Quinazoline-sulfonamides with potent inhibitory activity against the α-carbonic anhydrase from Vibrio cholerae

Article abstract of DOI:10.1016/j.bmc.2014.08.015

Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were synthesized by simple, eco-friendly procedures. These compounds were tested for their ability to inhibit the α-carbonic anhydrases (CA, EC 4.2.1.1) from Vibrio cholerae (VchCA) as well as the human α-CA isoforms, hCA I and hCA II. Nine compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme VchCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic isoform II. The best VchCA inhibitor had a K_Iof 2.7 nM. Many of these developed compounds showed high selectivity for inhibition of the bacterial over the mammalian CA isoforms, with one compound possessing selectivity ratios as high as 97.9 against hCA I and 9.7 against hCA II. Compound 9d was another highly effective VchCA inhibitor presenting a selectivity ratio of 99.1 and 8.1 against hCA I and hCA II, respectively. These results suggest that sulfonamides with quinazoline backbone could be considered suitable tools to better understand the role of bacterial CAs in pathogenesis.

Full text of DOI:10.1016/j.bmc.2014.08.015

Bioorganic & Medicinal Chemistry 22 (2014) 5133–5140
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quinazoline–sulfonamides with potent inhibitory activity against the
a-carbonic anhydrase from Vibrio cholerae
b
a
c
Ahmed M. Alafeefy a, , Mariangela Ceruso , Abdul-Malek S. Al-Tamimi , Sonia Del Prete ,
⇑
Clemente Capasso c, Claudiu T. Supuran b,d,
⇑
a Department of Pharmaceutical Chemistry, College of Pharmacy, Salman Bin Abdulaziz University, PO Box 173, Alkharj 11942, Saudi Arabia
b Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Rm 188, Via della Lastruccia 3, I 50019 Sesto Fiorentino (Firenze), Italy
c Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy
d Università degli Studi di Firenze, Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Via U. 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:
Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were synthesized by sim-
Received 30 June 2014
Revised 8 August 2014
Accepted 13 August 2014
Available online 21 August 2014
ple, eco-friendly procedures. These compounds were tested for their ability to inhibit the
a-carbonic
anhydrases (CA, EC 4.2.1.1) from Vibrio cholerae (VchCA) as well as the human -CA isoforms, hCA I
a
and hCA II. Nine compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme
VchCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic isoform II.
The best VchCA inhibitor had a KI of 2.7 nM. Many of these developed compounds showed high selectivity
for inhibition of the bacterial over the mammalian CA isoforms, with one compound possessing selectiv-
ity ratios as high as 97.9 against hCA I and 9.7 against hCA II. Compound 9d was another highly effective
VchCA inhibitor presenting a selectivity ratio of 99.1 and 8.1 against hCA I and hCA II, respectively. These
results suggest that sulfonamides with quinazoline backbone could be considered suitable tools to better
understand the role of bacterial CAs in pathogenesis.
Keywords:
Carbonic anhydrase
Vibrio cholera
Sulfonamide
Enzyme inhibitor
Quinazoline
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
expression of its virulence genes in vitro which could be
explained by the bicarbonate induced alkalinity of the small
Cholera is an infectious human disease of the small intestine
and is caused by the gram negative bacterium Vibrio cholerae. It
is characterized by massive loss of water and electrolytes which
leads to severe dehydration and hypovolemic shock followed by
death if not well treated.1 Globally, it was estimated that it affects
3–5 million people and causes 100.000–130.000 deaths annually.
In developing countries, cholera spreads among victims mainly
through contaminated water sources, and countries without
proper sanitation techniques have greater incidence of such horri-
ble disastrous disease.2
intestine.4
Consequently, recently, it was reported that the intestinal hyp-
oxic conditions enables V. cholera to express a number of virulence
activators. It has also been shown that V. cholerae may sense intes-
tinal anoxic signals by multiple components to activate virulence.5
Another potential inducer of virulence gene expression is sodium
bicarbonate, which is present at a high concentration in the upper
small intestine. Consequently, bicarbonate is considered the first
positive effector for ToxT, the major direct transcription activator
of the virulence genes.6
It was reported that the growth of Vibrio cholera during
infection of a host initiates a complex regulatory cascade that
results in the production of a regulatory protein that directly
activates transcription of the genes encoding cholera toxins
and other virulence inducer genes.3 Although the in vivo signals
that induce V. cholerae virulence gene expression have not yet
been determined, V. cholerae has been shown to modulate the
Jeffrey and Basel reported that ethoxzolamide, a potent sulfon-
amide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor, outstandingly
controlled the bicarbonate-mediated virulence induction, suggest-
ing that conversion of CO2 into bicarbonate by carbonic anhydrase
plays a major role in virulence induction.1 Recently our group suc-
ceeded to clone an a-CA enzyme from this pathogen, Vibrio cholerae,
which was named VchCA.7 This enzyme showed a significant cata-
lytic activity for the hydration of CO2 to produce HCOÀ3 , and was
greatly inhibited by sulfonamide and sulfamate containing mole-
cules.7–11
⇑
Corresponding authors. Tel.: +966 537600100; fax: +966 011 5886060 (A.M.A.);
tel./fax: +39 055 4573005 (C.T.S.).
Based on the aforementioned results we proposed VchCA as a
new target for the antibiotic development program. In the present
E-mail addresses: a.alafeefy@sau.edu.sa (A.M. Alafeefy), claudiu.supuran@unifi.it
(C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2014.08.015
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

5134
A.M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 5133–5140
paper we continued our efforts in developing sulfonamide CAIs as
potential antibacterial agents, and report the synthesis of certain
quinazoline derivatives crowned with sulfonamide moiety. In
addition to the bacterial enzyme VchCA, these novel compounds
were investigated for their ability to inhibit the physiologically
most important, cytosolic isoforms CA I and II.
2. Results and discussion
2.1. Sequence and phylogenic analysis
Figure 2. Phylogenetic analyses of VchCA and other
a-CAs from human and
bacterial sources. The phylogenetic tree was constructed using the program PhyML
3.0. Legend: for VchCA, hCA I and hCA II see Figure 1; NgoCA, Neisseria gonorrhoeae
(accession no. CAA72038.1); HphyCA, Helicobacter pylori (accession no.
NP_223829.1); SspCA, Sulfurihydrogenibium yellowstonense YO3AOP1 (accession
no. ACD66216.1).
An alignment of the amino acid sequences of VchCA with hCAs I
and II is shown in Figure 1 in order to identify salient features of
this bacterial enzyme. It may be observed that, like the other inves-
tigated
a-CAs, VchCA has the conserved three His ligands, which
coordinate the Zn(II) ion crucial for catalysis (His94, His96, and
His119, hCA I numbering system). The proton shuttle residue
(His64) is also conserved in all these enzymes. This residue assists
the rate-determining step of the catalytic cycle, transferring a pro-
ton from the water coordinated to the Zn(II) ion to the environ-
ment with formation of zinc hydroxide nucleophilic species of
the enzyme. VchCA has also the gate-keeping residues (Glu106
and Thr199), which orientate the substrate for catalysis and are
also involved in the binding of inhibitors. The only unique macro
2.2. Chemistry
A large number of aromatic and heteroaromatic were shown to
possess significant CA inhibitory activity against many classes of
CAs and diverse isoforms.7,8 The nature of the aromatic/heterocy-
clic scaffold is crucial for the binding of these compounds to the
enzyme, as the sulfonamide moiety (in deprotonated form) coordi-
nates the Zn(II) from the enzyme active site, whereas the organic
scaffold participates in interactions (positive or negative ones)
with amino acid residues and water molecules from the cavity,
which may lead to a stabilization (in case of positive interactions)
or destabilization (in case of clashes) of the enzyme-inhibitor com-
plex. As a consequence, exploration of diverse heterocyclic scaf-
folds incorporating sulfamoyl moieties may lead to interesting
compounds, both in terms of efficacy for the inhibition of the var-
ious CA isoforms, as well as in terms of selectivity for some iso-
forms over other ones which may be off targets, such as for
example the house-keeping, widespread human isoforms hCA I
and II.7,8 Thus, the rationale for the drug design presented here
was to explore compounds incorporating a ring system rarely
investigated for inhibition of these enzymes, that is, the quinazo-
lin-4-one.
feature in the primary structure of the bacterial a-CAs with respect
to the mammalian enzymes was the absence of four amino acid
loops indicated in Figure 1 by the ‘- - -’ symbols. This feature is typ-
ical of all bacterial
loops makes the bacterial proteins smaller and more compact.
