2
O
O
O2N
Cl
N
H
NH
O2N
O2N
+
O
N
DMA, 160 oC
AcOH,reflux
N
N
NH2
O
5a
7b
8a
6j
Sulfanilamide 5a (0.4 g, 1.0 equiv) was treated with 7b
(1.0 equiv) in dry DMA (25 ml) according to general procedure e,
previously reported. Reaction quenched with 10 ml of 1 M aqueous
HCl, obtained precipitate was filtered and washed with Et2O
4-(2-Aminoethyl)benzenesulfonamide 5b (0.15 g, 1.0 equiv)
was treated with 4e (1.0 equiv)in glacial acetic acid (3 ml) accord-
ing to the general procedure c, previously reported. Reaction was

M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
7761
(3 ꢀ 10 ml) and dried under vacuum to obtain the title compound
8a as an orange solid.
DMSO-d6) 2.64 (3H, s), 3.13 (2H, t. J 8.0), 4.33 (2H, t, J 8.0), 7.37
(2H, s, exchange with D2O, SO2NH2), 7.53 (2H, d, J 8.4), 7.82 (3H,
m), 8.57 (1H, dd, J 2.8, 9.2), 8.87 (1H, d, J 2.8); dC (100 MHz,
DMSO-d6) 24.0, 33.9, 46.5, 120.8, 123.3, 126.9, 129.2, 129.3,
130.3, 143.1, 143.6, 145.6, 151.9, 160.0, 161.3; m/z (ESI positive)
389.17 [M+H]+.
4-(2-Methyl-6-nitro-4-oxoquinazolin-3(4H)-yl)benzenesulfon-
amide 8a: yield 63%, silica gel TLC Rf = 0.12 (Ethyl acetate/n-hexane
80% v/v); mp 280–281 °C (decomp.); dH (400 MHz, DMSO-d6) 2.25
(3H, s), 7.60 (2H, s, exchange with D2O, SO2NH2), 7.78 (2H, d, J 8.8),
7.93 (1H, d, J 8.4), 8.07 (2H, d, J 8.8), 8.64 (1H, dd, J 2.8, 8.4), 8.84
(1H, d, J 2.8); dC (100 MHz, DMSO-d6) 25.3, 121.5, 123.3, 128.1,
129.5, 129.6, 130.1, 140.9, 145.7, 145.8, 152.4, 158.9, 161.5; m/z
(ESI negative) 359.17 [MꢁH]ꢁ.
4.1.31. Synthesis of 4-(2-(6-methoxy-2-methyl-4-oxoquinazolin
-3(4H)-yl)ethyl)benzenesulfonamide 8d
4.1.29. Synthesis of 4-(2-(2-methyl-4-oxoquinazolin-3(4H)-yl)
SO2NH2
ethyl)benzenesulfonamide 8b
O
O
+
O
N
SO2NH2
NH2
SO2NH2
O
N
O
N
5b
7c
+
O
N
DMA,120 oC
SO2NH2
O
N
NH2
5b
O
N
DMA, 120 o
C
7a
8b
4-(2-Aminoethyl)benzenesulfonamide 5b (0.4 g, 1.0 equiv) was
treated with 7a (1.0 equiv) in dry DMA (20 ml) according to gen-
eral procedure e, previously reported Reaction quenched with
slush, obtained precipitate was filtered and washed with H2O
(3 ꢀ 10 ml) and dried under vacuum to obtain the title compound
8b as a white solid.
4-(2-(2-Methyl-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesul-
fonamide 8b: yield 45%; silica gel TLC Rf = 0.2 (Ethyl acetate/n-hex-
ane 30% v/v); mp 265–266 °C; dH (400 MHz, DMSO-d6) 2.58 (3H, s),
3.11 (2H, t, J 8.0), 4.30 (2H, t, J 8.0), 7.37 (2H, s, exchange with D2O,
SO2NH2), 7.53 (3H, m), 7.63 (1H, d, J 8.0), 7.83 (3H, m), 8.17 (1H, d, J
8.0); dC (100 MHz, DMSO-d6) 23.7, 34.3, 46.1, 120.9, 126.9, 127.1,
127.2, 127.5, 130.2, 135.3, 143.4, 143.5, 147.9, 155.8, 162.0; m/z
(ESI positive) 344.17 [M+H]+.
