Inhibition of carbonic anhydrases from the extremophilic bacteria Sulfurihydrogenibium yellostonense (SspCA) and S. azorense (SazCA) with a new series of sulfonamides incorporating aroylhydrazone-, [1,2,4]triazolo[3,4-b][1, 3,4]thiadiazinyl- or 2-(cyanophenylmethylene)-1,3,4-thiadiazol-3(2H)-yl moieties

Article abstract of DOI:10.1016/j.bmc.2013.11.042

A series of new sulfonamides was prepared starting from 2-oxo-N′-(4-sulfamoylphenyl)-propanehydrazonoyl chloride, a sulfanilamide derivative, which was reacted with aroylhydrazides, amines, or thiols. A library of derivatives incorporating aroylhydrazone, [1,2,4]triazolo[3,4-b][1,3,4] thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thiadiazol-3(2H)-yl moieties was thus synthesized. The new compounds were investigated as inhibitors of four α-carbonic anhydrases (CAs, EC 4.2.1.1), the human (h) isoforms hCA I and II, and the bacterial ones recently isolated from the extremophilic bacteria Sulfurihydrogenibium yellostonense (SspCA) and Sulfurihydrogenibium azorense (SazCA). Low nanomolar activity was observed against hCA II (K_Is of 0.56-17.1 nM) whereas hCA I was less inhibited by these compounds (K _Is of 86.4 nM-32.8 μM). The bacterial CAs were also effectively inhibited by these derivatives (K_Is in the range of 0.77-234 nM against SazCA, and of 6.2-89.1 against SspCA, respectively), with several low nanomolar/subnanomolar inhibitors detected against both of them. As SspCA and SazCA are among the most thermostable and catalytically active CAs, it is of interest to find modulators of their activity for potential biotechnologic applications.

Full text of DOI:10.1016/j.bmc.2013.11.042

Bioorganic & Medicinal Chemistry 22 (2014) 141–147
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of carbonic anhydrases from the extremophilic bacteria
Sulfurihydrogenibium yellostonense (SspCA) and S. azorense (SazCA)
with a new series of sulfonamides incorporating aroylhydrazone-,
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenylmethyl-
ene)-1,3,4-thiadiazol-3(2H)-yl moieties
Ahmed M. Alafeefy a, , Hatem A. Abdel-Aziz b,c, Daniela Vullo d, Abdul-Malek S. Al-Tamimi a,
⇑
Nabila A. Al-Jaber e, Clemente Capasso f, Claudiu T. Supuran d,g,
⇑
a Department of Pharmaceutical Chemistry, College of Pharmacy, Salman bin Abdulaziz University, PO Box 173, Alkharj 11942, Saudi Arabia
b Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia
c Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
d Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
e Chemistry Department, Faculty of Science, King Saud University, Riyadh, Saudi Arabia
f Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italy
g Università degli Studi di Firenze, Neurofarba Dept., 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
A series of new sulfonamides was prepared starting from 2-oxo-N0-(4-sulfamoylphenyl)-propanehyd-
razonoyl chloride, a sulfanilamide derivative, which was reacted with aroylhydrazides, amines, or thiols.
A library of derivatives incorporating aroylhydrazone, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cya-
nophenyl-methylene)-1,3,4-thiadiazol-3(2H)-yl moieties was thus synthesized. The new compounds
Article history:
Received 14 October 2013
Revised 18 November 2013
Accepted 21 November 2013
Available online 1 December 2013
were investigated as inhibitors of four a-carbonic anhydrases (CAs, EC 4.2.1.1), the human (h) isoforms
hCA I and II, and the bacterial ones recently isolated from the extremophilic bacteria Sulfurihydrogenibium
yellostonense (SspCA) and Sulfurihydrogenibium azorense (SazCA). Low nanomolar activity was observed
against hCA II (KIs of 0.56–17.1 nM) whereas hCA I was less inhibited by these compounds (KIs of
Keywords:
Carbonic anhydrase
Alpha-class enzyme
Sulfonamide
86.4 nM–32.8 lM). The bacterial CAs were also effectively inhibited by these derivatives (KIs in the range
of 0.77–234 nM against SazCA, and of 6.2–89.1 against SspCA, respectively), with several low nanomolar/
subnanomolar inhibitors detected against both of them. As SspCA and SazCA are among the most
thermostable and catalytically active CAs, it is of interest to find modulators of their activity for potential
biotechnologic applications.