A phylogenetic analysis of VchCA and other -CAs (such as
human and bacterial -CAs) is shown in Figure 2. It may be
a-CAs investigated so far. Deletion of these
a
a
observed that VchCA is most closely related to the Neisseria
gonorrhoeae (NgoCA). The next closest relatives of these enzymes
are the pathogenic bacterium, Helicobacter pylori and the thermo-
philic bacterium enzyme SspCA, isolated from the extremophile
Sulfurihydrogenibium yellowstonense. The human enzymes clus-
tered together in the upper branches of the radial dendrogram
shown in Figure 2.
Figure 1. Alignment of VchCA, hCA I and hCA II amino acid sequences. The proton shuttle residue (His64), the zinc ligands (His 94, 96 and 119) and the gate keeper residues
(Glu106 and Thr199) are conserved in the bacterial and mammalian sequences. The hCA I numbering system was used. The asterisk (*) indicates identity at all aligned
positions; the symbol (:) relates to conserved substitutions, while (.) means that semi-conserved substitutions are observed. Multialignment was performed with the program
Clustal W, version 2.1. Legend: VchCA, Vibrio cholerae (accession number: AFC59768.1); hCA I, Homo sapiens, isoform I (accession no. NP_001158302.1); hCA II, Homo sapiens,
isoform II (accession no. AAH11949.1).

A.M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 5133–5140
5135
The approach to prepare the target potentially bioactive quinaz-
2.3. Carbonic anhydrase inhibition
oline derivatives 8a–h and thioxoquinazolin-4-one analogues 9a–e
was via a generalized route as depicted in Schemes 1–3. The
starting materials, thioxoquinazoline derivatives 3a–h and their
2,4-dithioxo isosteres 4a–e, were prepared according to reported
literature procedure.12,13 The appropriate anthranilic acid deriva-
tives 1a–e was reacted with the selected isothiocyanate derivative
in boiling ethanol containing catalytic amount of triethylamine.
The structure of these intermediates was confirmed by IR, NMR
and MS spectra (see Section 4 for details).
It is thought that in certain instances, conversion of the oxoqui-
nazoline derivatives to the thioxo isosteres could contribute to the
biological activity.14 Recently, and after in silico prediction, certain
thioxoquinazoline derivatives showed inhibitory potencies at sub-
micromolar levels against the catalytic domain of PDE7A1.15 Such
consideration led us to explore some selected thioxoquinazoline
isomers as possible inhibitors of VchCA isoform. Consequently,
the 2-thioxoquinazolin-4-thione derivatives 4a–e were obtained
by treatment of compounds 3a–e with phosphorus penta sulfide
in dry pyridine. 2-Chloro-N-(4-sulfamoylphenyl)-acetamide was
prepared by acylation of sulfanilamide at room temperature under
basic condition. Alkylation of both quinazoline derivatives 3a–h
and 4a–e with 2-chloro-N-(4-sulfamoylphenyl)-acetamide was
carried out afterwards to afford the target derivatives endowed
with the sulfonamide functionality 8a–h, and 9a–e, respectively.
The structure of the intermediate and target molecules were con-
firmed by elemental analyses, IR, NMR and MS spectra and were
in accordance with the suggested structure.
The I.R. spectra of compounds 8a–h showed two absorption
bands at around 3350 and 3250 cmÀ1 due to NH and NH2. Two
strong carbonyl bands ranging from 1725–1685 cmÀ1 were a com-
mon feature of these derivatives. Their 1H NMR showed two D2O
exchangeable signals of NH and NH2 groups around the regions d
11.00 and d 6.30, respectively. The 13C spectra confirmed the pres-
ence of the corresponding carbons at their expected shift values
specially the two carbonyl peaks at around 160–168 ppm. Conver-
sion of the thioxoquinazolin-4-one 3a–e derivatives to their corre-
sponding di-thioxo 4a–e isosteres was confirmed by the absence of
one of the carbonyl absorption band characteristic for compounds
3a–e in the IR and 13C spectra and the concomitant appearance of
the new C@S bands instead. Conversely, compounds 9a–e forma-
tion was evidenced by the same tools, where the final compounds
exhibited the C@S peaks at d values above 175 ppm. However, gen-
erally the NH, CH2 and C@O is a common functionality for N-(4-
sulfamoylphenyl)-acetamido containing compounds such as 8a–h
and 9a–e as evidenced by their IR, 1H and 13C NMR spectra. Their
mass spectra revealed, in each case, a peak corresponding to the
molecular ion in addition to the base peak.
The inhibition studies of the new quinazolin-4-one sulfonamide
derivatives 8a–h and their 4-thioxo bioisosteres 9a–e reported
here, against the human (h) CA isoforms hCA I, II, and the bacterial
enzyme VchCA, are reported in Table 1. Data for the selectivity
ratios of the dominant and physiologically relevant human car-
bonic anhydrase isoforms (hCA I and II) over the bacterial VchCA
enzyme with these compounds are also included in Table 1, as they
may be the main off-targets in case they are very efficient VchCA
inhibitors. The following structure-activity relationship (SAR) can
be observed from data of Table 1, for the inhibition of the VchCA
investigated here with the new group of quinazoline–sulfonamide
derivatives 8a–h and 9a–e:
(i) Generally, sulfonamide derivatives with quinazolin-4-one
backbone exhibited inhibitory activity towards VchCA
comparable to that obtained by those derivatives with
quinazolin-4-thione derivatives except for compound 8c.
Compounds 8–e, 8–f, 8–h and 9–e, were poorly effective as
VchCA inhibitors (KIs >10,000 nM) and noticed no great
difference between allyl or benzyl group as 3-substituents.
(ii) Substitution at the 5, 6 and or 8 positions of the quinazoline
nucleus led to compounds with varying degrees of activities.
The 6-methyl substituted derivatives were more active than
the 5-substituted methyl derivatives and the later ones were
more active than their 8-methyl substituted quinazoline sul-
fonamide congeners. Compound 8g with 6-nitro-3-allyl
showed very good inhibitory activity but the 6-nitro-3-ben-
zyl 8h exhibited moderate activity the same as the its dith-
ioxo congener 9e.
(iii) In both series the 6-methyl-3-benzyl derivatives (8–c, 9–b)
were mostly more active than the standard drug, however
the oxoquinazoline series could be considered inferior to
the thioxoquinazolines based on the better selectivity ratio
of the latter.
(iv) Against the cytosolic slow isoform hCA I, the new quinazo-
line derivatives and their bioisosteres reported here were
generally ineffective inhibitors, with inhibition constants
ranging between 674–5432 nM. Only compounds 8a–c
showed medium inhibition activity against this isoform,
with KIs of 86.5–105 nM (Table 1).
(v) The physiologically dominant human isoform hCA II was
high effectively inhibited by 8a–c, which showed KIs in
the range of 1.3–1.7 nM, with better inhibition activity
than the clinically used acetazolamide AAZ (KI of 12 nM).
Derivative 8g showed high inhibition potency against this
cytosolic isoform with KI of the same order of magnitude
O
S
R1
CO2H
Ethanol
N
N
P2S5 /Xylene
reflux/5 hour
R NCS
R
+
R
R
1
reflux/1 hour
N
H
S
N
H
S
NH2
1a-c
1-
2a, b
3a-h
4a-e
3-
a
b
c
d
R1
2-
4-
R
R
R1
R
CH2CH=CH2.
CH2CH=CH2.
CH2C6H5.
a
b
c
a
a
b
c
5-CH3.
6-CH3.
6-CH3.
8-CH3
6-CH3.
5-CH3.
3-CH3.
3-OCH3.
CH2CH=CH2.
CH2C6H5.
5-CH3.
6-CH3.
8-CH3.
8-OCH3.
b
CH2C6H5.
d
d
CH2C6H5.
e 6-OCH3.
f 8-OCH3.
e 5-NO2.
e 6-NO2.
CH2C6H5.
CH2CH=CH2.
CH2C6H5.
6-NO2.
g
h
6-NO2.
Scheme 1. Synthesis of 3,5,6 and/or 8 substituted-2-thio-4-oxoquinazoline and 2-thio-4-thioxoquinazoline derivatives 3a–h and 4a–e.

5136
A.M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 5133–5140
Acetone/K2CO3
0 °C/ 0.5 hr
Cl
HN
O
SO NH
2 2
ClCOCH Cl
2
H NO S
NH
2
+
+
2
2
5
6
7
O
SO NH
2 2
O
R1
R1
N
DMF/K2CO3
r.t. 12 hrs
N
R
7
R
N
S
N
H
S
NH
3a-h
8a-h
O
3-
a
b
c
d
e
8-
R
R1
R
R1
a
b
c
d
e
f
5-CH3. CH2CH=CH2.
6-CH3. CH2CH=CH2.
6-CH3. CH2C6H5.
5-CH3. CH2CH=CH2.
6-CH3. CH2CH=CH2.
6-CH3. CH2C6H5.