8d
4-(2-Aminoethyl)benzenesulfonamide 5b (0.5 g, 1.0 equiv) was
treated with 7c (1.0 equiv) in dry DMA (20 ml) according to general
procedure e, previously reported Reaction quenched with slush
and acidified with 1 M aqueous HCl, obtained precipitate was fil-
tered and washed with Et2O (3 ꢀ 10 ml) and then filtered and dried
under vacuum to obtain title compound 8d as a beige solid.
4-(2-(6-Methoxy-2-methyl-4-oxoquinazolin-3(4H)-yl)ethyl)
benzenesulfonamide 8d: yield 40%, silica gel TLC Rf = 0.2 (Ethyl
acetate/n-hexane 80% v/v); mp 245–246 °C; dH (400 MHz, DMSO-
d6) 2.55 (3H, s), 3.10 (2H, t, J 8.0), 3.91 (3H, s), 4.30 (2H, t, J 8.0),
7.37 (2H, s, exchange with D2O, SO2NH2), 7.45 (1H, dd, J 2.8, 8.8),
7.51 (2H, d, J 8.0), 7.54 (1H, d, J 2.8), 7.58 (1H, d, J 8.8), 7.81 (2H,
d, J 8.0); dC (100 MHz, DMSO-d6) 23.4, 34.3, 46.2, 56.5, 106.9,
121.6, 124.9, 126.8, 129.2, 130.2, 142.5, 143.4, 143.5, 153.3,
158.3, 161.8; m/z (ESI positive) 374.25 [M+H]+.
4.1.30. Synthesis of 4-(2-(2-methyl-6-nitro-4-oxoquinazolin-3
(4H)-yl)ethyl)benzenesulfonamide 8c
SO2NH2
O
4.1.32. Synthesis of 4-(2-(6-methoxy-4-oxoquinazolin-3(4H)-yl)
O2N
ethyl)benzenesulfonamide 8e
+
O
N
NH2
SO2NH2
5b
7b
O
SO2NH2
O
O
N
+
O
O2N
N
N
DMA, 120 o
C
NH2
5b
7d
8c
SO2NH2
O
N
O
4-(2-Aminoethyl)benzenesulfonamide 5b (0.1 g, 1.0 equiv) was
treated with 7b (1.0 equiv) in dry DMA (6 ml) according to general
procedure e, previously reported Reaction was quenched with
slush, extracted with ethyl acetate (3 ꢀ 20 ml) and the combined
organic layers were washed with H2O (3 ꢀ 20 ml), dried over
Na2SO4, filtered and concentrated in vacuum to give desired
compound 8c as a yellow solid.
N
pyridine, 120 o
C
8e
4-(2-(2-Methyl-6-nitro-4-oxoquinazolin-3(4H)-yl)ethyl)ben-
zenesulfonamide 8c: yield 65%, silica gel TLC Rf = 0.13 (Ethyl
acetate/n-hexane 80% v/v); 263–264 °C (decomp.); dH (400 MHz,
4-(2-Aminoethyl)benzenesulfonamide 5b (0.3 g, 1.0 equiv) was
treated with 7d (1.0 equiv) in dry pyridine (10 ml) according to
general procedure e. Reaction quenched with slush and neutralized

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M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
with 1 M aqueous HCl solution, extracted with ethyl acetate
(3 ꢀ 20 ml) and the combined organic layers were washed with
H2O (3 ꢀ 20 ml), dried over Na2SO4, filtered and concentrated in
vacuum to give title compound 8e as a beige solid.
