Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhydrase I, II and IX inhibitory activity and cytotoxic effects against breast cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2016.05.016

A series of 4-(thiazol-2-ylamino)-benzenesulfonamides was synthesized and screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory and cytotoxic activity on human breast cancer cell line MCF-7. Human (h) CA isoforms I, II and IX were included in the study. The new sulfonamides showed excellent inhibition of all three isoforms, with K_Is in the range of 0.84-702 nM against hCA I, of 0.41-288 nM against hCA II and of 5.6-29.2 against the tumor-associated hCA IX, a validated anti-tumor target, with a sulfonamide (SLC-0111) in Phase I clinical trials for the treatment of hypoxic, metastatic solid tumors overexpressing CA IX. The new compounds showed micromolar inhibition of growth efficacy against breast cancer MCF-7 cell lines.

Full text of DOI:10.1016/j.bmc.2016.05.016

Accepted Manuscript
Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhy-
drase I, II and IX inhibitory activity and cytotoxic effects against breast cancer
cell lines
Nagwa M. Abdel Gawad, Noha H. Amin, Mohammed T. Elsaadi, Fatma M.M.
Mohamed, Andrea Angeli, Viviana De Luca, Clemente Capasso, Claudiu T.
Supuran
PII:
S0968-0896(16)30339-X
http://dx.doi.org/10.1016/j.bmc.2016.05.016
BMC 13000
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 April 2016
4 May 2016
11 May 2016
Please cite this article as: Abdel Gawad, N.M., Amin, N.H., Elsaadi, M.T., Mohamed, F.M.M., Angeli, A., Luca,
V.D., Capasso, C., Supuran, C.T., Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhydrase
I, II and IX inhibitory activity and cytotoxic effects against breast cancer cell lines, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.016
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Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhydrase
I, II and IX inhibitory activity and cytotoxic effects against breast cancer cell
lines
Nagwa M. Abdel Gawad,a Noha H. Amin,*b Mohammed T. Elsaadi,b Fatma
M.M. Mohamed,b Andrea Angeli,c Viviana De Luca,d Clemente Capasso,d and
Claudiu T. Supuran*c
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, 11562,
Egypt.
b*Department of Medicinal Chemistry, Faculty of Pharmacy, Beni Suef University, Beni Suef, 62514,
Egypt.
cUniversità degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy; E-mail:
claudiu.supuran@unifi.it; Fax: +39055 457 3385; Tel: +39055 457 3005
dIstituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 81, Napoli, Italy.
Abstract. A series of 4-(thiazol-2-ylamino)-benzenesulfonamides was synthesized
and screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory and cytotoxic
activity on human breast cancer cell line MCF-7. Human (h) CA isoforms I, II and IX
were included in the study. The new sulfonamides showed excellent inhibition of all
three isoforms, with KIs in the range of 0.84-702 nM against hCA I, of 0.41 – 288 nM
against hCA II and of 5.6 – 29.2 against the tumor-associated hCA IX, a validated
anti-tumor target, with a sulfonamide (SLC-0111) in Phase I clinical trials for the
treatment of hypoxic, metastatic solid tumors overexpressing CA IX. The new
compounds showed micromolar inhibitory of growth efficacy against breast cancer
MCF-7 cell lines.
Keywords: Carbonic anhydrase; inhibitor; sulfonamide, antitumor, cytotoxic agent.
______
* Corresponding authors: E-mail:hani.noha@gmail.com (NHA); Phone: +39-055-
4573005; fax: +39-055-4573385; E-mail: claudiu.supuran@unifi.it (CTS).

1. Introduction
Cancer is a life threatening group of diseases in which deregulated proliferation of
cells invades and disrupts the surrounding tissues, leading to dysfunctional metabolic,
angiogenetic and immunologic functions.1 According to World Health Organization
(WHO), cancer figures among the leading cause of death worldwide, accounting for
8.2 million deaths in both developed and developing countries in 2012. Although a
large number of anticancer drugs were launched ultimately, mainly kinase inhibitors,2
their limited success, the drug resistance problems and severe side effects indicate that
there is an imperative need of novel strategies for cancer management.2 Thus,
increasing efforts are being done towards developing novel such agents, with higher
safety margins, possibly targeting novel pathways which exploit the particular
biology/biochemistry of tumor cells.3 Among them, hypoxia, a hallmark of many
cancer types, leads to the overexpression of a host of proteins that drive adaptation of
the cells to the hypoxic and acidotic conditions.4 A large number of proteins
(specifically overexpressed in tumors and with a limited diffusion in normal cells)
participate in these processes, among which two isoforms of the metalloenzyme
carbonic anhydrases (CA, EC 4.2.1.1), i.e., CA IX and XII.5
The CAs are a group of metalloenzymes involved in many physiologic processes,
among which pH buffering of extra- and intracellular spaces, by catalyzing the
reversible hydration of carbon dioxide to bicarbonate and a proton.5-10 The family of
human CAs (hCAs) comprises 16 different α-isoforms, of which several are cytosolic
(CA I-III, CA VII and CA XIII), five are membrane-bound isoforms (CA IV, IX, XII,
XIV and XV), two are mitochondrial (CA VA and VB), and one is secreted into saliva
(CA VI). These enzymes are involved in various biochemical/metabolic processes,
such as gluconeogenesis, lipogenesis, and ureagenesis.5-10 Abnormal levels or
activities of many CA isoforms have been associated with different diseases such as
cancer (overexpression of CA IX/XII due to the hypoxia cascade activation),4,5
epilepsy (abnormal levels/activities of brain CA isoforms),6 obesity (dysregulation of
the mitochondrial isoforms CA VA/B, involved among others in lipogenesis),7
glaucoma (upregulation and high activity levels of CA II, IV and XII).8,9 Furthermore,
the renal excretion of anions is also mediated by several CA isoforms, and their
inhibition with sulfonamides (the main class of CA inhibitors, CAIs)4b,11,12 leads to a
strong diuretic effect.10 Although many sulfonamides are in clinical use as diuretics,
antiglaucoma, antiepilepsy/antiobesity agents for decades,4-12 only recently a