8-CH3
CH2C6H5.
8-CH3
CH2C6H5.
6-OCH3. CH2C6H5.
8-OCH3. CH2C6H5.
6-OCH3. CH2C6H5.
8-OCH3. CH2C6H5.
f
g 6-NO2. CH2CH=CH2.
g 6-NO2. CH2CH=CH2.
6-NO2. CH2C6H5.
6-NO2. CH2C6H5.
h
h
Scheme 2. Synthese of substituted sulfa derivative 7 and 2,3,5,6 and/or 8-substituted-4-oxoquinazoline derivatives 8a–h.
(vi) The bacterial enzyme VchCA was generally highly inhibited
by the quinazolines reported here, with KIs in the range of
2.7–8.5 nM. Several low nanomolar VchCA inhibitors were
detected here. However, the best VchCA inhibitor was deriv-
ative 8c (KI of 2.7 nM), which was also a very strong inhibitor
of hCA II with KI of 1.7 nM and a medium potency inhibitor
of the cytosolic isoform hCA I with KI of 86.5 nM. The least
effective VchCA inhibitors were 8e–f, 8g and 9e, which
showed KIs higher than 10,000 nM. Although most of the
compounds reported here inhibited efficiently VchCA, no
significant change of KI values has been observed. Therefore,
SAR is almost impossible to delineate as all substitution pat-
terns lead to highly effective inhibitors of this bacterial CA,
VchCA.
S
S
SO NH
2
2
N
C H
N
6
5
DMF/K2CO3
r.t. 12 hrs
R
R
7
+
S
S
N
H
N
NH
4a-e
9a-e
O
4-
9-
R
R
a
b
c
a
b
c
5-CH3.
6-CH3.
8-CH3.
5-CH3.
6-CH3.
8-CH3.
d
d
8-OCH3.
8-OCH3.
e 6-NO2.
e 6-NO2.
Scheme 3. Synthesis of 2,3,5,6 and/or 8-substituted-4-thioxoquinazoline deriva-
tives 9a–e.
(vii) Some of the potent VchCA inhibitors reported here also
showed a high selective inhibition of the bacterial CA over
the human isoforms. Indeed, compounds 8d, 9b and 9c pos-
sessing selectivity ratios ranging from 97.9 to 266.9 against
hCA I and 9.7 to 25.7 against hCA II, represents the most
interesting CAIs reported here (Table 1). Furthermore,
another highly effective VchCA inhibitor presenting similar
selectivity ratios to the previous compound as high as 99.1
against hCA I and 8.1 against hCA II was observed for com-
pound 9d.
Table 1
Inhibition data against human (h) isoforms hCA I, II (cytosolic), and bacterial enzyme
VchCA of derivates 8a–h, 9a–e and acetazolamide AAZ (as standard inhibitor) by a
stopped-flow CO2 hydrase assay17
Compound
KI* (nM)
Selectivity ratios
VchCA/hCA I VchCA/hCA II
hCA I
hCA II
VchCA
8-a
8-b
8-c
8-d
8-e
8-f
8-g
8-h
9-a
9-b
9-c
9-d
9-e
AAZ
105
119
1.3
1.8
1.7
208.4
233.8
208.9
12.1
122.9
67.5
78.26
114.2
65.3
96.0
12.0
8.1
6.0
2.7
8.5
>10,000
>10,000
6.6
>10,000
7.7
5.5
8.7
7.9
>10,000
6.8
13.0
14.7
10.7
256.6
—
—
83.27
—
514.5
97.9
266.9
99.1
—
0.2
0.2
0.2
25.7
—
—
1.5
—
8.3
9.7
14.1
8.1
—
86.5
2078
4761
5432
674
3371
4168
793
2162
803
4549
250
Overall the compounds presented better selectivity inhibition
ratios against hCA I than hCA II and some of them were low selec-
tive inhibitor for the bacterial isoform over the human ones such as
8a–c. It is also interesting to notice that acetazolamide AAZ pos-
sesses lower selectivity ratios for inhibiting VchCA against hCA I
and II (36.7 and 1.76, respectively) than the derivatives 8d–8h
reported here, although it acts as an efficient VchCA inhibitor.
3. Conclusion
36.7
1.76
*
Mean from 3 different assays, by a stopped flow technique (errors were in the
A series of quinazolin-4-one and quinazolin-4-thione deriva-
tives endowed with the biologically interesting benzenesulfon-
amide moiety were designed and synthesized as potential CAIs.
The CA inhibitory activity of these compounds was assessed
against the human pathogenic bacterium Vibrio cholera VchCA
and the human cytosolic isoforms hCA I and II. Most of the novel
tested sulfonamide derivatives showed interesting activities
against VchCA enzyme and three of them potently inhibited hCA
II at a subnanomolar concentration and were more potent than
range of 5–10% of the reported values).
as acetazolamide. Some of the remaining derivatives, such as
9a–b, 9d and 9e, were medium potency hCA II inhibitors (KIs
in the range of 44.0–91.9 nM). The other compounds
reported here 8d–8f, 8h and 9c showed ineffective hCA II
inhibitory activity, with KIs in the range of 114.2–233.8 nM
(Table 1).

A.M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 5133–5140
5137
acetazolamide AAZ (as standard inhibitor). According to the results
obtained in the current study, many of the highly effective VchCA
inhibitors reported here also possessed excellent selectivity ratios
for inhibiting the bacterial over the human enzymes.
(C12H12N2OS, 232.30) C, 62.04 (61.87); H, 5.21 (5.37); N, 12.06
(11.79); S, 13.80 (13.89).
4.1.1.3.
4(1H)-one 3c12,13
3284 (NH), 1686 (C@O), 1263 (C@S) cmÀ1
3-Benzyl-2,3-dihydro-6-methyl-2-thioxoquinazolin-
Yield (86%); mp 168–166 °C; IR (KBr)
1H NMR (400 MHz,
.
m:
;
4. Experimental
DSMO-d6) d 2.35 (s, 3H, CH3), 4.45–4.44 (d, 2H, J = 3.4 Hz, NCH2),
7.63–7.62 (m, 3H, Ar-H), 7.67–7.64 (d, J = 7.6, 2H, Ar-H), 7.88–
7.86 (d, J = 8.6, 2H, Ar-H), 8.15 (s, 1H, Ar-H), 11.54 (s, D2O
exchangeable, 1H, NH); 13C NMR (DSMO-d6) d 15.9 (CH3), 45.3
(NCH2), 121.2, 125.7, 126.4, 127.2, 128.8, 129.1, 133.5, 136.7,
137.1, 152.0 (Ar-C), 161.6 (C@O), 178.3 (C@S). MS (m/z): 282
[M+]. Anal. (C16H14N2OS, 282.36) C, 68.06 (68.19); H, 5.00 (4.79);
N, 9.92 (10.16); S, 11.36 (11.17).
4.1. Chemistry
Melting points (°C, uncorrected) were determined in open
capillaries on
a Gallenkamp melting point apparatus (Sanyo
Gallenkamp, Southborough, UK) and were uncorrected. Precoated
silica gel plates (silica gel 0.25 mm, 60G F254; Merck, Germany)
were used for thin layer chromatography, dichloromethane/
methanol (9.5:0.5) mixture was used as a developing solvent
system and the spots were visualized by ultraviolet light and/
or iodine. Infra red spectra were recorded in KBr discs using
IR-470 Shimadzu spectrometer (Shimadzu, Tokyo, Japan). 1H
NMR spectra were recorded on Bruker AC-400 Ultra Shield
4.1.1.4.
4(1H)-one 3d12,13
3287 (NH), 1683 (C@O), 1266 (C@S) cmÀ1
3-Benzyl-2,3-dihydro-8-methyl-2-thioxoquinazolin-
Yield (92%); mp 167–169 °C; IR (KBr)
1H NMR (400 MHz,
.
m:
;
DSMO-d6) d 2.32 (s, 3H, CH3), 4.60–4.59 (d, 2H, J = 3.6 Hz, NCH2),
7.67–7.65 (m, 3H, Ar-H), 7.68–7.65 (d, J = 7.5, 2H, Ar-H), 8.02–
8.01 (m,3H, Ar-H), 11.14 (s, D2O exchangeable, 1H, NH); 13C NMR
(DSMO-d6) d 16.3 (CH3), 41.5 (NCH2), 121.4, 125.1, 126.8, 127.7,
128.2, 129.1, 133.0, 136.5, 138.7, 152.7 (Ar-C), 160.7, (C@O),
179.3 (C@S). MS (m/z): 282 [M+]. Anal. (C16H14N2OS, 282.36) C,
68.06 (68.23); H, 5.00 (4.81); N, 9.92 (10.25); S, 11.36 (11.14).