Sulfurihydrogenibium spp.
Extremophiles
Inhibition study
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The CA inhibitors (CAIs) of the sulfonamide type constitute one
of the most important categories of such drugs.1a,11a Sulfonamide
Primary sulfonamides of the type RSO2NH2, where R is an ali-
phatic, aromatic or heterocyclic moiety, constitute an important
class of drugs, comprising several types of pharmacological agents
with anti-carbonic anhydrase (CA, EC 4.2.1.1),1–3 antibacterial,1b,4–7
antiviral,8,9 diuretic,8a,10–12 protease inhibitory,13a cyclooxigenase 2
(COX2)13b inhibitory and anticancer6a,13c,d activities among others.
It is interesting to note that the toolbox of medicinal chemists is
composed by a fairly small number of privileged building blocks,
recurrently found in potent drug molecules, and the SO2NH2 moi-
ety is one of them.1–13
CAIs were and are still used as antiglaucoma systemically acting
drugs,14 but they are employed for the treatment of other condi-
tions, such as neuromuscular disease,15 altitude sickness,16 or as
diuretics.8a,17 Furthermore, antiepileptic drugs,18 antipsychotic
acting as dopamine receptor antagonists,19 antiobesity agents20
or antitumor13d/antimetastatic21 primary sulfonamides have been
reported or are in clinical use. However targeting CAs in bacterial
pathogens with sulfonamide CAIs started to be investigated only
recently.1b,4–7
We have recently reported the discovery of two a
-CAs,4a,22 from
two extremophilic bacteria, that is, Sulfurihydrogenibium yellow-
stonense YO3AOP1 (the enzyme was denominated SspCA)4a as well
as the one from related species, Sulfurihydrogenibium azorense,22
SazCA, which is one of the most efficient enzymes known to date
(and surely the CA with the highest catalytic activity for the
⇑
Corresponding authors. Tel.: +966 50 706 9896 (A.M.A.); tel.: +39 055 457 3005;
fax: +39 055 4573385 (C.T.S.).
E-mail addresses: alafeefy@hotmail.com (A.M. Alafeefy), claudiu.supuran@
unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.042

142
A. M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 141–147
physiologic, CO2 hydration reaction).4a,22 Both enzymes are highly
thermostable: even after heating at around 100 °C for several
hours, they preserve a significant catalytic activity.4a,22 This is
probably due to the fact that bacteria belonging to the genus
Sulfurihydrogenibium comprise chemolithotrophic species able to
survive in extremely harsh conditions of temperature (up to
110 °C) and in the presence of high concentrations of hydrogen sul-
On the other hand, the treatment of 1 with 4-amino-5-(methyl/
phenyl)-4H-1,2,4-triazole-3-thiol 6a, b gave the 1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine derivatives 7a, b, respectively, (Scheme 2).
Their 1H NMR spectra exhibited the D2O-exchangeable signal of
hydrazone NH at d 9.20 in addition to the D2O exchangeable signal
of sulfonamide NH2 at d 6.65. These data are in complete agree-
ment with those of reported such derivatives.4b,5b,6b
fide (between 1 and 100
l
M) or other sulfur-containing inorganic
In order to obtain a different type of derivatization, we per-
formed the reaction of 1 with thioanilide derivatives 8a–d in the
presence of triethylamine, which afforded the 1,3,4-thiadiazoles
10a–d, as depicted in Scheme 2. The IR spectra of the isolated prod-
ucts revealed, in each case, the appearance of nitrile absorption
band around the region 2225 cmꢀ1. Their mass spectra revealed,
in each case, a peak corresponding to the molecular ion. The latter
reaction proceeded via loss of hydrogen chloride to form the non-
isolable intermediate 9a–d which cyclized by elimination of ani-
line molecule to form the isolable 1,3,4-thiadiazole derivatives
10a–d.7b,8b,9b
anions,23–25 and as their names indicate, they live in the Yellow-
stone National Park or the Azore Islands.23–25 Due to the potential
of such enzymes in biotechnologic applications of CO2 capture
processes,26 it is of relevance to investigate modulators of their cat-
alytic activity, such as inhibitors or activators. In this paper we
report the synthesis of a new family of sulfonamides incorporating
benzenesulfonamide and aroylhydrazone, [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thia-
diazol-3(2H)-yl moieties, as well as their inhibitory properties
against the two enzymes from extremophilic bacteria, SazCA and
SspCA.