ureidosulfonamide acting as potent CA IX/XII inhibitor, SLC-0111 (A, Fig. 1),
entered in Phase I clinical trials for the treatment of advanced solid, metastatic
tumors.12 (Fig.1). SLC-0111 A and some of its congeners B-E (Fig. 1) are one of the
many examples of isoform-selective sulfonamide CAIs discovered in the last period
by using the tail approach.11-15
O
H
N
SO2NH2
N
R
H
A: R = 4-F-C6H4 (SLC-0111); KI = 960 nM
B: R = C6F5; KI = 50 nM
C: R= 2(i-Pr-)-C6H4; KI = 3.3nM
D: R = 3-O2N-C6H4; KI = 15 nM
E: R = cyclopentyl; KI = 226 nM
Figure 1. Substituted ureido benzenesulfonamides A-E, some of which possessing
substantial antitumor activity in vitro and/or in vivo, and their hCA II inhibitory
activity.13
The structural requirements for the sulfonamide derivatives to show high affinity
for CAs, are fully understood.11-15 The sulfonamide moiety incorporates the essential
nitrogen atom that is a zinc binding group (ZBG) (as sulfonamidate anion) and binds
to the metal ion within the active site of the enzyme in a stable tetrahedral geometry
of the zinc.11-15 Furthermore, residues Thr 199 and Glu 106 in its neighborhood
participate in hydrogen bonds to both with sulfonamide NH and oxygen atoms. In
addition to that, the organic scaffold that is usually an aliphatic, aromatic, heterocyclic
or sugar moiety interacts both with the hydrophilic and hydrophobic halves of the CA
active site.11-15
Continuing our interest in sulfonamide CAIs, in the present study we report the
design of novel 4-/5-substituted thiazolylbenzenesulfonamides, in an attempt to obtain
selective inhibitors for the tumor-associated isoform CA IX, which were further
explored for their cytotoxic effects on human breast cancer (MCF-7) cell lines, which
overexpress CA IX.16
2. Results and discussion
2.1. Chemistry

The drug design strategy of our work used the SLC-0111 series of compounds
(Fig. 1) as leads, in which the ureido moiety present in A-B was replaced by a five-
membered heterocylic scaffold, the thiazole/ thiazolidinone, which incorporates the
isosteric thioureido moiety present in the lead compounds within the heterocyle. In
fact a recent X-ray/kinetic study of thioureido analog of SLC-0111 showed that this
isosteric replacement may lead to highly effective CAIs for the tumor-associated
isoforms.17 Thus, we designed various strategies for synthesis of -benzenesulfonamide
derivatives incorporating thiazole/ thiazolidine scaffolds, which are shown in
Schemes 1-4. Three key intermediates, were synthesized, the first of which was a
thiazolidinone, i.e., 4- ((4-oxo-4,5-dihydrothiazol-2-yl)amino)benzenesulfonamide 3.
It has been prepared by treating previously prepared chloroacetamide derivative 218
with ammonium thiocyanate in ethanol via an intramolecular cyclization
rearrangement reaction.18 Furthermore, compound 3 underwent a nucleophilic
substitution reaction with phosphorus oxychloride in presence of pyridine to yield 4-
(4-Chloro-thiazol-2-ylamino)-benzenesulfonamide 5.19 Coupling reactions were
conducted between previously prepared diazonium salts of selected primary aromatic
amines (e.g., sulfanilamide, para-chloroaniline, para-aminobenzoic acid or
sulfaguanidine) and C5 active thiazole methylene group of intermediate 3 in order to
synthesize 5-phenylhydrazonothiazol-2-yl-aminobenzenesulfonamides 4a-d. (Scheme
1).
Compound 5, which possesses a reactive chlorine atom in position 4 of the
heterocyclic ring, was refluxed with commercially available hydrazine hydrate in
ethanol to yield 4-((4-hydrazinylthiazol-2-yl)amino)benzenesulfonamide 7. By
refluxing compound 5 with selected amines (sulfanilamide, para-benzoic acid or
para- fluoro aniline) or piperidine in the presence of triethylamine, the 4-
phenylthiazol-2-yl-aminobenzenesulfonamides 6a-6d, were obtained (Scheme 2).

Cl
O
S
O
N
HN
HN
NH2
(b)
(a)
SO2NH2
SO2NH2
SO2NH2
3
1
2
R
NCl
N
a R=SO2NH2
b R=Cl
(d)
(c)
c R=COOH
d R=SO2NC(NH2)2
H
S
R
N
Cl
N
N
HN
S
O
HN
N
SO2NH2
5
SO2NH2
4a-d
4a R=SO2NH2
4b R=Cl
4c R=COOH
4d R=SO2NC(NH2)2
Scheme 1. Synthesis of target compounds 2-5: (a) chloroacetyl chloride, dry DMF, rt,
2 h; (b) ammonium thiocyanate, absolute ethanol, reflux, 1 h; (c) 2N HCl, sodium
acetate, rt, 18 h; (d) phosphorus oxychloride, pyridine, reflux, 3 h.

S
Cl
N
HN
SO2NH2
5
(c)
(b)
(a)
S
S
S
NH
NH
NH2
N
N
N
HN
HN
N
HN
R
SO2NH2
SO2NH2
SO2NH2
6a-c
6d
7
6a R=SO2NH2
6b R=COOH
6c R=F
Scheme 2. Synthesis of target compounds 6 and 7: (a) sulfanilamide or p-amino
benzoic acid or p-fluoro aniline, TEA, DMF, reflux, 24 h; (b) piperidine, TEA, dry
DMF, reflux, 24 h; (c) hydrazine hydrate 99.9%, absolute ethanol, reflux, 24 h.
A multistep reaction was then applied for obtaining sulfonamides 11 (Scheme 1).
The preparation of 2-benzamidoacetic acid 9a or 2-(4-chlorobenzamido)acetic acid 9b
from glycine 8 was done by benzoylation with benzoyl chloride or para-
chlorobenzoyl chloride, followed by Erlenmeyer reaction with appropriate aldehydes
(benzaldehyde, para- chlorobenzaldehyde or para- dimethylaminobenzaldehyde) in
acetic anhydride to yield oxazolone derivatives 10a-f.20 Compounds 10a-f were
reacted with the hydrazinyl derivative 7 and dry dimethylformamide in glacial acetic
acid to give imidazolones 11a-f (Scheme 3).

OH
O
R2
O
O
NH
O
N
OH
(b)
(a)
H2N
O
R1
9a,b
R1
8
9a R1=H
9b R1=Cl
10a-f
10a R1= H, R2= H
10b R1= H, R2=Cl
10c R1= H, R2= N(CH3)2
10d R1= Cl, R2= H
10e R1= Cl, R2= Cl
10f R1= Cl, R2= N(CH3)2
S
NH
NH2
N
HN
(c)
SO2NH2
7
O
O
S
R2
R2
S
HN
N
N
HN
N
N
N
N
N
N
H
H
H2NO2S
H2NO2S
R1
R1
11a-c
11d-f
11a R1= H, R2= H
11b R1= H, R2=Cl
11d R1= Cl, R2= H
11e R1= Cl, R2= Cl
11c R1= H, R2= N(CH3)2
11f R1= Cl, R2= N(CH3)2
Scheme 3. Synthesis of target compounds 9-11: (a) benzoyl chloride or p-chloro
benzoyl chloride, 10% NaOH, rt, 2 h; (b) benzaldehyde or p-chlorobenzaldehyde or p-
dimethylaminobenzaldehyde, fused sodium acetate , acetic anhydride, boiling water
bath, 2 h; (c) glacial acetic acid, dry DMF, boiling water bath, 10 h.
The one pot ternary condensation reaction of aldehydes, ethyl cyanoacetate and
thiourea was first reported by Kambe et al. to yield carbonitriles.21 We applied this
reaction by using equimolar amounts of benzaldehyde/ para- substituted
benzaldehydes and thiourea to obtain the new carbonitriles 13a-c (Scheme 4).
Reaction of carbonitriles 13a-c with intermediates 7 and 5, led to 4-{4-[N'-(5-Cyano-
6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-yl)-hydrazino]-thiazol-2-ylamino}-
benzenesulfonamide derivatives 14a-c and 4-[4-(5-Cyano-6-oxo-4-phenyl-1,6-