NMR spectrometer (Bruker, Flawil, Switzerland,
d ppm) at
300 MHz for 1H and 75 MHz for 13C, using TMS as internal stan-
dard and peak multiplicities are designed as follows: s, singlet;
d, doublet; t, triplet; m, multiplet. Electron Impact Mass Spectra
were recorded on
a Shimadzu GC-MS-QP 5000 instrument
(Shimadzu, Tokyo, Japan). Elemental analyses were performed,
on Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau,
Germany), at the Micro-analytical Unit, Faculty of Science, Cairo
University, Cairo, Egypt, and the found results were within 0.4%
of the theoretical values.
4.1.1.5. 3-Benzyl-2,3-dihydro-6-methoxy-2-thioxoquinazolin-
4(1H)-one 3e12,13
.
Yield (86%); mp 165–167 °C; IR (KBr)
m:
3287 (NH), 1681 (C@O), 1269 (C@S) cmÀ1 1H NMR (400 MHz,
;
DSMO-d6) d 3.73 (s, 3H, OCH3), 4.26–4.25 (d, J = 7.0 Hz, 2H,
NCH2), 7.65–7.63 (m, 3H, Ar-H), 7.66–7.65 (d, J = 7.5 Hz, 2H, Ar-
H), 7.83–7.82 (d, J = 7.3 Hz, 2H, Ar-H), 8.11 (s, 1H, Ar-H), 10.99 (s,
D2O exchangeable, 1H, NH); 13C NMR (DSMO-d6) d 43.0 (NCH2),
55.6 (OCH3), 121.7, 125.2, 127.0, 127.4, 128.4, 129.1, 133.4,
135.7, 151.5, (Ar-C), 162.5 (C@O), 184 (C@S). MS (m/z): 298 [M+].
Anal. (C16H14N2O2S, 298.35) C, 64.41 (64.72); H, 4.73 (4.96); N,
9.39 (9.11); S, 10.75 (10.59).
4.1.1. General procedure for preparation of 2-thioxo-3-
substituted-5, 6 or 8-substituted-3H-quinazolin-4-ones 3a–
h12,13
A mixture of the appropriate anthranilic acid derivative, 1a–e
(0.01 mol), the appropriate isothiocyanate derivative (0.012 mol),
and TEA (2 mL) in absolute ethanol (50 mL) was heated under
reflux for 1 h. The reaction mixture was then cooled and the sol-
vent was evaporated under vacuum. The obtained residue was
washed with water, filtered, dried and crystallized from absolute
ethanol to give the title compounds.
4.1.1.6. 3-Benzyl-2,3-dihydro-8-methoxy-2-thioxoquinazolin-
4(1H)-one 3f12,13
.
Yield (89%); mp 163–165 °C; IR (KBr)
m:
3289 (NH), 1681 (C@O), 1264 (C@S) cmÀ1 1H NMR (400 MHz,
;
DSMO-d6) d 3.77 (s, 3H, OCH3), 4.27–4.26 (d, J = 6.5 Hz, 2H,
NCH2), 7.67–7.64 (m, 4H, Ar-H), 7.67–7.66 (d, J = 7.6, 2H, Ar-H),
7.88–7.86 (d, J = 8.7, 2H, Ar-H), 11.14 (s, D2O exchangeable, 1H,
NH); 13C NMR (DSMO-d6) d 43.1 (NCH2), 55.5 (OCH3), 121.3,
125.1, 126.6, 127.3, 128.7, 129.2, 133.7, 135.1, 151.5 (Ar-C),
161.1, (C@O), 184.3 (C@S). MS (m/z): 298 [M+]. Anal. (C16H14N2O2S,
298.35) C, 64.41 (64.62); H, 4.73 (4.89); N, 9.39 (9.17); S, 10.75
(10.94).
4.1.1.1.
4(1H)-one 3a12,13
3268 (NH), 1685 (C@O), 1271 (C@S) cmÀ1
3-Allyl-2,3-dihydro-5-methyl-2-thioxoquinazolin-
Yield (90%); mp 172–174 °C; IR (KBr)
1H NMR (400 MHz,
.
m:
;
DMSO-d6) d 2.44 (s, 3H, CH3), 4.42–4.41 (d, 2H, J = 4.1 Hz, NCH2),
5.12–5.11 (d, 2H, J = 4.5 Hz, Allyl-CH2), 5.91–5.90 (m, 1H, Allyl-
CH), 6.72–6.71 (d, J = 7.0 Hz, 1H, ArH), 6.98–6.99 (d, J = 7.1 Hz,
1H, ArH), 7.25–7.26 (t, J = 7.2 Hz, 1H, ArH), 11.75 (s, 1H, NH), 13C
NMR (100 MHz, DMSO-d6) d 17.9 (CH3), 40.6 (NCH2), 116.9,
125.1, 125.2, 125.9, 127.9, 127.7, 128.8, 137.4, 138.6 (Ar-C), 160.2
(C@O), 176.5 (C@S). MS (m/z): 232 [M+]. Anal. (C12H12N2OS,
232.30) C, 62.04 (62.21); H, 5.21 (4.88); N, 12.06 (12.19); S, 13.80
(14.03).
4.1.1.7. 3-Allyl-2,3-dihydro-6-nitro-2-thioxoquinazolin-4(1H)-
one 3g12,13
.
Yield (82%); mp 197–199 °C; IR (KBr)
m: 3277
(NH), 1680 (C@O), 1258 cmÀ1 1H NMR (400 MHz, DSMO-d6) d
;
3.82–3.81 (d, 2H, J = 3.6 Hz, NCH2), 5.15–5.14 (d, 2H, J = 4.9 Hz,
Allyl-CH2), 5.88–5.87 (m, 1H, Allyl-CH), 7.62–7.61 (dd, J1 = 7.0 Hz,
J2 = 3.8 Hz, 2H, Ar-H), 7.98 (s, 1H, Ar-H), 10.87 (s, D2O exchange-
able, 1H, NH); 13C NMR (DSMO-d6) d 39.0 (NCH2), 122.5, 123.7,
127.1, 129.5, 133.6, 134.5, 136.3, 137.2, 154.9 (Ar-C), 159.8,
(C@O), 185.7 (C@S). MS (m/z): 263 [M+]. Anal. (C11H9N3O3S,
263.27) C, 50.18 (49.98); H, 3.45 (3.69); N, 15.96 (16.17); S,
12.18 (11.89).
4.1.1.2.
4(1H)-one 3b12,13
3271 (NH), 1690 (C@O), 1258 (C@S) cmÀ1
3-Allyl-2,3-dihydro-6-methyl-2-thioxoquinazolin-
Yield (92%); mp 183–185 °C; IR (KBr)
1H NMR (400 MHz,
.
m:
;
DSMO-d6) d 2.39 (s, 3H, CH3), 4.48–4.47 (d, 2H, J = 3.2 Hz, NCH2),
5.13–5.12 (d, 2H, J = 4.6 Hz, Allyl-CH2), 5.90 (m, 1H, Allyl-CH),
7.62–7.61 (d, J = 7.2, 1H, Ar-H), 7.85–7.84 (d, J = 6.7, 1H, Ar-H),
8.11 (s, 1H, Ar-H), 11.55 (s, D2O exchangeable, 1H, NH); 13C NMR
(DSMO-d6) d 16.2 (CH3), 41.1 (NCH2), 117.3, 120.5, 122.1, 123.0,
127.4, 129.5, 132.7, 134.9, 136.4, 137.8, 141.6, 144.7, 154.3
(Ar-C), 160.7 (C@O), 178.1 (C@S). MS (m/z): 232 [M+]. Anal.
4.1.1.8.
4(1H)-one 3h12,13
3295 (NH), 1684 (C@O), 1252 (C@S) cmÀ1
DSMO-d6) 4.34 (s, 2H, NCH2), 7.60–7.59 (m, 3H, Ar-H),
3-Benzyl-2,3-dihydro-6-nitro-2-thioxoquinazolin-
Yield (89%); mp 185–183 °C; IR (KBr)
1H NMR (400 MHz,
.
m:
;
d

5138
A.M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 5133–5140
7.67–7.65 (d, J = 7.0, 2H, Ar-H), 7.98–7.97 (d, J = 7.7, 2H, Ar-H), 8.11
(s, 1H, Ar-H), 10.98 (s, D2O exchangeable, 1H, NH); 13C NMR
(DSMO-d6) d 44.3 (NCH2), 121.5, 125.7, 126.4, 127.2, 128.5, 129.1,
133.7, 136.2, 138.3, 152.5 (Ar-C), 161.8 (C@O), 184.5. MS (m/z):
313 [M+]. Anal. (C15H11N3O3S, 313.33) C, 57.50 (57.36); H, 3.54
(3.77); N, 13.41 (3.28); S, 10.23 (10.14).