2.2. Carbonic anhydrase inhibition studies
2. Results and discussion
2.1. Chemistry
The library of new sulfonamides reported here has been inves-
tigated for the inhibition of two mammalian (hCA I and hCA II,
h = human isoform) and two bacterial (SazCA and SspCA)
enzymes,29 in order to establish their inhibition profiles and poten-
tial usefulness in biotechnologic applications.
As seen from data of Table 1, sulfonamides 3a–10d (and aceta-
zolamide, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ, as
standard inhibitor) showed interesting inhibitory properties
against all four investigated CAs. The following structure–activity
relationship (SAR) can be delineated:
In the present study we aim to report the utility of 2-oxo-
N0-(4-sulfamoylphenyl)propanehydrazonoyl chloride (1) in the
synthesis of new compounds linked to the sulfonamide function.
In connection with our previous studies in the chemistry of
bis-hydrazones,1b,2b,3b we used the key intermediate 1 as starting
material for synthesizing the new sulfonamides reported here.
The rationale behind this synthesis was that the carbonyl
functionality and the chlorine atom from 1 possess a good
reactivity towards various nucleophiles, and this can be exploited
for introducing chemical diversity in the molecules of benzenesulf-
onamides possessing elongated tails. In fact it has been shown by
means of X-ray crystallography27,28 that apart the sulfonamide
moiety of a CAI, which binds to the metal ion from the enzyme
active site, the tail moiety is highly relevant for both the potency
and isoform-selectivity of such a compound. Indeed, many such
tails interact with amino acid residues towards the exit of the
active site cavity, and those are the least conserved regions in these
proteins, allowing thus a certain discrimination and isoform selec-
tivity profile. In this particular case, the first approach for derivati-
zation of 1 consisted in the formation of Schiff’s bases by reaction
with aroylhydrazides, followed by nuclepohilic substitution
reactions of the chlorine atom with amines or benzenesulfinate.
Thus, compound 1 was reacted with carbohydrazides 2a–d in
refluxing ethanol to give (1Z,2E)-2-(2-arylhydrazono)-N0-(4-sul-
famoylphenyl)propanehydrazonoyl chlorides 3a–d, respectively,
(Scheme 1). IR spectra of the latter products showed a carbonyl
absorption band around 1668 cmꢀ1 in addition to the absorption
(i) The slow cytosolic isoform hCA I was moderately inhibited
by sulfonamides 3a–3d, 4a–4d, 5a–5d and 7a, 7b, with inhibition
constants in the range of 86.4–753 nM, and weakly inhibited by
derivatives 10a–10d (KIs in the range of 6.54–32.8 lM). It is inter-
esting to note that the Schiff’s bases 3a–3d showed a rather similar
behavior as hCA I inhibitors, with a relatively small variation of the
inhibition constants irrespective of the nature of moiety Ar. The
strongest inhibitor in the subseries was the phenyl derivative 3a,
but its substitution with 4-Cl, 4-MeO groups, or its replacement
by a thienyl scaffold, led to a slight diminution of the inhibitor
potency in compounds 3b–3d. For piperidines 4a–4d, the range
of inhibition was already higher compared to derivatives 3, with
the thienyl derivative 4d being the most active in the subseries
(KI of 91.3 nM) and the 4-methoxyphenyl one 4c the least active
(KI of 247 nM, Table 1). Sulfones 5 also showed a significant varia-
tion of the inhibitory power with the nature of the Ar group, with
the best inhibitor being the phenyl derivative 5a (KI of 130 nM) and
the least effective one the thienyl derivative 5d (KI of 753 nM). For
compounds 7, the methyl derivative 7a showed effective hCA I
inhibitory properties (this was the best inhibitor of this isoform
among the reported derivatives, being almost 3-fold more effective
than the clinically used sulfonamide AAZ) whereas the bulkier Ph
derivative 7b was much less effective as inhibitor of this isoform.
Even a stronger loss of activity was then observed for sulfonamides
10, which probably due to their more rigid scaffold (the thiadiazole
ring is directly connected to the benzene one in these compounds)
can be less well accommodated within the restricted space of the
hCA I active site.28d
bands of 2NH and NH2 groups around the region d 3320 cmꢀ1
.