DMSO-d6) 34.5, 43.6, 113.9, 120.1, 125.6, 126.7, 129.3, 130.1,
136.8, 138.3, 143.3, 143.8, 151.7, 161.4; m/z (ESI positive) 329.0
[M+H]+.
4-(2-(6-Methoxy-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesul-
fonamide 8e: yield 60%, silica gel TLC Rf = 0.11 (Ethyl acetate/n-
hexane 80% v/v); mp 244–245 °C; dH (400 MHz, DMSO-d6) 3.15
(2H, t, J 7.6), 3.92 (3H, s), 4.29 (2H, t, J 7.6), 7.35 (2H, s, exchange
with D2O, SO2NH2), 7.46 (3H, m), 7.59 (1H, d, J 3.2), 7.64 (1H, d, J
8.8), 7.78 (2H, d, J 8.4), 8.13 (1H, s); dC (100 MHz, DMSO-d6) 34.9,
47.8, 56.6, 106.9, 123.3, 124.7, 126.8, 129.8, 130.2, 143.0, 143.2,
143.4, 146.5, 158.9, 160.8; m/z (ESI positive) 360.2 [M+H]+.
4.1.35. Synthesis of 3-(quinazolin-4-ylamino)benzenesulfona-
mide 10c
Cl
H2N
SO2NH2
TEA
HN
SO2NH2
+
N
EtOH, reflux
N
N
N
4.1.33. Synthesis of 4-(quinazolin-4-ylamino)benzenesulfona-
mide 10a
5c
9
10c
A solution of 3-aminobenzenesulfonamide 5c (0.2 g, 1 equiv)
was treated with 4-chloroquinazoline (1.0 equiv) in EtOH
SO2NH2
SO2NH2
9
Cl
(10 ml), followed by the addition of TEA (1.2 equiv).The reaction
mixture was treated according to the general procedure f, previ-
ously reported, to give the title compound 10c as a white solid.
3-(Quinazolin-4-ylamino)benzenesulfonamide 10c: yield 63%;
mp >300 °C; dH (400 MHz, DMSO-d6) 7.44 (2H, s, exchange with
D2O, SO2NH2), 7.64 (2H, m), 7.72 (1H, t, J 8.0), 7.87 (1H, d, J 8.0),
7.94 (1H, d, J 8.0), 8.22 (1H, m), 8.44 (1H, s), 8.64 (1H, d, J 8.0),
8.70 (1H, s,), 10.10 (1H, s, exchange with D2O, NH); dC (100 MHz,
DMSO-d6) 116.6, 121.0, 122.8, 124.5, 127.5, 128.8, 129.1, 131.3,
135.6, 141.0, 145.5, 150.8, 155.9, 159.6; m/z (ESI positive) 301.1
[M+H]+.
TEA
HN
+
N
EtOH, reflux
N
N
NH2
N
5a
9
10a
A solution of sulfanilamide 5a (0.2 g, 1.0 equiv) in EtOH (10 ml)
was treated with 4-chloroquinazoline 9 (1.0 equiv), followed by
the addition of TEA (1.2 equiv). The reaction mixture was treated
according to the general procedure f, to obtain the title compound
10a as a white solid.
4-(Quinazolin-4-ylamino)benzenesulfonamide 10a: yield 63%,
silica gel TLC Rf = 0.18 (Ethyl acetate/n-hexane 30% v/v); mp 195–
196 °C; dH (400 MHz, DMSO-d6) 7.48 (2H, s, exchange with D2O,
SO2NH2), 7.96 (6H, m), 8.16 (1H, t, J 8.0), 8.88 (1H, d, J 8.0), 9.02
(1H, s), 11.59 (1H, br s, exchange with D2O, NH); dC (100 MHz,
DMSO-d6) 115.1, 121.6, 126.0, 126.8, 128.4, 131.2, 138.7, 140.3,
141.3, 142.9, 152.6, 161.9; m/z (ESI positive) 301.1 [M+H]+.