4.1.2.5. 3-Benzyl-2,3-dihydro-6-methoxy-2-thioxoquinazolin-
4(1H)-thione 4e12,13
Yield (46%); mp 160–162 °C; IR (KBr)
: 3274 (NH), 1275, 1222 (2C@S) cmÀ1 1H NMR (400 MHz,
.
m
;
DSMO-d6) d 3.73 (s, 3H, OCH3), 4.22–4.23 (d, J = 7.0 Hz, 2H,
NCH2), 7.63–7.61 (m, 3H, Ar-H), 7.68–7.69 (d, J = 7.1 Hz, 2H, Ar-
H), 7.84–7.82 (d, J = 9.3 Hz, 2H, Ar-H), 8.15 (s, 1H, Ar-H), 11.35 (s,
D2O exchangeable, 1H, NH); 13C NMR (DSMO-d6) d 44.6 (NCH2),
55.8 (OCH3), 121.3, 125.4, 127.1, 127.4, 128.1, 129.4, 133.5,
135.6, 151.1, (Ar-C), 172.9, 180.2 (2C@S). MS (m/z): 314 [M+]. Anal.
(C16H14N2OS2, 298.35) C, 61.12 (60.91); H, 4.49 (4.31); N, 8.91
(9.20); S, 20.40 (20.67).
4.1.2. General procedure for preparation of 2-thioxo-3-
substituted-5, 6 or 8-substituted-3H-quinazolin-4-thiones 4a–
e12,13
A
mixture of 2-thioxo-3-benzyl-3H-quinazolin-4-one 3a–e
derivative, 1a–e (0.015 mol) and phosphorus penta-sulfide (4.0 g,
0.018 mol) in xylene (50 ml) was heated under reflux for 5 h. The
reaction mixture was filtered while hot, the filtrate was cooled and
the separated solid was washed with water, filtered, dried and crys-
tallized from dimethylformamide to give the title compounds.
4.1.3. Synthesis of 2-chloro-N-(4-sulfamoylphenyl)-acetamide
716
To a solution of sulfanilamide 5 (1.72 gm, 0.01 mol) and K2CO3
(2.76 g, 0.02 mol) in acetone (20 mL), 2-chloroacetyl chloride 6
(1.354 g, 0.012 mol) was added drop-wise. The reaction mixture
was stirred at 0 °C for 0.5 h then left to warm to room temperature.
The progress of the reaction was monitored by TLC till completion.
The reaction mixture was filtered and the filtrate was evaporated
under reduced pressure to afford the crude product, which was
washed with water and crystallized from ethanol to afford analyt-
ically pure product of compound 7.
4.1.2.1.
4(1H)-thione 4a12,13
: 3261 (NH), 1285, 1235 (2C@S) cmÀ1
3-Allyl-2,3-dihydro-5-methyl-2-thioxoquinazolin-
Yield (54%); mp 207–209 °C; IR (KBr)
1H NMR (400 MHz, DMSO
.
m
;
-d6) d 2.42 (s, 3H, CH3), 4.41–4.40 (d, 2H, J = 4.5 Hz, NCH2), 5.13–
5.12 (d, 2H, J = 4.8 Hz, Allyl-CH2), 5.93–5.92 (m, 1H, Allyl-CH),
6.75–6.74 (d, J = 7.2 Hz, 1H, ArH), 6.96–6.97 (d, J = 7.5 Hz, 1H,
ArH), 7.21–7.22 (t, J = 7.0 Hz, 1H, ArH), 11.92 (s, 1H, NH), 13C
NMR (100 MHz, DMSO-d6) d 18.1 (CH3), 41.5 (NCH2), 118.0,
125.5, 125.6, 126.2, 127.1, 127.7, 128.0, 138.5, 139.5 (Ar-C),
176.5, 179.5 (2C@S). MS (m/z): 248 [M+]. Anal. (C12H12N2S2,
248.30) C, 58.03 (57.81); H, 4.87 (4.65); N, 11.28 (10.99); S, 25.82
(26.05).
Compound 7: Yield (95%); mp 236–238 °C; IR
m 3347 (NH), 1682
(C@O) cmÀ1 1H NMR (400 MHz, DMSO-d6): 4.33 (s, 2H, CH2), 7.31
;
(s, 2H, SO2NH2), 8.01–8.03 (J = 9.0, 2H, Ar-H), 8.07–8.09 (J = 8.5, 2H,
Ar-H), 10.66 (s, D2O exchangeable, 1H, NH); 13C NMR (DSMO-d6) d
15.8 (CH3), 27.6 (SCH2), 44.6 (NCH2), 120.1, 127.8, 140.1, 142.4 (Ar-
C), 166.3 (C@O); MS m/z (Rel. Int.) 248 (M+). Anal. (C8H9ClN2O3S,
248.68) C, 38.64 (38.35); H, 3.65 (3.81); N, 11.26 (11.05); S,
12.89 (13.06).
4.1.2.2.
4(1H)-thione 4b12,13
(KBr)
: 3260 (NH), 1282, 1240 (2C@S) cmÀ1
3-Allyl-2,3-dihydro-6-methyl-2-thioxoquinazolin-
Yield (42%); mp 211–213–185 °C; IR
1H NMR (400 MHz,
.
m
;
4.1.4. General procedure for preparation of 2-substitiuted
thioxo-3-substituted-5, 6 or 8-substituted-3H-quinazolin-4-
ones 8a–h and 9a–e
To a solution of compound 7 (2.48 g, 0.01 mol) in DMF (10 mL),
the appropriate thioxoquinazolin-4-one (0.01 mol) and anhydrous
potassium carbonate (1.38 g, 0.01 mol) were added and the mix-
ture was stirred at room temperature for 12 h. Water (15 mL)
was added and the mixture was stirred for further 30 min. The sep-
arated solid was filtered, washed with cold water, dried and crys-
tallized from absolute ethanol.
DMSO-d6) d 2.42 (s, 3H, CH3), 4.41–4.40 (d, 2H, J = 4.8 Hz, NCH2),
5.13–5.11 (d, 2H, J = 4.3 Hz, Allyl-CH2), 5.93–5.92 (m, 1H, Allyl-
CH), 6.77–6.75 (d, J = 10.1 Hz, 1H, ArH), 6.96–6.97 (d, J = 7.4 Hz,
1H, ArH), 7.21–7.23 (t, J = 7.7 Hz, 1H, ArH), 11.95 (s, 1H, NH), 13C
NMR (100 MHz, DMSO-d6) d 18.0 (CH3), 41.3 (NCH2), 118.2,
125.1, 125.6, 126.0, 127.0, 127.7, 128.4, 138.4, 139.5 (Ar-C),
174.5, 179.5 (2C@S). MS (m/z): 248 [M+]. Anal. (C12H12N2S2,
248.30) C, 58.03 (57.85); H, 4.87 (4.69); N, 11.28 (11.19); S, 25.82
(26.02).
4.1.2.3.
4(1H)-thione 4c12,13
: 3284 (NH), 1286, 1253 (2C@S) cmÀ1
3-Benzyl-2,3-dihydro-6-methyl-2-thioxoquinazolin-
Yield (46%); mp 162–164 °C; IR (KBr)
1H NMR (400 MHz,
4.1.4.1. N-4-Aminosulphonylphenyl-2-(3-allyl-4-oxo-6-methyl-
.
quinazolin-2ylthio)-acetamide 8-a.
Yield (72%); mp 287–
m
;
289 °C; IR
m
3343–3297 (NH, NH2), 1714, 1688 (2C@O) cmÀ1
;
1H
DSMO-d6) d 2.37 (s, 3H, CH3), 4.45–4.43 (d, 2H, J = 3.8 Hz, NCH2),
7.64–7.63 (m, 3H, Ar-H), 7.67–7.66 (d, J = 4.6, 2H, Ar-H), 7.88–
7.87 (d, J = 8.6, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 11.52 (s, D2O
exchangeable, 1H, NH); 13C NMR (DSMO-d6) d 17.1 (CH3), 44.5
(NCH2), 120.8, 125.1, 126.6, 127.8, 128.5, 129.4, 133.1, 136.9,
137.5, 151.8 (Ar-C), 176.3, 178.7 (2C@S). MS (m/z): 298 [M+]. Anal.
(C16H14N2S2, 282.36) C, 64.39 (64.22); H, 4.73 (4.52); N, 9.39
(9.24); S, 21.49 (21.65).