The 1H NMR of compounds 3a–d showed two D2O exchangeable
signals of 2NH and NH2 groups in the regions d 9.15–9.35 and d
6.60–6.85, respectively. We previously reported the X-ray dif-
fraction structure for an analogue of compound 3, which con-
firmed the (1Z,2E)-configuration of these bis-hydrazones in
solid state.1b
Furthermore, the reaction of bis-hydrazones 3a–d with piperi-
dine in ethanol led to the formation of piperidine derivatives
4a–d (Scheme 1). The 1H NMR of 4a–d revealed the protons of
piperidine moiety in the region at d 1.55 and 3.54–3.60 while their
13C NMR showed the signals of five sp3 carbons at around 25.0 and
52.0, respectively. Moreover, bis-hydrazones 3a–d reacted with
sodium benzenesulfinate to afford the corresponding sulphones
5a–d, respectively, on the basis of their spectral data (Scheme 1).
(ii) All new compounds reported here were excellent inhibitors
of the physiologically dominant (in humans) isoform hCA II.
Indeed, only 3a showed an inhibition constant of 17.1 nM (compa-
rable to that of acetazolamide, 12.1 nM) whereas all the remaining
derivatives were sub-nanomolar or low nanomolar hCA II inhibi-
tors, with KIs in the range of 0.55–8.6 nM. The SAR is thus almost
impossible to delineate as all these substitution patterns seem to

A. M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 141–147
143
Cl
H
O
N
N
H2N
O
Me
S
1
O
O
NH2
Ar
Cl
N
H
2
O
H
N
N
N
N
Ar
PhSO2Na
NH
N
H
H2N
O
Me
S
3
O
O
Ph
N
N
O
O
S
O
H
N
H
N
2-5 Ar
N
N
N
Ar
N
H
Ar
H
a
b
c
C6H5
H2N
O
Me
H2N
Me
S
4
4-ClC6H4
S
5
O
O
4-CH3OC6H4
2-thienyl
O
d
Scheme 1. Synthesis of compounds 3a–d, 4a–d and 5a–d.
Cl
CN
CN
O
H
H
N
N
O
R
Ph
N
R
R
Ph
O
NH
H2N
O
Me
SH
8
S
HN
N
S
NH2
S
1
O
O
N
N
Me
9
SH
N
- PhNH2
O
NH2
6
NH2
S
O
R
N
N
S
Me
N
Me
O
N
N
H
N
N
N
S
NC
10
10
a
R
R
H2N
7
S
CN
O
O
b
c
EtOCO
PhCO
7
R
a
Me
Ph
d
NH2SO2C6H4NHCO
b
Scheme 2. Synthesis of compounds 7a, b and 10a–d.
be highly favorable for obtaining tight-binding hCA II inhibitors.
Among the best such compounds were the Schiff’s base 3d (incor-
porating the thienyl moiety), the piperidine 4b (incorporating a
4-chlorophenyl moiety), the sulphones 5b and 5c, incorporating
4-Cl- and 4-MeO-phenyl moieties, the bulky phenyl-substituted
7b as well as the 1,3,4-thiadiazoles 10c and 10d. All these com-
pounds showed inhibition constants <1 nM, being among the most
effective hCA II inhibitors reported up until now.
Table 1
Inhibition data against the human isoforms hCA I and II, and the bacterial ones SazCA
and SspCA with sulfonamides 3–10 reported here by a stopped-flow, CO2 hydrase
assay29
Compound
KI* (nM)
hCA I
134
185
180
193
151
132
247
hCA II
SazCA
SspCA
(iii) SazCA was also effectively inhibited by most of the sulfon-
amides investigated here. Thus, compounds 3a–3d, 4a–4d, 5d, 7a
and 7b, were highly effective SazCA inhibitors, with KIs in the range
of 0.77–24.3 nM (Table 1). For the Schiff’s bases 3, the best inhibi-
tor was the thienyl-containing one 3d, whereas the phenyl- and
substituted-phenyl derivatives showed a very similar behavior as
SazCA inhibitors. The same trend was in fact observed also for
the piperidines 4, with the 2-thienyl-derivative 4d being a subn-
anomolar SazCA inhibitor, and together with acetazolamide, the
most potent such inhibitor reported to date to this highly effective
enzyme. It is also interesting to note that compounds 7 incorporat-
ing a rather bulky tail were also effective SazCA inhibitors
(KIs > 25 nM) whereas the sulphones 5 as well as the thiadiazoles
10 showed much weaker such properties, with inhibition
constants in the range of 59.1–234 nM (only the thienyl derivative
5d was an effective SazCA inhibitor in these subseries, with a KI of
21.9 nM). It is thus obvious that small modifications in the scaf-
folds of these inhibitors lead to drastic changes in the affinity for
the enzyme, a phenomenon also observed for the other CAs
discussed here.