dihydro-pyrimidin-2-ylsulfanyl)-thiazol-2-ylamino]-benzenesulfonamide
15a-c,
respectively, by procedures already reported in the literature22 (Scheme 4).
O
CN
HN
OCH2CH3
NC
(a)
S
N
O
H
R
12
13a-c
13a R= H
13b R= Cl
13c R= N(CH3)2
S
NH
NH2
S
Cl
N
HN
N
HN
(b)
(c)
SO2NH2
SO2NH2
7
5
S
S
H
NH
HN
S
O
N
N
H
N
HN
N
HN
O
N
CN
N
CN
SO2NH2
14a-c
SO2NH2
15a-c
R
R
14a R= H
14b R=Cl
14c R= NCH3)2
15a R= H
15b R=Cl
15c R= NCH3)2
Scheme 4. Synthesis of target compounds 12-15: (a) benzaldehyde or p-
chlorobenzaldehyde or p-dimethylaminobenzaldehyde, thiourea, potassium carbonate,
absolute ethanol, reflux, 5h; (b) TEA, dry DMF, reflux, 24 h; (c) TEA, dry DMF,
reflux, 24h.
All compounds were extensively characterized by physico-chemical and
spectroscopic procedures which confirmed their structures (see Experimental for
details).
2.2. CA inhibition
The CA inhibition assay was conducted on human (h) CA isoforms hCA I, II and
IX with a selection of the new sulfonamides reported here, i.e., compounds 4a-d, 6a-

d, 11b, 14a, and acetazolamide (AAZ) as a standard inhibitor, by a stopped- flow CO2
hydrase assay23 and the results, as inhibition constants KI (nM), are shown in Table 1.
Table 1. Inhibition data of human (h) CA isoforms hCA I, II and IX with compounds
4a-d, 6a-d, 11b, 14a and acetazolamide (AAZ)/SLC-0111 as standard inhibitors, by a
stopped-flow CO2 hydrase assay. 23
KI (nM)*
hCA I
hCA
II
hCA
IX
Compound
250
5080
6.4
12.1
25.8
45.0
5.8
7.3
6.4
8.5
7.3
8.8
5.6
6.7
29.2
6.5
AAZ
960
5.5
SLC-0111
4a
4b
4c
7.9
4.1
7.6
4.4
0.85
6.5
0.53
0.41
0.62
0.58
0.47
288
7.1
4d
6a
6.6
6b
6c
3.2
0.84
702
41
6d
11b
14a
* Mean from 3 different assays, errors in the range of ± 10 % of the reported values
(data not shown).
The following structure activity relationship (SAR) is evident from the data of
Table 1:
(i) The slow isoform hCA I was highly inhibited by the investigated sulfonamides,
with KIs ranging between 0.84 and 41 nM, except 11b which was a poor inhibitor (KI
of 702 nM). AAZ and SLC-0111, poorly inhibit this isoform (KI of 250 nM for AAZ
and of > 5 µM for the second standard). As the physiological role of hCA I is poorly
understood to date,11,12 the sulfonamides reported here may be considered interesting

tools to better understand whether hCA I inhibition in some biological systems has
physiologic consequences. For the congeneric subseries of compounds 4, it is
interesting to note that a second primary sulfonamide moiety (in addition to the
sulfanilamide one), as in 4a, does not improve the CA inhibitory properties, whereas
the best inhibitor was 4d, which has a sulfaguanidine such moiety (which is not a
good ZBG for CAIs).11,12 Compounds 6c and 14a, although possessing bulkier
scaffolds compared to derivatives 4, showed similar inhibitory activity, whereas 11b
is a poor inhibitor probably due to the fact that its scaffold became too bulky.
(ii) A similar inhibition profile with these sulfonamides was also observed against
the second cytosolic isoform, hCA II, with compound 11b a poor inhibitor (KI of 288
nM) and the remaining ones highly effective CAIs, with KIs ranging between 0.41 and
5.5 nM. Thus all the substitution patterns present in these derivatives led to highly
effective, low nanomolar or subnanomolar inhibitors. Probably the rigid thiazole
scaffold present in the new derivatives reported here (compared to the
ureido/thioureido-substituted such componds reported earlier,13,17 which have a
greater rotational flexibility) leads to the observed incerase in hCA II inhibitory
properties (but also to a lack of selectivity for inhibiting hCA IX over hCA II, see
Table 1).
(iii) The tumor-associated, transmembrane isoform hCA IX was effectively
inhibited by all the investigated sulfonamides, with KIs ranging between 5.6 and 29.2
nM (AAZ, a compound used clinically which showed antitumor effects in some
models, has a KI of 25 nM). Thus, as for hCA I and II inhibition, all substitution
patterns present in these sulfonamides lead to low nanomolar hCA IX inhibitors,
except 11b, which has a potency similar to that of AAZ, with a KI of 29.2 nM (Table
1).
(iv) The main drawback of these compounds is that they potently inhibit both
cytosolic as well as transmembrane CA isoforms, with no isoform selectivity at all
being present for any member of the series.
2.3. Cytotoxicity studies
The newly obtained compounds were screened for their cytotoxic activity against
human breast cancer cell line MCF-7,24 obtained from the American Type Culture
Collection (ATCC, Rockville, MD), which has been known to be a good predictor of
clinically useful drugs. Concentrations required to cause cytotoxic effects in 50% of