NMR (DSMO-d6) d 2.23 (s, 3H, CH3), 3.72 (s, 2H, SCH2), 3.83–3.82
(d, 2H, J = 4.3 Hz, NCH2), 5.14–5.13 (d, 2H, J = 4.7 Hz, Allyl-CH2),
5.92–5.90 (m, 1H, Allyl-CH), 6.28 (s, D2O exchangeable, 1H, NH2),
7.64–7.62 (m, 3H, Ar-H), 7.94–7.93 (d, J = 8.7, 2H, Ar-H), 8.13–
8.12 (d, J = 7.0, 2H, Ar-H), 10.72 (s, D2O exchangeable, 1H, NH);
13C NMR (DSMO-d6) d 15.8 (CH3), 27.6 (SCH2), 42.5 (NCH2), 117.7,
120.3, 122.4, 123.9, 127.5, 129.8, 132.4, 134.4, 136.7, 137.4,
141.0, 144.5, 154.7 (Ar-C), 161.6, 165.8 (2C@O); MS m/z (Rel. Int.)
444 (M+). Anal. (C20H20N4O4S2, 444.52) C, 54.04 (54.15); H, 4.53
(4.28); N, 12.60 (12.73); S, 14.43 (14.26).
4.1.2.4.
4(1H)-thione 4d12,13
: 3271 (NH), 1277, 1251 (2C@S) cmÀ1
3-Benzyl-2,3-dihydro-8-methyl-2-thioxoquinazolin-
Yield (45%); mp 160–162 °C; IR (KBr)
1H NMR (400 MHz,
.
m
;
4.1.4.2. N-4-Aminosulphonylphenyl-2-(3-allyl-4-oxo-8-methyl-
DSMO-d6) d 2.36 (s, 3H, CH3), 4.46–4.44 (d, 2H, J = 3.1 Hz, NCH2),
7.74–7.72 (m, 3H, Ar-H), 7.87–7.85 (d, J = 5.5, 2H, Ar-H), 7.93–
7.92 (d, J = 8.2, 2H, Ar-H), 8.15 (s, 1H, Ar-H), 11.83 (s, D2O
exchangeable, 1H, NH); 13C NMR (DSMO-d6) d 17.1 (CH3), 44.4
(NCH2), 120.2, 125.0, 126.5, 127.1, 128.6, 129.3, 133.4, 136.8,
137.4, 152.1 (Ar-C), 177.3, 179.7 (2C@S). MS (m/z): 298 [M+]. Anal.
(C16H14N2S2, 282.36) C, 64.39 (64.25); H, 4.73 (4.57); N, 9.39
(9.26); S, 21.49 (21.61).
quinazolin-2yl-thio)-acetamide 8b.
Yield (89%); mp 283–
285 °C; IR
m
3345–3291 (NH, NH2), 1725, 1690 (2C@O) cmÀ1
;
1H
NMR (DSMO-d6) d 2.22 (s, 3H, CH3), 3.73 (s, 2H, SCH2), 3.88 (d,
2H, J = 3.2 Hz, NCH2), 5.16 (d, 2H, J = 4.8 Hz,Allyl-CH2), 5.92 (m,
1H, Allyl-CH), 6.27 (s, D2O exchangeable, 1H, NH2), 7.65–7.62 (d,
J = 7.5, 2H, Ar-H), 7.95–7.94 (d, J = 8.6, 2H, Ar-H), 8.13–8.11 (d,
J = 8.4, 2H, Ar-H), 8.26 (s, 1H, Ar-H), 10.56 (s, D2O exchangeable,
1H, NH); 13C NMR (DSMO-d6) d 15.5 (CH3), 26.9 (SCH2), 40.8

A.M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 5133–5140
5139
(NCH2), 117.3, 120.5, 122.1, 123.0, 127.4, 129.5, 132.7, 134.9, 136.4,
137.8, 141.6, 144.7, 154.3 (Ar-C), 160.7, 166.8 (2C@O); MS m/z (Rel.
Int.) 444 (M+, 100). Anal. (C20H20N4O4S2, 444.52) C, 54.04 (53.88);
H, 4.53 (4.78); N, 12.60 (12.49); S, 14.43 (14.57).
6.29 (s, D2O exchangeable, 1H, NH2), 7.65–7.63 (d, J = 7.3, 2H, Ar-
H), 7.96–7.95 (d, J = 8.6, 2H, Ar-H), 8.13–8.12 (d, J = 8.0, 2H, Ar-H),
8.26 (s, 1H, Ar-H), 10.56 (s, D2O exchangeable, 1H, NH); 13C NMR
(DSMO-d6) d 28.0 (SCH2), 38.9 (NCH2), 118.1, 121.0, 122.3, 123.5,
127.3, 129.6, 133.1, 134.2, 136.7, 137.2, 141.0, 144.3, 154.9
(Ar-C), 159.8, 166.1 (2C@O); MS m/z (Rel. Int.) 475 (M+, 82). Anal.
(C19H17N5O6S2, 475.49) C, 47.99 (48.16); H, 3.60 (3.82); N, 14.73
(14.96); S, 13.49 (13.37).
4.1.4.3.
methyl-quinazolin-2yl-thio)-acetamide 8c.
mp 258–260 °C; IR 3346–3295 (NH, NH2), 1728, 1685 (2C@O)
cmÀ1 1H NMR (DSMO-d6) d 2.26 (s, 3H, CH3), 3.83 (s, 2H, SCH2),
N-4-Aminosulphonylphenyl-2-(3-benzyl-4-oxo-6-
Yield (86%);
m
;
4.47 (s, 2H, NCH2), 6.27 (s, D2O exchangeable, 2H, NH2), 7.67–
7.64 (m, 5H, Ar-H), 7.67–7.64 (d, J = 7.6, 2H, Ar-H), 7.98–7.97 (d,
J = 8.6, 2H, Ar-H), 8.12–8.10 (d, J = 8.5, 2H, Ar-H), 8.25 (s, 1H, Ar-
H), 10.77 (s, D2O exchangeable, 1H, NH); 13C NMR (DSMO-d6) d
15.6 (CH3), 29.1 (SCH2), 46.8 (NCH2), 119.3, 120.5, 121.3, 125.4,
126.7, 127.5, 128.7, 129.0, 133.4, 136.0, 138.1, 142.5, 144.8, 154.3
(Ar-C), 161.6, 168.5 (2C@O); MS m/z (Rel. Int.) 494 (M+, 71). Anal.
(C24H22N4O4S2, 494.58) C, 58.28 (58.35); H, 4.48 (4.27); N, 11.33
(11.40); S, 12.97 (13.22).
4.1.4.8. N-4-Aminosulphonylphenyl-2-(3-benzyl-4-oxo-6-nitro-
quinazolin-2yl-thio)-acetamide 8h.
Yield (69%); mp 221–
223 °C; IR
m
3346–3295 (NH, NH2), 1728, 1685 (2C@O) cmÀ1
;
1H NMR (DSMO-d6) d 3.81 (s, 2H, SCH2), 4.36 (s, 2H, NCH2),
6.25 (s, D2O exchangeable, 1H, NH2), 7.66–7.64 (m, 5H, Ar-H),
7.67–7.65 (d, J = 7.0, 2H, Ar-H), 7.98–7.97 (d, J = 7.7, 2H, Ar-H),
8.12–8.10 (d, J = 6.6, 2H, Ar-H), 8.24 (s, 1H, Ar-H), 10.77 (s, D2O
exchangeable, 1H, NH); 13C NMR (DSMO-d6) d 28.2 (SCH2), 44.9
(NCH2), 119.4, 120.5, 121.3, 125.4, 126.7, 127.5, 128.7, 129.0,
133.4, 136.0, 138.1, 142.5, 144.8, 154.3 (Ar-C), 161.6, 168.5
(2C@O); MS m/z (Rel. Int.) 525 (M+, 82). Anal. (C23H19N5O6S2,
525.56) C, 52.56 (52.67); H, 3.64 (3.85); N, 13.33 (13.52); S,
12.20 (11.95).
4.1.4.4.
methyl-quinazolin-2yl-thio)-acetamide 8d.
mp 252–254 °C; IR 3345–3298 (NH, NH2), 1723, 1689 (2C@O)
cmÀ1 1H NMR (DSMO-d6) d 2.22 (s, 3H, CH3), 3.89 (s, 2H, SCH2),
N-4-Aminosulphonylphenyl-2-(3-benzyl-4-oxo-8-
Yield (81%);
m
;
4.67 (s, 2H, NCH2), 6.24 (s, D2O exchangeable, 1H, NH2), 7.67–
7.65 (m, 3H, Ar-H), 7.68–7.65 (d, J = 7.5, 2H, Ar-H), 7.87–7.85 (d,
J = 9.0, 2H, Ar-H), 8.12–8.10 (d, J = 8.5, 2H, Ar-H), 8.25–8.23
(m,3H, Ar-H), 10.94 (s, D2O exchangeable, 1H, NH); 13C NMR
(DSMO-d6) d 16.3 (CH3), 28.5 (SCH2), 42.9 (NCH2), 119.7, 120.2,
121.4, 125.1, 126.8, 127.7, 128.2, 129.1, 133.0, 136.5, 138.7,
142.3, 143.9, 152.7 (Ar-C), 160.7, 165.9 (2C@O); MS m/z (Rel. Int.)