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
17.1
6.4
3.1
0.94
5.8
0.57
1.1
0.89
8.6
0.97
0.55
4.5
1.0
0.93
4.2
24.3
18.5
21.2
14.9
13.5
8.1
6.2
51.1
45.2
6.4
21.9
80.6
62.8
20.5
42.9
68.3
78.2
74.5
28.3
47.9
89.1
38.7
76.8
71.0
4.5
4.0
91.3
0.77
94.5
59.1
169
21.9
18.3
9.7
116
193
234
101
0.90
130
367
162
753
5c
5d
7a
86.4
7b
390
16,300
27,500
32,800
6540
10a
10b
10c
10d
AAZ
5.4
0.91
0.56
12.1
250
The standard, clinically used sulfonamide acetazolamide (AAZ) was also included in
the assay for comparison reasons.
*
Mean from three different assays. Errors were in the range of 10% of the reported
data (not shown).

144
A. M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 141–147
(iv) SspCA showed a rather diverse inhibition profile compared
2-Oxo-N0-(4-sulfamoylphenyl)propanehydrazonoyl
chloride
to SazCA although the two enzymes show a rather high degree of
homology. Thus, only two compounds (except acetazolamide
AAZ) showed KIs < 10 nM, that is, 3a and 3d. The remaining deriv-
atives had a compact behavior of medium potency SspCA inhibi-
tors, with KIs in the range of 20.5–89.1 nM. Although the X-ray
structure of this enzyme (complexed with AAZ) was recently
reported,30 it is difficult to rationalize these results, that is, to
explain why two compounds act as very effective SspCA inhibitors
whereas the remaining ones have around 10 times a lower affinity.
(v) The inhibition profiles of the four CAs investigated here with
this class of sulfonamides are very different of each other, leading
thus to the evidencing of isoform-selective inhibitors. For example,
5b and 10d show a good selectivity for inhibiting hCA II over hCA I,
SazCA and SspCA. Compound 4d is selective for hCA II and SazCA
over hCA I and SspCA.
(1), carbohydrazides 2a–d, 4-amino-5-(methyl/phenyl)-4H-1,2,4-
triazole-3-thiol 6a, b, thioanilides 8a–c, 2-cyano-3-mercapto-3-
(phenylamino)-N-(4-sulfamoylphenyl)acrylamide (8d)10b,13b were
prepared in our laboratory according to reported procedures. Other
chemicals and solvents employed in the present work were
purchased from Sigma Aldrich GmbH, Sternheim, Germany or
obtained from commercial sources and were used without further
purification.
4.1.2. Synthesis of (1Z,2E)-2-(2-arylhydrazono)-N0-(4-
sulfamoylphenyl)propanehydrazonoyl chloride 3a–d
A
mixture of 2-oxo-N0-(4-sulfamoylphenyl)propanehydrazo-
noyl chloride (1) (2.76 g, 10 mmol) and carbohydrazides 2a–d
(10 mmol) in absolute ethanol (50 mL) was refluxed for 9 h. Then
left to cool, the formed solid was filtered off, washed with ethanol
and crystallized from EtOH/DMF to afford the corresponding
hydrazones 3a–d, respectively.
3. Conclusion
4.1.2.1.
nyl)propanehydrazonoyl chlorides (3a).