intact cells (IC50), were estimated from graphic plots and shown in Table 2. The
results of table 2 reveal that compounds 4a-d had IC50 ranging between 2.79 and 11.9
μM, whereas 6a-d had IC50 of 12 to 24.3 μM. On the other hand compounds 11a-f,
14a-c and 15a-c had IC50 ranging between 36.2 to and ›150 μM. The reference drug
5-fluorouracil (5-FU) with an IC50 of 11.1 μM, was also included in the tests. One of
the most active compounds, 4a (IC50 of 2.79 μM), was four times as active as 5-FU. A
closer look at compounds 4a-d has revealed that superior cytotoxic activity due to
substitution on the phenyl ring at C5 of thiazole was owed to the sulfamoyl moiety or
its isosteric COOH group as in compounds 4a and 4c, respectively, whereas
substitution with electron withdrawing chloride group (4b) decreased the activity,
which is further decreased if substituted with a bulkier less acidic sulfaguanidino
moiety (4d). As for the C4 ring substitution of the thiazole ring, maximum activity
was observed for the phenylamino derivatives 6a-c and the piperdinyl one 6d,
followed by the heterocyclic 5-membered imidazolohydrazono derivatives 11a-f and
finally the heterocyclic 6-membered dihydropyrimidino derivatives 14a-c and 15a-c.
A closer look at compounds 6a-d has shown that the fluorophenyl derivative 6c
possessed superior cytotoxic activity within this class. Also, it was worthy to highlight
the effect of increasing the bulkiness of the thiazole substituents, which was not in
favor for the cytotoxic activity (poor CA IX inhibitory effects), as proved by
significant cytotoxic activity decline generally noticed in the derivatives of
compounds 11, 14 and 15 if compared to simpler C5 or C4 derivatives of compounds
4 and 6. Investigating other steric concepts, ring contraction of the 4-oxo-pyrimidine
ring 14a-c to imidazol-5(4H)-one gave more promising analogues. Thus, the best CA
IX inhibitors were also the most cytotoxic derivatives, according to data of tables 1
and 2.
Table 2: IC50 values of 5-fluorouracil (5-FU) and all the newly synthesized
compounds against human breast cancer cell line (MCF7).24
Compound
IC50
(μM)
11.1
2.79
10.7
5.45
11.9
21.5
22.1
5-FU
4a
4b
4c
4d
6a
6b

12
24.3
45.87
›150
38.9
›150
136.2
44.5
›150
›150
89.7
›150
86.6
85
6c
6d
11a
11b
11c
11d
11e
11f
14a
14b
14c
15a
15b
15c
Pharmacological screening and
evaluation summarize that
biological
most of the tested compounds have good cytotoxic activity, with compounds 4a-d and
6a-d showing the best cytotoxic activity, comparable to 5-fluorouracil as a reference
drug (Fig. 1), where compounds 4a-c have shown more cytotoxic activity against
breast cancer cell line (MCF-7) than 5-fluorouracil, while compounds 4d and 6a-d
showed slightly less cytotoxic activity against breast cancer cell line (MCF-7) than 5-
fluorouracil. Accordingly, 5-phenylhydrazonothiazol-2-yl-aminobenzenesulfonamide
derivatives 4a-d and 4-phenylthiazol-2-yl-aminobenzenesulfonamide derivatives 6a-d
could be considered to be useful building blocks for future research aiming at the
synthesis of effective cytotoxic agents.
Fig. 1. The order of potency for the most active compounds 4a-d and 6a-d,
compared to 5-fluorouracil as a reference drug, showing the in- vitro screening IC50
values.
3. Conclusions

A series of 4-(thiazol-2-ylamino)-benzenesulfonamides was synthesized and screened
for their CA inhibitory and cytotoxic activity on human breast cancer cell line MCF-7.
hCA isoforms I, II and IX were included in the study. The new sulfonamides showed
excellent inhibition of all three isoforms, with KIs in the range of 0.84-702 nM against
hCA I, of 0.41 – 288 nM against hCA II and of 5.6 – 29.2 nM against the tumor-
associated hCA IX, a validated anti-tumor target, with a sulfonamide (SLC-0111) in
phase I clinical trials for the treatment of hypoxic, metastatic solid tumors over
expressing CA IX. The new compounds showed micromolar inhibitory of growth
efficacy against breast cancer MCF-7 cell lines.
4. Experimental
4.1. Chemistry
All chemicals were purchased from VWR International Merck, Germany or
Sigma-Aldrich and used without further purification. Melting Points were carried out
by open capillary tube method using IA 9100MK-Digital Melting Point Griffin
Apparatus and they are uncorrected. Elemental Microanalysis was carried out at the
Regional Micro Analytical Center for Mycology and Biotechnology, Al-Azhar
University. Infrared spectra were recorded on BRUKER Vector 22 (Japan) and
expressed in wave number (cm-1) using potassium bromide discs at the
1
13
microanalytical Center, Faculty of Science, Cairo University. H-NMR and C-
1
NMR Spectra were recorded on a Bruker Avance III 400 MHz for H and 100
MHz for 13C (Bruker AG, Switzerland) with BBFO Smart Probe and Bruker
400 AEON Nitrogen-Free at the microanalytical Center, Faculty of Pharmacy,
Beni Suef University. Chemical shifts were expressed in δ units and were related to
that of the solvents. As for the proton magnetic resonance, D2O was carried out for
NH and OH exchangeable protons. Mass Spectra were recorded using Fennigan
MAT, SSQ 7000, Mass spectrometer, at 70 eV (EI) at the microanalytical
Center, Faculty of Science, Cairo University and Waters Micromass Q-Tof
Micro mass spectrometer (ESI) and Waters Acquity Ultra Performance LC with
ZQ detector in ESI mode. All the compounds were named according to the
IUPAC system using CS Chem. Draw Ultra version 12.0. Compounds 2,18 3,18
5,19 9,20 10a-f,20 13a-c,21 were prepared according to reported methods, while