494 (M+, 71). Anal. (C24H22N4O4S2, 494.58) C, 58.28 (58.31); H,
4.48 (4.59); N, 11.33 (11.14); S, 12.97 (12.71).
4.1.4.9.
methyl-quinazolin-2yl-thio)-acetamide 9a.
mp 255–57 °C; IR 3344–3288 (NH, NH2), 1681 (C@O), 1273
(C@S), cmÀ1 1H NMR (DSMO-d6) d 2.29 (s, 3H, CH3), 3.83 (s,
N-4-Aminosulphonylphenyl-2-(3-benzyl-4-thioxo-5-
Yield (70%);
m
;
2H, SCH2), 4.33 (s, 2H, NCH2), 6.23 (s, D2O exchangeable, 1H,
NH2), 7.62–7.61 (m, 3H, Ar-H), 7.66–7.65 (d, J = 8.1, 2H, Ar-H),
7.95–7.94 (d, J = 7.0, 2H, Ar-H), 8.11–8.09 (d, J = 7.1, 2H, Ar-
H), 8.25–8.23 (m, 3H, Ar-H), 10.77 (s, D2O exchangeable, 1H,
NH); 13C NMR (DSMO-d6)
d 16.2 (CH3), 27.3 (SCH2), 45.5
(NCH2), 118.7, 119.6, 120.8, 126.7, 127.0, 127.6, 128.6, 129.1,
138.6, 141.8, 146.9, 151.2, 159.5 (Ar-C), 160.0, 166.2 (2C@O);
MS m/z (Rel. Int.) 510 (M+, 68). Anal. (C24H22N4O3S3, 510.65)
C, 56.45 (56.59); H, 4.34 (4.51); N, 10.97 (11.18); S, 18.84
(18.97).
4.1.4.5. N-4-Aminosulphonylphenyl-2-(3-benzyl-4-oxo-6-meth-
oxy-quinazolin-2yl-thio)-acetamide 8e.
259–261 °C; IR 3346–3296 (NH, NH2), 1727, 1685 (2C@O)
cmÀ1 1H NMR (DSMO-d6) d 3.70 (s, 3H, OCH3), 3.82 (s, 2H, SCH2),
Yield (86%); mp
m
;
4.27 (s, 2H, NCH2), 6.27 (s, D2O exchangeable, 1H, NH2), 7.66–
7.63 (m, 5H, Ar-H), 7.66–7.65 (d, J = 7.5, 2H, Ar-H), 7.86–7.85 (d,
J = 7.3, 2H, Ar-H), 8.11–8.10 (d, J = 7.4, 2H, Ar-H), 8.25 (s, 1H, Ar-
H), 10.92 (s, D2O exchangeable, 1H, NH); 13C NMR (DSMO-d6) d
28.8 (SCH2), 43.6 (NCH2), 55.9 (OCH3), 119.3, 121.0, 121.7, 126.0,
127.1, 127.6, 128.4, 129.5, 133.8, 135.1, 141.8, 151.3, 155.2
(Ar-C), 161.4, 166.2 (2C@O); MS m/z (Rel. Int.) 510 (M+, 75). Anal.
(C24H22N4O5S2, 510.58) C, 56.46 (56.30); H, 4.34 (4.57); N, 10.97
(10.85); S, 12.56 (12.24).
4.1.4.10. N-4-Aminosulphonylphenyl-2-(3-benzyl-4-thioxo-6-
methyl-quinazolin-2yl-thio)-acetamide 9b.
mp 242–244 °C; IR 3348–3298 (NH, NH2), 1688 (C@O), 1275
(C@S) cmÀ1 1H NMR (DSMO-d6) d 2.36 (s, 3H,CH3), 3.77 (s, 2H,
Yield (73%);
m
;
SCH2), 3.85 (s, 2H, NCH2), 7.65–7.63 (m, 5H, Ar-H), 7.66–7.64 (d,
J = 7.6, 2H, Ar-H), 7.95–7.93 (d, J = 8.0, 2H, Ar-H), 8.12–8.11 (d,
J = 6.5, 2H, Ar-H), 8.24 (s, 1H, Ar-H), 10.79 (s, D2O exchangeable,
1H, NH); 13C NMR (DSMO-d6) d 16.1 (CH3), 27.3 (SCH2), 47.5
(NCH2), 118.7, 119.6, 120.8, 126.7, 127.0, 127.6, 128.6, 129.1,
138.6, 141.8, 146.9, 151.2, 159.5 (Ar-C), 160.0 (C@O), 186.5
(C@S); MS m/z (Rel. Int.) 510 (M+, 100). Anal. (C24H22N4O3S3,
510.65) C, 56.45 (56.60); H, 4.34 (4.15); N, 10.97 (11.13); S, 18.84
(18.99).
4.1.4.6. N-4-Aminosulphonylphenyl-2-(3-benzyl-4-oxo-8-meth-
oxy-quinazolin-2yl-thio)-acetamide 8f.
263–265 °C; IR 3339–3299 (NH, NH2), 1724, 1685 (2C@O)
cmÀ1 1H NMR (DSMO-d6) d 3.79 (s, 3H, OCH3), 3.83 (s, 2H, SCH2),
Yield (86%); mp
m
;
4.29 (s, 2H, NCH2), 6.27 (s, D2O exchangeable, 1H, NH2), 7.67–
7.64 (m, 3H, Ar-H), 7.67–7.66 (d, J = 7.6, 2H, Ar-H), 7.88–7.86 (d,
J = 8.7, 2H, Ar-H), 8.12–8.11 (d, J = 8.6, 2H, Ar-H), 8.24–8.22 (m,
3H, Ar-H), 10.95 (s, D2O exchangeable, 1H, NH); 13C NMR (DSMO-
d6) d 28.9 (SCH2), 43.2 (NCH2), 56.3 (OCH3), 119.1, 121.0, 121.6,
125.9, 127.0, 127.5, 128.3, 129.7, 133.9, 135.7, 142.4, 150.3, 153.2
(Ar-C), 161.1, 166.7 (2C@O); MS m/z (Rel. Int.) 510 (M+, 71). Anal.
(C24H22N4O5S2, 510.58) C, 56.46 (56.53); H, 4.34 (4.47); N, 10.97
(11.20); S, 12.56 (12.74).
4.1.4.11. N-4-Aminosulphonylphenyl-2-(3-benzyl-4-thioxo-8-
methyl-quinazolin-2yl-thio)-acetamide 9c.
mp 265–67 °C; IR 3344–3295 (NH, NH2), 1686 (C@O), 1279
(C@S) cmÀ1 1H NMR (DSMO-d6) d 2.25 (m, 3H, CH3), 3.75 (s, 2H,
Yield (76%);
m
;
SCH2), 3.86 (s, 2H, NCH2), 7.64–7.60 (m, 3H, Ar-H), 7.65–7.64 (d,
J = 7.0, 2H, Ar-H), 7.95–7.94 (d, J = 7.1, 2H, Ar-H), 8.12–8.10 (d,
J = 7.3, 2H, Ar-H), 8.27–8.25 (m, 3H, Ar-H), 10.83 (s, D2O exchange-
able, 1H, NH); 13C NMR (DSMO-d6) d 16.5 (CH3), 27.2S(CH2), 46.1
(NCH2), 119.2, 119.9, 121.4, 126.1, 127.1, 127.7, 128.5, 129.2,
138.9, 142.0, 147.3, 151.2, 159.4 (Ar-C), 163.4 (C@O), 184.9
(C@S); MS m/z (Rel. Int.) 510 (M+, 100). Anal. (C24H22N4O3S3,
510.65) C, 56.45 (56.54); H, 4.34 (4.44); N, 10.97 (10.81); S, 18.84
(19.05).
4.1.4.7.
quinazolin-2yl-thio)-acetamide 8g.