Experimental in agreement with reported data.34b
4.1.36. Synthesis of 4-(quinazolin-4-yloxy)benzenesulfonamide
10d
SO2NH2
SO2NH2
Cl
TEA
O
N
+
N
EtOH, reflux
N
N
OH
4.1.34. Synthesis of 4-(2-(quinazolin-4-ylamino)ethyl)benzene-
sulfonamide 10b
5d
10d
9
A solution of 4-hydroxybenzenesulfonamide 5d (0.2 g, 1 equiv)
was treated with 4-chloroquinazoline (1.0 equiv) in EtOH
(10 ml), followed by the addition of TEA (1.2 equiv).The reaction
mixture was treated according to the general procedure f, to give
the title compound 10d as a white solid.
4-(Quinazolin-4-yloxy)benzenesulfonamide 10d: yield 34%;sil-
ica gel TLC Rf = 0.12 (Ethyl acetate/n-hexane 50% v/v); mp 212–
213 °C; dH (400 MHz, DMSO-d6) 7.49 (2H, s, exchange with D2O,
SO2NH2), 7.62 (2H, d, J 8.4), 7.86 (1H, t, J 7.6), 7.99 (2H, d, J 8.4),
8.10 (2H, m), 8.45 (1H, d, J 7.6), 8.79 (1H, s); dC (100 MHz,
DMSO-d6) 117.1, 124.5, 125.1, 128.9, 129.4, 130.3, 136.9, 142.7,
152.5, 155.3, 156.3, 167.9; m/z (ESI positive) 302.1 [M+H]+.
SO2NH2
SO2NH2
9
Cl
TEA
HN
N
+
N
EtOH, reflux
N
N
NH2
5b
10b
9
A solution of 4-(2-aminoethyl)benzenesulfonamide 5b (0.2 g,
1.0 equiv) in EtOH (10 ml) was treated with 4-chloroquinazoline
9 (1.0 equiv), followed by the addition of TEA (1.2 equiv). The reac-
tion mixture was treated according to the general procedure f, to
obtain the title compound 10b as a white solid.
4.2. CA inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity.25 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 con-
stant the ionic strength), following the initial rates of the CA-cat-
alyzed CO2 hydration reaction for a period of 10–100 s. The CO2
4-(2-(Quinazolin-4-ylamino)ethyl)benzenesulfonamide
yield 63%; silica gel TLC Rf = 0.1 (Ethyl acetate/n-hexane 80% v/v);
mp 215–216 °C (decomp.); dH (400 MHz, DMSO-d6) 3.17 (2H, t, J
10b:
8.0), 4.02 (2H, q,
J 8.0), 7.35 (2H, s, exchange with D2O,
SO2NH2),7.51 (2H, d, J 8.4), 7.78 (2H, d, J 8.4), 7.82 (1H, t, J 8.0),
7.92 (1H, d, J 8.0), 8.08 (1H, t, J 8.0), 8.63 (1H, d, J 8.0), 8.95
(1H, s), 10.59 (1H, t, J 8.0, exchange with D2O, NH); dC (100 MHz,

M. Bozdag et al. / Bioorg. Med. Chem. 23 (2015) 7751–7764
7763
10. (a) Lopez, M.; Paul, B.; Hofmann, A.; Morizzi, J.; Wu, Q.; Charman, S. A.;
Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2009, 52,
6421; (b) Lopez, M.; Salmon, A. J.; Supuran, C. T.; Poulsen, S. A. Curr. Pharm. Des.
2010, 16, 3277; (c) Lopez, M.; Trajkovic, J.; Bornaghi, L. F.; Innocenti, A.; Vullo,
D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1481; (d) Buller, F.;
Steiner, M.; Frey, K.; Mircsof, D.; Scheuermann, J.; Kalisch, M.; Bühlmann, P.;
Supuran, C. T.; Neri, D. ACS Chem. Biol. 2011, 6, 336; (e) Supuran, C. T.; Casini, A.;
Mastrolorenzo, A.; Scozzafava, A. Mini-Rev. Med. Chem. 2004, 4, 625.