compounds 14a-c and 15a-c were prepared according to modified procedures from
reported methods.22
General procedure for the synthesis of compounds 4a-d.
A solution of sodium nitrite (0.18 g, 2.56 mmol) in water (3 mL) was added drop
wise to a stirred and previously cooled solution of the appropriate amino derivative
(2.56 mmol) in 2N hydrochloric acid (3 mL) in an ice bath. The mixture was stirred
for one hour at 0-5°C. Then a solution of 3 (0.7 g, 2.56 mmol) in 2N hydrochloric
acid (6 mL) was added and pH 7 was adjusted with sodium acetate and left to stir for
18 h. at room temperature. The product was then filtered, dried, and recrystallized
from dioxane.
4-(2-(4-oxo-2-((4-sulfamoylphenyl)amino)thiazol-5(4H)-ylidene)hydrazinyl)
benzenesulfonamide (4a). Yield 70%; m.p. 260-262 °C; IR (KBr) (ʋmax,/ cm-1): 3468,
1
3280, 3109 (2NH2, 2NHs), 3025 (CH aromatic), 1696 (C=O), 1396, 1145 (SO2); H-
NMR (CDCl3, 400 MHz) δ (ppm): 7.17 (2H, s, ex, NH2), 7.35-7.88 (4H, m, Ar-Hs),
7.46 (2H, s, ex, NH2), 7.98 (2H, d, Ar-Hs, J = 8.2 Hz), 8.11 (d, 2H, Ar-Hs, J = 8.2
Hz), 10.05, 10.73 (2H, 2s, ex, 2 NH); 13C NMR (DMSO- d6, 100 MHz) δ (ppm):
113.8, 126.8, 127.7, 127.9, 129.8, 137.1, 137.9 and 144.3 (aromatic Cs), 146.87
(thiazole C5), 150.5 (thiazole C2), 162.1 (C=O). MS m/z: 454 (M+). Anal. Calcd. For
C15H14N6O5S3 (454.50): C, 39.64; H, 3.10; N, 18.49. Found: C, 39.89; H, 3.17; N,
18.19.
4-((5-(2-(4-chlorophenyl)hydrazono)-4-oxo-4,5-dihydrothiazol-2-
yl)amino)benzenesulfonamide (4b). Yield 65%. m.p. 250-252 °C; IR (KBr) (ʋmax,/ cm-
1): 3308, 3211 (NH2, 2NHs), 3090 (CH aromatic),1639 (C=O), 1326, 1155 (SO2); 1H
NMR (DMSO-d6, 400 MHz) δ (ppm): 7.08-7.78 (4H, m, Ar-Hs), 7.50 (2H, s, ex,
NH2), 7.86 (2H, d, Ar-Hs, J = 8.6 Hz), 8.01 (2H, d, Ar-Hs, J = 8.6 Hz), 10.01, 10.53
(2H, 2s, ex, 2NHs); MS m/z: 409 (M+). Anal. Calcd. For C15H12ClN5O3S2 (409.87): C,
43.96; H, 2.95; N, 17.09. Found: C, 44.19; H, 2.94; N, 17.22.
4- (2- (4- oxo- 2- ((4 -sulfamoylphenyl) amino )thiazol- 5(4H)- ylidene
)hydrazinyl) benzoicacid (4c). Yield 60%. m.p. 248-250 °C; IR (KBr) (ʋmax,/ cm-1):
3356 (OH), 3262, 3228 (NH2, 2NHs), 3065 (CH aromatic), 1720,1 607 (2C=O), 1375,
1160 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 6.96-7.66 (4H, m, Ar-Hs), 7.20

(1H, s, ex, NH), 7.49 (2H, s, ex, NH2), 7.77 (2H, d, Ar-Hs, J = 8.8 Hz), 7.96 (2H, d,
Ar-Hs, J = 8.8 Hz), 10.06 (1H, s, ex, NH), 11.00 (s, 1H, ex, OH); MS m/z: 419 (M+).
Anal. Calcd. For C16H13N5O5S2 (419.43): C, 45.82; H, 3.12; N, 16.70. Found: C,
46.03; H, 3.17; N, 16.87.
N-(diaminomethylene)-4-(2-(4-oxo-2-((4-sulfamoylphenyl)amino)thiazol-5(4H)-
ylidene)hydrazinyl)benzenesulfonamide (4d). Yield 75%; m.p. 252-254 °C. IR (KBr)
(ʋmax,/ cm-1): 3390, 3329, 3272,3141 (3NH2, 2NHs), 3039 (CH aromatic), 1611
(C=O), 1341, 1163 (SO2); 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 6.67 (4H, s, ex, 2
NH2), 7.33-7.75 (4H, m, Ar-Hs), 7.49 (2H, s, ex, NH2), 7.85 (2H, d, Ar-Hs, J = 8.0
Hz), 7.95 (2H, d, Ar-Hs, J = 8.0 Hz), 10.04, 10.71 (2H, 2s, ex, 2NHs); MS m/z:
496.31 (M+). Anal. Calcd. For C16H16N8O5S3 (496.54): C, 38.70; H, 3.25; N, 22.57.
Found: C, 38.88; H, 3.23; N, 22.31.
General procedure for the synthesis of compounds 6a-d. The appropriate
amine or piperidine (2.1 mmol) and triethylamine (5 mL) were added to a solution of
5 (0.58 g, 2 mmol) in dry DMF (10 mL). The mixture was heated under reflux for 24
h. Then the solution was poured onto ice water; the product was collected, dried, and
recrystallized from ethanol.
4, 4ˊ-(Thiazole-2,4-diylbis(azanediyl))dibenzenesulfonamide (6a). Yield 50%;
m.p. 240-242 °C; IR (KBr) (ʋmax,/ cm-1): 3255-3098 (2NH2, 2NHs), 3010 (CH
1
aromatic), 1321, 1156 (SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 5.82 (1H, s,
ex, NH), 6.89 (1H, s, thiazole CH), 6.98-7.82 (8H, m, Ar-Hs), 7.23 (2H, s, ex, NH2),
10.87 (1H, s, ex, NH), 11.60 (2H, s, ex, NH2); 13C NMR (DMSO- d6, 100 MHz)
δ(ppm): 112.3 (thiazole C5), 121.2, 126.7, 127.4, 127.7, 129.7, 133.1, 133.4, 135.2
and 140.1 (aromatic Cs), 152.8 (thiazole C2); MS m/z: 425(M+). Anal. Calcd. For
C15H15N5O4S3 (425.51): C, 42.34; H, 3.55; N, 16.46. Found: C, 42.49; H, 3.52; N,
16.62.
4-((2-((4-Sulfamoylphenyl)amino)thiazol-4-yl)amino)benzoic acid (6b). Yield
55%; m.p. 237-239 °C; IR (KBr) (ʋmax,/ cm-1): 3294-3100 (OH, NH2, 2NHs), 3045
1
(CH aromatic), 1710 (C=O), 1338, 1159 (SO2); H NMR (DMSO-d6, 400 MHz) δ
(ppm): 6.50 (1H, s, thiazole CH), 7.09-7.72 (8H, m, Ar-Hs), 7.10 (2H, s, ex, NH2),