299 °C; IR
3347–3295 (NH, NH2), 1727, 1687 (2C@O) cmÀ1
N-4-Aminosulphonylphenyl-2-(3-allyl-4-oxo-6-nitro-
Yield (82%); mp 297–
1H
m
;
NMR (DSMO-d6) d 3.72 (s, 2H, SCH2), 3.85 (d, 2H, J = 3.8 Hz,
NCH2), 5.15 (d, 2H, J = 4.9 Hz, Allyl-CH2), 5.88 (m, 1H, Allyl-CH),

5140
A.M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 5133–5140
4.1.4.12. N-4-Aminosulphonylphenyl-2-(3-benzyl-4-thioxo-8-
methoxy-quinazolin-2yl-thio)-acetamide 9d. Yield (76%);
mp 245–47 °C; IR 3344–3298 (NH, NH2), 1689 (C@O), 1276
(C@S) cmÀ1 1H NMR (DSMO-d6) d 3.76 (s, 3H, OCH3), 3.81 (s, 2H,
SCH2), 4.31 (s, 2H, NCH2), 6.25 (s, D2O exchangeable, 1H, NH2),
7.65–7.63 (m, 3H, Ar-H), 7.66–7.65 (d, J = 7.5, 2H, Ar-H), 7.88–
7.87 (d, J = 8.1, 2H, Ar-H), 8.12–8.11 (d, J = 8.3, 2H, Ar-H), 8.26–
8.25 (m, 3H, Ar-H), 10.91 (s, D2O exchangeable, 1H, NH); 13C
NMR (DSMO-d6) d 28.1 (SCH2), 43.0 (NCH2), 55.4 (OCH3), 120.6,
121.1, 121.9, 125.5, 127.1, 127.7, 128.3, 129.6, 133.2, 135.7,
142.6, 151.0, 154.6 (Ar-C), 160.7 (C@O), 188.5 (C@S); MS m/z
(Rel. Int.) 526 (M+, 74). Anal. (C24H22N4O4S3, 526.65) C, 54.73
(54.50); H, 4.21 (4.40); N, 10.64 (10.45); S, 18.27 (17.98).
References and notes
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Supuran, C. T. Expert Opin. Drug Discov. 2013, 8, 793; (c) Supuran, C. T. Bioorg.
Med. Chem. Lett. 2010, 20, 3467; (d) Harju, A. K.; Bootorabi, F.; Kuuslahti, M.;
Supuran, C. T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2013, 28, 231.
8. (a) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (b) Alterio, V.; Di
Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112,
4421; (c) Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 677; (d) Pinard, M. A.;
Boone, C. D.; Rife, B. D.; Supuran, C. T.; McKenna, R. Bioorg. Med. Chem. 2013, 21,
7210; (e) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; (f) Capasso,
C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379.
;
4.1.4.13. N-4-Aminosulphonylphenyl-2-(3-benzyl-4-thioxo-6-
9. (a) Del Prete, S.; Isik, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.;
Supuran, C. T.; Capasso, C. J. Med. Chem. 2012, 55, 10742; (b) De Del Prete, S.;
Luca, V.; Scozzafava, A.; Carginale, V.; Supuran, C. T.; Capasso, C. J. Enzyme Inhib.
Med. Chem. 2014, 29, 23.
nitro-quinazolin-2yl-thio)-acetamide 9e.
239–241 °C; IR 3341–3296 (NH, NH2), 1687 (C@O),1277 (C@S),
cmÀ1 1H NMR (DSMO-d6) d 3.49 (s, 2H, SCH2),4.67 (s, 2H, NCH2),
Yield (76%); mp
m
;
10. (a) Çavdar, H.; Ekinci, D.; Talaz, O.; Saraçog˘lu, N.; Sßentürk, M.; Supuran, C. T. J.
Enzyme Inhib. Med. Chem. 2012, 27, 148; (b) Chohan, Z. H.; Scozzafava, A.;
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Mincione, F.; Somma, T.; Scozzafava, G.; Galassi, F.; Masini, E.; Impagnatiello,
F.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 138; (d) Ekinci, D.;
Karagoz, L.; Ekinci, D.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem.
2013, 28, 283; (e) Carta, F.; Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
2012, 22, 79; (f) Masini, E.; Carta, F.; Scozzafava, A.; Supuran, C. T. Expert Opin.
Ther. Pat. 2013, 23, 705.
11. (a) Vullo, D.; Isik, S.; Del Prete, S.; De Luca, V.; Carginale, V.; Scozzafava, A.;
Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2013, 23, 1636; (b) Alp, C.;
Maresca, A.; Alp, N. A.; Gültekin, M. S.; Ekinci, D.; Scozzafava, A.; Supuran, C. T.
J. Enzyme Inhib. Med. Chem. 2013, 28, 294; (c) Carta, F.; Scozzafava, A.; Supuran,
C. T. Expert Opin. Ther. Pat. 2012, 22, 747; (d) Supuran, C. T. Expert Opin. Ther.
6.24 (s, D2O exchangeable, 1H, NH2), 7.67–7.65 (m, 5H, Ar-H),
7.68–7.65 (d, J = 7.5, 2H, Ar-H), 7.87–7.85 (d, J = 9.0, 2H, Ar-H),
8.12–8.10 (d, J = 8.5, 2H, Ar-H), 8.26 (s, 1H, Ar-H), 10.94 (s, D2O
exchangeable, 1H, NH); 13C NMR (DSMO-d6) d 28.5 (SCH2), 42.9
(NCH2), 119.7, 120.2, 121.4, 125.1, 126.8, 127.7, 128.2, 129.1,
133.0, 136.5, 138.7, 142.3, 143.9, 152.7 (Ar-C), 160.7 (C@O),
187.2 (C@S); MS m/z (Rel. Int.) 541 (M+, 68). Anal. (C23H19N5O5S3,
541.65) C, 51.00 (51.24); H, 3.54 (3.48); N, 12.93 (13.10); S, 17.76
(17.51).
4.2. CA inhibition studies
ˇ
Pat. 2013, 23, 677; (e) Supuran, C. T.; Maresca, A.; Gregán, F.; Remko, M. J.
Enzyme Inhib. Med. Chem. 2013, 28, 289; (f) Chohan, Z. H.; Scozzafava, A.;
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2002, 17, 261.
12. Al-Omar, M. A.; Abdel-Hamide, S. G.; Hamad, A. A.; El-Subbagh, I. E. Saudi
Pharm. J. 2004, 12, 63.
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31, 1503.
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Hernández, J.; Pérez, C.; Cuenca, J.; Pérez-Castillo, A.; Martínez, A.; Huertas, O.;
Gelpí, J. L.; Luque, F. J.; Ke, H.; Gil, C. ChemMedChem 2009, 4, 866.
15. Sánchez, A. I.; Martínez-Barrasa, V.; Burgos, C.; Vaquero, J. J.; Alvarez-Builla, J.;
Terricabras, E.; Segarra, V. Bioorg. Med. Chem. 2013, 21, 2370.
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18. (a) Supuran, C. T.; Mincione, F.; Scozzafava, A.; Briganti, F.; Mincione, G.; Ilies,
M. A. Eur. J. Med. Chem. 1998, 33, 247; (b) Carta, F.; Supuran, C. T. Expert Opin.
Ther. Pat. 2013, 23, 681; (c) Scozzafava, A.; Supuran, C. T.; Carta, F. Expert Opin.
Ther. Pat. 2013, 23, 725; (d) Gieling, R. G.; Parker, C. A.; De Costa, L. A.;
Robertson, N.; Harris, A. L.; Stratford, I. J.; Williams, K. J. J. Enzyme Inhib. Med.
Chem. 2013, 28, 360.
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity.17 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 7.4) and 20 mM NaBF4 (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 deter-
mined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (10 mM) were prepared in DMSO
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 RT prior to assay, in order to allow for the formation
of the E–I complex. The inhibition constants were obtained by non-
linear least-squares methods using PRISM 3, and the Cheng–Prus-
off equation, as reported earlier,18–20 and represent the mean from
at least three different determinations. All CAs were recombinant
proteins obtained as reported earlier by these groups.18–20
19. (a) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Vullo, D.;
Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2007, 50, 1651; (b) Bonneau, A.;
Maresca, A.; Winum, J. Y.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28,
397; (c) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib.
Med. Chem. 2013, 28, 388; (d) Alp, C.; Özsoy, S.; Alp, N. A.; Erdem, D.; Gültekin,
_
M. S.; Küfreviog˘lu, Ö. I.; Sßentürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem.
2012, 27, 818.
20. (a) Supuran, C. T. Curr. Med. Chem. 2012, 19, 44; (b) Kazancıog˘lu, E. A.; Güney,
M.; Sßentürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 880; (c) Liu,
F.; Martin-Mingot, A.; Lecornué, F.; Maresca, A.; Thibaudeau, S.; Supuran, C. T. J.
Enzyme Inhib. Med. Chem. 2012, 27, 886; (d) Maresca, A.; Scozzafava, A.; Vullo,
D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 384; (e) Koz, O.; Ekinci,
D.; Perrone, A.; Piacente, S.; Alankus-Caliskan, O.; Bedir, E.; Supuran, C. T. J.
Enzyme Inhib. Med. Chem. 2013, 28, 412.
Acknowledgments
This research was financed by the National Plan of Science,
Technology and Innovation (Grant No. 11-MED1874-02), King
Saud University, Riyadh (to AF).