181–83 °C; IR
3385–3257 (2NH + NH2), 1664 (C@O) cmꢀ1
(1Z,2E)-2-(2-Benzoylhydrazono)-N0-(4-sulfamoylphe-
Yield (74%); mp
1H
Several series of new sulfonamides were prepared by reaction
of 2-oxo-N0-(4-sulfamoylphenyl)-propanehydrazonoyl chloride
with aroylhydrazides, amines, and/or thiols. The library of new
derivatives incorporated aroylhydrazone, [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thia-
diazol-3(2H)-yl moieties. The compounds were investigated as
m
;
NMR (DMSO-d6) d 2.40 (s, 3H, CH3), 6.62 (s, D2O exchangeable,
2H, NH2), 7.0–7.66 (m, 9H, ArH), 9.15 (s, D2O exchangeable, 1H,
NH), 9.33 (s, D2O exchangeable, 1H, NH); 13C NMR (DMSO-d6) d
10.2 (CH3), 116.5, 126.4, 128.3, 130.3, 131.0, 132.3, 132.9, 145.0,
147.4, 153.4 (Ar-C), 162.9 (C@O); MS m/z (Rel Int.) 395 (M++2,
6.5), 393 (M+, 21.0). Anal. (C16H16ClN5O3S, 393.85) C, H, N. Calcd/
Found: C, 48.79 (49.04); H, 4.09 (3.85); Cl, 9.00 (8.72); N, 17.78
(18.11); S, 8.14 (7.90).
inhibitors of four
a-CAs, the human isoforms hCA I and II, and
the bacterial ones isolated from the extremophilic bacteria Sulfu-
rihydrogenibium yellostonense (SspCA) and S. azorense (SazCA).
Most of these compounds showed very interesting inhibitory
properties against these enzymes. Low nanomolar activity was
observed against hCA II (KIs of 0.56–17.1 nM) whereas hCA I
4.1.2.2. (1Z,2E)-2-(2-(4-Chlorobenzoyl)hydrazono)-N0-(4-sulfa-
was less inhibited (KIs of 86.4 nM–32.8 lM). The bacterial
moylphenyl)propanehydrazonoyl
(76%); mp 237–39 °C; IR 3382–3256 (2NH + NH2), 1669
(C@O) cmꢀ1 1H NMR (DMSO-d6) d 2.41 (s, 3H, CH3), 6.62 (s, D2O
chloride
(3b).
Yield
enzymes were also effectively inhibited by these sulfonamides,
with KIs in the range of 0.77–234 nM against SazCA, and of
6.2–89.1 against SspCA, respectively. Thus, several low
nanomolar/subnanomolar inhibitors were detected against both
extreme-CAs investigated here. As SspCA and SazCA are among
the most thermostable and catalytically active such enzymes, it
is of interest to find modulators of their activity for potential bio-
technologic applications.
m
;
exchangeable, 2H, NH2), 7.0–7.72 (m, 8H, ArH), 9.15 (s, D2O
exchangeable, 1H, NH), 9.30 (s, D2O exchangeable, 1H, NH); 13C
NMR (DMSO-d6) d 11.0 (CH3), 116.6, 128.1, 130.5, 131.1, 137.5,
144.3, 146.5, 153.7 (Ar-C), 163.5 (C@O); MS m/z (Rel Int.) 428
(M++2, 9.5), 427 (M+, 49.6). Anal. (C16H15Cl2N5O3S, 428.29) C, H,
N. Calcd/Found: C, 44.87 (44.99); H, 3.53 (3.78); N, 16.35 (16.62);
S, 7.49 (7.71).
4. Experimental
4.1. Chemistry
4.1.2.3. (1Z,2E)-2-(2-(4-Methoxybenzoyl)hydrazono)-N0-(4-sul-
famoylphenyl)propanehydrazonoyl chloride (3c).
(70%); mp 221–23 °C; IR 3385–3259 (2NH + NH2), 1672
(C@O) cmꢀ1 1H NMR (DMSO-d6) d 2.40 (s, 3H, CH3), 3.56 (s, 3H,
Yield
m
4.1.1. General
;
Melting points (°C, uncorrected) were determined in open cap-
illaries on a Gallenkamp melting point apparatus (Sanyo Gallenk-
amp, Southborough, UK). Pre-coated 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 (Shima-
dzu, Tokyo, Japan). 1H NMR spectra were recorded on Bruker
AC-300 Ultra Shield NMR spectrometer (Bruker, Flawil, Switzer-
land, d ppm) at 300 MHz for 1H and 75 MHz for 13C, using TMS
as internal standard and peak multiplicities are designed as fol-
lows: 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 Microanalytical Unit, Faculty of Science,
Cairo University, Cairo, Egypt, and the found results were within
0.4% of the theoretical values.