9.64 (2H, s, ex, 2NHs), 13.01 (1H, s, ex, OH); MS m/z: 390(M+). Anal. Calcd. For
C16H14N4O4S2 (390.44): C, 49.22; H, 3.61; N, 14.35. Found: C, 49.51; H, 3.67; N,
14.51.
4-((4-((4-Fluorophenyl)amino)thiazol-2-yl)amino)benzenesulfonamide (6c). Yield
60%; m.p. 220-222 °C; IR (KBr) (ʋmax,/ cm-1): 3425-3303(NH2, 2NHs), 3077 (CH
1
aromatic), 1308, 1154 (SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.10 (1H, s,
thiazole CH), 7.15-8.03 (8H, m, Ar-Hs), 7.28 (2H, s, ex, NH2), 8.79, 10.93 (2H, 2s,
ex, 2NHs); MS m/z: 364 (M+). Anal. Calcd. For C15H13FN4O2S2 (364.42): C, 49.44;
H, 3.60; N, 15.37. Found: C, 49.68; H, 3.71; N, 15.44.
4-((4-(Piperidin-1-yl)thiazol-2-yl)amino)benzenesulfonamide (6d). Yield 49%;
m.p. 245-247 °C; IR (KBr) (ʋmax,/ cm-1): 3335 (NH2, NH), 3085 (CH aromatic),1315,
1
1150 (SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 1.54-1.68 (m, 2H, piperidine),
2.89-3.17 (m, 4H, piperidine), 3.49-3.62 (m, 4H, piperidine), 7.30 (2H, s, ex, NH2),
7.75-8.15 (5H, m, Ar-Hs+ thiazole CH), 10.94 (1H, s, ex, NH); MS m/z: 338 (M+).
Anal. Calcd. For C14H18N4O2S2 (338.45): C, 49.68; H, 5.36; N, 16.55. Found: C,
49.87; H, 5.44; N, 16.43.
General procedure for the synthesis of compound 7. Hydrazine hydrate (3.16 g,
3.1 mL, 100 mmol) was added to a suspension of 5 (2.9 g, 10 mmol) in ethanol (35
mL). The reaction mixture was heated under reflux for 24 h. Then the mixture was
cooled, the product collected, washed with ethanol, dried and recrystallized from
dioxane.
4-((4-Hydrazinylthiazol-2-yl)amino)benzenesulfonamide (7). Yield: 66%. m.p.:
290-292°C. IR (KBr) (ʋ, cm-1): 3355-3058 (2NH2, 2NHs), 3005 (CH aromatic), 1321,
1
1156 (SO2). H-NMR (DMSO-d6- 400 MHz) δ (ppm): 7.09 (2H, s, ex, NH2), 7.52-
7.66 (m, 5H, Ar-H), 9.62, 13.00 (2H, s, ex, 2NHs), 13.25 (2H, s, ex, NH2). MS m/z:
285 (M+). Anal. Calcd. For C9H11N5O2S2 (285.35): C, 37.88; H, 3.89; N, 24.54.
Found: C, 37.66; H, 4.11; N, 24.43.
General procedure for the synthesis of compounds 11a-f. A mixture of
equimolar amounts of compound 7 and the appropriate oxazolone 10a-f (10 mmol) in
glacial acetic acid (10 mL) containing few drops of dry DMF was heated in boiling

water bath at 100 °C for 10 h. Then the solution was poured onto ice water; the
product was collected, dried, and recrystallized from dioxane.
4-((4-((4-benzylidene-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)amino)thiazol-2-yl)amino) benzenesulfonamide (11a). Yield: 49%; m.p.: 258-260
°C;IR (KBr) (ʋmax,/ cm-1): 3452, 3263, 3192 (NH2, 2NHs), 3087 (CH aromatic), 1644
1
(C=O), 1338, 1167 (SO2); H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.11 (1H, s,
thiazole CH), 7.17 (2H, s, ex, NH2), 7.40-7.71 (7H, m, Ar-Hs+ methine H), 7.91 (2H,
d, Ar-Hs, J = 8.2 Hz), 8.12 (2H, d, Ar-Hs, J = 8.2 Hz), 8.21 (2H, d, Ar-Hs, J = 8.6
Hz), 8.34 (2H, d, Ar-Hs, J = 8.6 Hz), 9.73, 10.05 (2H, 2s, ex, 2NHs); 13C NMR
(DMSO- d6, 100 MHz) δ (ppm): 113.1 (thiazole C5), 113.9 (methine C), 117.4- 150.7
(aromatic Cs), 153.5 (pyrazolone C2), 162.1 (thiazole C2), 168.1 (C=O); MS m/z: 516
(M+). Anal. Calcd. For C25H20N6O3S2 (516.59): C, 58.12; H, 3.90; N, 16.27. Found:
C, 58.40; H, 3.94; N, 16.29.
4-((4-((4-(chlorobenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)amino)thiazol-2-yl)amino)benzenesulfonamide (11b). Yield: 53%; m.p.: 262-264
°C; IR (KBr) (ʋmax,/ cm-1): 3412, 3216 (NH2, 2NHs), 3095 (CH aromatic), 1678
1
(C=O), 1389, 1138 (SO2); H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.08 (1H, s,
thiazole CH), 7.11 (2H, s, ex, NH2), 7.41-7.93 (10H, m, Ar-Hs+ methine H), 8.13
(2H, d, Ar-Hs, J = 8.0 Hz), 8.33 (2H, d, Ar-Hs, J = 8.0 Hz), 9.69, 10.03 (2H, 2s, ex,
2NHs); MS m/z: 551 (M+). Anal. Calcd. For C25H19ClN6O3S2 (551.04): C, 54.49 ; H,
3.48; N, 15.25. Found: C, 54.67; H, 3.51; N, 15.37.
4-((4-((4-(4-(dimethylamino)benzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-
imidazol-1-yl)amino)thiazol-2-yl)amino)benzenesulfonamide (11c). Yield: 55%; m.p.:
270-272 °C; IR (KBr) (ʋmax,/ cm-1): 3417, 3262 (NH2, 2NHs), 3060 (CH aromatic),
2929 (CH Aliphatic) 1634 (C=O), 1320, 1154 (SO2); 1H-NMR (DMSO-d6, 400 MHz)
δ (ppm): 3.17 (6H, s, 2CH3), 6.58 (1H, s, thiazole CH), 7.03 (2H, s, ex, NH2), 7.24-
7.99 (10H, m, Ar-Hs+ methine H), 8.11 (2H, d, Ar-Hs, J = 8.6 Hz), 8.24 (2H, d, Ar-
Hs, J = 8.6 Hz), 10.31, 10.46 (2H, 2s, ex, 2NHs); MS m/z: 559 (M+). Anal. Calcd. For
C27H25N7O3S2 (559.66): C, 57.94; H, 4.50; N, 17.52. Found: C, 58.18 ; H, 4.40; N,
17.73.
4-((4-((4-benzylidene-2-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-imidazol-1-
yl)amino)thiazol-2-yl)amino)benzenesulfonamide (11d). Yield: 48%; m.p.: 249-251
°C; IR (KBr) (ʋmax,/ cm-1): 3450 (NH2, 2NHs), 3075 (CH aromatic), 1644 (C=O),