11. (a) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran,
C. T. Chem. Commun. 2012, 1868; (b) De Simone, G.; Di Fiore, A.; Supuran, C. T.
Curr. Pharm. Des. 2008, 14, 655.
12. (a) De Simone, G.; Alterio, V.; Supuran, C. T. Expert Opin. Drug Discov. 2013, 8,
793; (b) Di Fiore, A.; Maresca, A.; Alterio, V.; Supuran, C. T.; De Simone, G.
Chem. Commun. 2011, 11636.
13. (a) Güzel-Akdemir, Ö.; Biswas, S.; Lastra, K.; McKenna, R.; Supuran, C. T.
Bioorg. Med. Chem. 2013, 21, 6674; (b) Pala, N.; Micheletto, L.; Sechi, M.;
Aggarwal, M.; Carta, F.; McKenna, R.; Supuran, C. T. ACS Med. Chem. Lett.
2014, 5, 927.
14. (a) Bozdag, M.; Ferraroni, M.; Nuti, E.; Vullo, D.; Rossello, A.; Carta, F.;
Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 334; (b) D’Ambrosio,
K.; Vitale, R. M.; Dogné, J. M.; Masereel, B.; Innocenti, A.; Scozzafava, A.; De
Simone, G.; Supuran, C. T. J. Med. Chem. 2008, 51, 3230.
15. (a) Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.;
McKenna, R.; Supuran, C. T. Chem. Commun. 2010, 8371; (b) Carta, F.; Garaj, V.;
Maresca, A.; Wagner, J.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna,
R.; Supuran, C. T. Bioorg. Med. Chem. 2011, 19, 3105.
16. (a) Biswas, S.; Aggarwal, M.; Guzel, O.; Scozzafava, A.; McKenna, R.; Supuran, C.
T. Bioorg. Med. Chem. 2011, 19, 3732; (b) Biswas, S.; Carta, F.; Scozzafava, A.;
McKenna, R.; Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 2314; (c) Bozdag, M.;
Pinard, M.; Carta, F.; Masini, E.; Scozzafava, A.; McKenna, R.; Supuran, C. T. J.
Med. Chem. 2014, 57, 9673; (d) Carta, F.; Di Cesare Mannelli, L.; Pinard, M.;
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1828, 2015, 23.
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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 at
room temperature prior to assay, in order to allow for the forma-
tion 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,35–37 and represent
the mean from at least three different determinations. All CA iso-
forms were recombinant ones obtained in-house as reported ear-
lier.35–37 The enzyme concentrations in the assay system were:
8.3 nM for hCA I; 4.8 nM for hCA II; 5.1 nM for hCA IX and
10.4 nM for hCA XII. When highly potent inhibitors were detected,
the enzyme concentrations were lowered up to the pM range and
the time to register the reaction increased. This allowed the calcu-
lation of inhibition constants in the picomolar range.
Acknowledgments
This research was financed in part by the National Plan of
Science, Technology and Innovation (Grant No. 11-MED-1874-2),
King Saud University, Riyadh, Saudi Arabia (to A.M.A.) and by
two European Union Grants, Dynano and Metoxia (to A.S. and C.
T.S.).
18. (a) Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran,
C. T. J. Med. Chem. 2010, 53, 335; (b) Abellán-Flos, M.; Tanç, M.; Supuran, C. T.;
Vincent, S. P. Org. Biomol. Chem. 2015, 13, 7445.
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Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20,
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2014, 57, 9152.
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29, 292.
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2012, 22, 267; (b) Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. 2012, 20, 2266; (c) Carta, F.; Vullo, D.; Maresca, A.; Scozzafava, A.;
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Arechederra, R. L.; Waheed, A.; Sly, W. S.; Supuran, C. T.; Minteer, S. D. Bioorg.
Med. Chem. 2013, 21, 1544; (c) Scozzafava, A.; Supuran, C. T.; Carta, F. Expert
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