1
1340, 1171 (SO2); H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.65 (1H, s, thiazole
CH), 7.23 (2H, s, ex, NH2), 7.32-7.89 (10H, m, Ar-Hs+ methine H), 8.22 (2H, d, Ar-
Hs, J = 8.8 Hz), 8.34 (2H, d, Ar-Hs, J = 8.8 Hz), 9.98, 10.42 (2H, 2s, ex, 2NHs); MS
m/z: 551 (M+). Anal. Calcd. For C25H19ClN6O3S2 (551.04): C, 54.49; H, 3.48 ; N,
15.25. Found: C, 54.62; H, 3.46; N, 15.41.
4-((4-((4-(4-chlorobenzylidene)-2-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-
imidazol-1-yl)amino)thiazol-2-yl)amino)benzenesulfonamide (11e). Yield: 51%; m.p.:
268-270 °C; IR (KBr) (ʋmax,/ cm-1): 3417 (NH2, 2NHs), 3093 (CH aromatic), 1627
1
(C=O), 1323, 1151 (SO2); H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.08 (1H, s,
thiazole CH), 7.13-8.07 (9H, m, Ar-Hs+ methine H), 7.55 (2H, s , ex, NH2), 8.14
(2H, d, Ar-Hs, J = 8.2 Hz), 8.38 (2H, d, Ar-Hs, J = 8.2 Hz), 9.95, 12.66 (2H, 2s, ex,
2NHs); MS m/z: 585 (M+). Anal. Calcd. For C25H18Cl2N6O3S2 (585.48): C, 51.29; H,
3.10; N, 14.35. Found: C, 51.57; H, 3.18; N, 14.52.
4-((4-((2-(4-chlorophenyl)-4-(4-(dimethylamino)benzylidene)-5-oxo-4,5-dihydro-
1H-imidazol-1-yl)amino)thiazol-2- yl)amino)benzenesulfonamide (11f). Yield: 45%;
m.p.: 253-255 °C; IR (KBr) (ʋmax,/ cm-1): 3427 (NH2, 2NHs), 3065 (CH aromatic),
1
2933 (CH Aliphatic), 1628 (C=O), 1325, 1155 (SO2); H-NMR (DMSO-d6- 400
MHz) δ (ppm): 3.08 (6H, s, 2CH3), 6.85 (1H, s, thiazole CH), 7.12 (2H, s, ex, NH2),
7.24-8.00 (7H, m, Ar-Hs+ methine H), 8.11 (2H, d, Ar-Hs, J = 8.4 Hz), 8.23 (2H, d,
Ar-Hs, J = 8.4 Hz), 8.34 (2H, d, Ar-Hs, J = 8.0 Hz), 9.38, 11.48 (2H, s, ex, 2NHs);
MS m/z: 594 (M+). Anal. Calcd. For C27H24ClN7O3S2 (594.11): C, 54.58; H, 4.07; N,
16.50. Found: C, 54.89; H, 4.03; N, 16.78.
General procedure for the synthesis of compounds 14a-c. A mixture of
equimolar amounts of compound 7 and the appropriate 2-thioxo-tetrahydropyrimidine
derivative 13a-c (1.6 mmol) in dry DMF (10 mL) containing few drops of
triethylamine was heated under reflux for 24 h. Then the solution was poured onto ice
water; the product was collected, dried and recrystallized from DMF/water.
4-((4-(2-(5-Cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl)hydrazinyl)thiazol-
2-yl)amino)benzenesulfonamide (14a). Yield 45%; m.p. 270-272 °C; IR (KBr) (ʋ, cm-
1): 3329, 3108 (NH2, NHs), 3082 (CH aromatic), 2203 (C≡N), 1680 (C=O), 1324,
1
1156 (SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.07 (2H, s, ex, 2NHs), 7.20
(2H, s, ex, NH2), 7.59-7.89 (6H, m, Ar-Hs+ thiazole CH), 8.00 (2H, d, Ar-Hs, J =

8.2 Hz), 8.16 (2H, d, Ar-Hs, J = 8.2 Hz), 9.52, 11.89 (2H, 2s, ex, 2NHs); MS m/z: 480
(M+). Anal. Calcd. For C20H16N8O3S2 (480.52): C, 49.99; H, 3.36; N, 23.32. Found: C,
50.12; H, 3.38; N, 23.67.
4-((4-(2-(4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-
yl)hydrazinyl)thiazol-2-yl)amino)benzenesulfonamide (14b). Yield 40%; m.p. 260-262
°C; IR (KBr) (ʋmax,/ cm-1): 3426 (NH2, NHs), 3098 (CH aromatic), 2213 (C≡N), 1610
1
(C=O), 1324, 1154 (SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.25 (2H, s, ex,
NH2), 7.42-8.12 (5H, m, Ar-Hs+ thiazole CH), 8.25 (2H, d, Ar-Hs, J = 8.4 Hz), 8.33
13
(2H, d, Ar-Hs, J = 8.4 Hz), 10.28, 11.16 (2s, 4H, ex, 4NHs); C NMR (DMSO- d6,
100 MHz) δ (ppm): 110.1 (pyrimidinone C5), 113.1 (thiazole C5), 113.1-151.9
(aromatic Cs+CN), 152.3 (pyrimidinone C2), 153.5 (thiazole C2), 168.1 (C=O), 170.1
(of pyrimidinone C4); MS m/z: 514 (M+). Anal. Calcd. For C20H15ClN8O3S2: (514.97):
C, 46.65; H 2.94; N, 21.76. Found: C, 46.87; H, 2.92; N, 21.99.
4-((4-(2-(5-Cyano-4-(4-(dimethylamino)phenyl)-6-oxo-1,6-dihydropyrimidin-2-
yl)hydrazinyl) thiazol-2-yl)amino)benzenesulfonamide (14c). Yield 55%; m.p. 275-
277 °C; IR (KBr) (ʋmax,/ cm-1): 3429 (NH2, NHs), 3090 (CH aromatic), 2932 (CH
1
Aliphatic), 2206 (C≡N), 1623 (C=O), 1326, 1153 (SO2); H NMR (DMSO-d6, 400
MHz) δ (ppm): 3.03 (6H, s, 2CH3), 6.81-7.95 (5H, m, Ar-Hs+ thiazole CH), 7.49 (2H,
s, ex, NH2), 8.05 (2H, d, Ar-Hs, J = 8.0 Hz), 8.17 (2H, d, Ar-Hs, J = 8.0 Hz), 10.02,
10.55, 12.95 (4H, 3s, ex, 4 NHs); MS m/z: 523 (M+). Anal. Calcd. For C22H21N9O3S2
(523.59): C, 50.47; H, 4.04; N, 24.08. Found: C, 50.62; H, 4.11; N, 24.46.
General procedure for the synthesis of compounds 15a-c. A mixture of
compound 5 (2.9 g, 10 mmol), the appropriate 2-thioxo-tetrahydropyrimidine
derivative XIIIa-c (10 mmol) and triethylamine (2 mL) was heated under reflux in
dry DMF (10 mL) for 24 h. Then the solution was poured onto ice water; the product
was collected, dried and recrystallized from DMF/water.
4-((4-((5-Cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl)thio)thiazol-2-
yl)amino) benzenesulfonamide (15a). Yield 60%; m.p. 255-257 °C; IR (KBr) (ʋmax,/
cm-1): 3477, 3382, 3318 (NH2, 2NHs), 3075 (CH aromatic), 2236 (C≡N), 1626
1
(C=O), 1309, 1150 (SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.21 (1H, s,

thiazole CH), 7.39-8.09 (5H, m, Ar-Hs), 7.66 (2H, s, ex, NH2), 8.18 (2H, d, Ar-Hs, J
= 8.4 Hz), 8.35 (2H, d, Ar-Hs, J = 8.4 Hz), 11.95, 12.65 (2H, 2s, ex, 2NHs); MS m/z:
482 (M+). Anal. Calcd. For C20H14N6O3S3 (482.56): C, 49.78; H, 2.92; N, 17.42.
Found: C, 49.97; H, 2.90; N, 17.58.
4-((4-((4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)thiazol-
2-yl)amino)benzenesulfonamide (15b). Yield 65%; m.p. 260-262 °C; IR (KBr) (ʋmax,/
cm-1): 3444, 3340, 3233 (NH2, 2NHs), 3055 (CH aromatic), 2165 (C≡N), 1629
1
(C=O), 1374, 1158 (SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.11 (1H, s,
thiazole CH), 7.22-8.06 (4H, m, Ar-Hs), 7.37 (2H, s, ex, NH2), 8.24 (2H, d, Ar-Hs, J
= 8.2 Hz), 8.37 (2H, d, Ar-Hs, J = 8.2 Hz),10.85, 11.20 (2H, 2s, ex, 2NHs); 13C NMR
(DMSO- d6, 100 MHz) δ (ppm): 113.1 (pyrimidinone C5), 113.8 (thiazole C5),
117.37- 146.8 (aromatic Cs+ CN group), 150.6 (thiazole C2), 153.5 (pyrimidinone
C2), 162.8 (C=O), 168.1 (pyrimidinone C4); MS m/z: 517 (M+). Anal. Calcd. For
C20H13ClN6O3S3 (517.00): C, 46.46; H, 2.53; N, 16.26. Found: C, 46.55; H, 2.56; N,
16.08.
4-((4-((5-Cyano-4-(4-(dimethylamino)phenyl)-6-oxo-1,6-dihydropyrimidin-2-
yl)thio)thiazol-2-yl)amino)benzenesulfonamide (15c). Yield 50%; m.p. 245-247 °C;
IR (KBr) (ʋmax,/ cm-1): 3420 (NH2, 2NHs), 3049 (CH aromatic), 2930 (CH Aliphatic),
1
2212 (C≡N), 1626 (C=O), 1321, 1153 (SO2); H NMR (DMSO-d6, 400 MHz) δ
(ppm): 3.17 (6H, s, 2CH3), 7.28 (2H, s, ex, NH2), 7.34-7.95 (5H, m, Ar-Hs+ thiazole
CH), 8.14 (2H, d, Ar-Hs, J = 8.0 Hz), 8.27 (2H, d, Ar-Hs, J = 8.0 Hz), 11.92 (2H, s,
ex, 2NHs); MS m/z: 525 (M+). Anal. Calcd. For C22H19N7O3S3 (525.63): C, 50.27; H,
3.64; N, 18.65. Found: C, 50.53; H, 3.78; N, 18.26.
4.2. Carbonic Anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the
CA catalyzed CO2 hydration activity.56 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) as buffer, and 20 mM Na2SO4 for maintaining constant the ionic
strength (this anion is not inhibitory and has a KI> 200 mM against these enzymes),30
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 measurement at least six
traces of the initial 5-10% of the reaction have been used for determining the initial

velocity, working with 10-fold decreasing inhibitor concentrations ranging
between0.1 nM and 10-100 µM (depending on the inhibitor potency, but at least 5
points at different inhibitor concentrations were employed for determining the
inhibition constants). 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 0.1 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 formation of the
E-I complex. The inhibition constants were obtained by non-linear least-squares
methods using the Cheng-Prusoff equation, and represent the mean from at least three
different determinations. The human isoforms hCA I, II and IX were recombinant
enzymes produced as described earlier in our laboratory. 25-28
4.3. Cytotoxicity studies
In vitro screening was carried out in accordance with the Regional Center for
Mycology and Biotechnology, Al-Azhar University, Egypt. All the targeted
compounds were screened for their cytotoxic activity against human breast cancer cell
line MCF-7, obtained from the American Type Culture Collection (ATCC, Rockville,
MD), which has been known to be a good predictor of clinically useful drugs. IC50,
the concentration required to cause toxic effects in 50% of intact cells, was estimated
from graphic plots, recorded and tabulated in table 2. In this protocol,24 the cells were
grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum
and 50μg/ ml gentamycin. The cells were maintained at 37ᵒC in a humidified
atmosphere with 5% CO2 and were subcultured two to three times a week. The
cytotoxic activity was evaluated on tumor cells. The cells were grown as monolayers
in growth RPMI-1640 medium supplemented with 10% inactivated fetal calf serum
and 50μg/ml gentamycin. The monolayers of 10,000 cells adhered at the bottom of the
wells in a 96-well microtiter plate incubated for 24h at 37ᵒC in a humidified incubator
with 5% CO2. The monolayers were then washed with sterile phosphate buffered
saline (0.01 M pH 7.2) and simultaneously the cells were treated with 100 μl from
different dilutions of tested sample in fresh maintenance medium and incubated at 37
ᵒC. A control of untreated cells was made in the absence of tested sample. A positive
control containing 5- Fluorouracil (5-FU) drug was also tested as reference drug for
comparison. Six wells were used for each concentration of the test sample. Every 24h

the observation under the inverted microscope was made. The number of the
surviving cells was determined by staining the cells with crystal violet,24 followed by
cell lysing using 33% glacial acetic acid and read the absorbance at 590nm using
ELISA reader (SunRise, TECAN, Inc, USA) after well mixing. The absorbance
values from untreated cells were considered as 100% proliferation. The number of
viable cells was determined using ELISA reader as previously mentioned before and
the percentage of viability was calculated as [1-(ODt/ODc)]x100% where ODt is the
mean optical density of wells treated with the tested sample and ODc is the mean
optical density of untreated cells. 50% inhibitory concentration (IC50), the
concentration required to cause toxic effects in 50% of intact cells, was estimated
from graphic plots.
Acknowledgements
The Authors would like to thank Prof. Dr. Hanan H. Georgey, Department of
pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Egypt, for giving
time and effort to add fruitful scientific comments to the research and the analytical
chemistry stuff at Regional microanalytical Center for Mycology & Biotechnology,
Al-Azhar University for carrying out the in vitro screening. Work from Supuran
laboratory was financed by two EU grants (Dynano and Metoxia).

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Synthesis of 4-(thiazol-2-yl)amino-benzenesulfonamides with potent carbonic
anhydrase I, II and IX inhibitory activity and cytotoxic effects against breast
cancer cell lines
Nagwa M. Abdel Gawad,a Noha H. Amin,*b Mohammed T. Elsaadi,b Fatma
M.M. Mohamed,b Andrea Angeli,c Viviana De Luca,d Clemente Capasso,d and
Claudiu T. Supuran*c