–OCH3), 6.81 (s, D2O exchangeable, 2H, NH2), 6.95–7.73 (m, 8H,
ArH), 9.15 (s, D2O exchangeable, 1H, NH), 9.31 (s, D2O exchange-
able, 1H, NH); 13C NMR (DMSO-d6) d 10.2 (CH3), 55.7 (OCH3),
115.3, 116.7, 125.8, 128.6, 130.3, 145.1, 146.7, 153.2, 162.8 (Ar-
C), 163.0 (C@O); MS m/z (Rel Int.) 425 (M++2, 21.5), 423 (M+,
65.2). Anal. (C17H18ClN5O4S, 423.87) C, H, N. Calcd/Found: C,
48.17 (47.85); H, 4.28 (3.96); N, 16.52 (16.22); S, 7.56 (7.39).
4.1.2.4.
bonyl)hydrazono)-propane-hydrazonoyl chloride (3d).
(68%); mp 190–92 °C; IR 3385–3252 (2NH + NH2), 1666
(C@O) cmꢀ1 1H NMR (DMSO-d6) d 2.38 (s, 3H, CH3), 6.61 (s, D2O
(1Z,2E)-N0-(4-Sulfamoylphenyl)-2-(2-(thiophene-2-car-
Yield
m
;
exchangeable, 2H, NH2), 7.06–8.15 (m, 7H, ArH), 9.14 (s, D2O
exchangeable, 1H, NH), 9.30 (s, D2O exchangeable, 1H, NH); 13C
NMR (DMSO-d6) d 11.1 (CH3), 116.0, 129.3, 130.0, 130.3, 130.8,
132.5, 137.9, 145.2, 146.5, 154.3 (Ar-C), 165.6 (C@O); MS m/z
(Rel Int.) 401 (M++2, 16.5), 399 (M+, 47.4). Anal. (C14H14ClN5O3S2,
399.88) C, H, N. Calcd/Found: C, 42.05 (41.88); H, 3.53 (3.71); N,
17.51 (17.39); S, 16.04 (15.78).

A. M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 141–147
145
4.1.3. Synthesis of 4-(2-(2-(2-aroylhydrazono)-1-(piperidin-1-
yl)propylidene)hydrazinyl)-benzenesulfonamides 4a–d
To a solution of propanehydrazonoyl chloride 3a–d (2 mmol) in
ethanol (50 mL), piperidine (0.16 g, 2 mmol) was added. The reac-
tion mixture was heated 2 h at 100 °C then left to cool at room
temperature overnight. The precipitated product was filtered off,
washed with ethanol and dried. Recrystallization from EtOH affor-
ded compounds 4a–d, respectively.
added. The mixture was refluxed for 18 h, then left to cool. The
reaction mixture was poured into cold water and the solid product
was filtered off, washed with water, dried and crystallized from
EtOH/DMF to give the sulfones 5a–d.
4.1.4.1. 4-(2-(2-(2-Benzoylhydrazono)-1-(phenylsulfonyl)propyl-
idene)hydrazinyl)benzene-sulfonamide (5a).
Yield (59%);
mp >300 °C; IR
m
3392–3261 (2NH + NH2), 1663 (C@O) cmꢀ1
;
1H
NMR (DMSO-d6) d 2.39 (s, 3H, CH3), 6.61 (s, D2O exchangeable,
2H, NH2), 6.90–7.66 (m, 14H, ArH), 9.12 (s, D2O exchangeable, 1H,
NH), 9.35 (s, D2O exchangeable, 1H, NH); 13C NMR (DMSO-d6) d
10.1 (CH3), 116.7, 127.1, 128.4, 128.9, 129.5, 130.1, 132.5, 133.8,
135.1, 139.4, 146.7, 151.1, 156.0 (Ar-C), 163.7 (C@O); MS m/z (Rel
Int.) 499 (M+, 3.0). Anal. (C22H21N5O5S2, 499.56) C, H, N. Calcd/
Found: C, 52.89 (53.04); H, 4.24 (3.95); N, 14.02 (13.85); S, 12.84
(13.03).
4.1.3.1. 4-(2-(2-(2-Benzoylhydrazono)-1-(piperidin-1-yl)propyl-
idene)hydrazinyl)-benzene sulfonamide (4a).
Yield (52%); mp
225–27 °C; IR
m
3385–3257 (2NH + NH2), 1667 (C@O) cmꢀ1; 1H NMR
(DMSO-d6) d 1.55 (m, 6H, 3CH2 of piperidine), 2.33 (s, 3H, CH3), 3.54
(m, 4H, 2CH2 of piperidine), 6.61 (s, D2O exchangeable, 2H, NH2),
7.01–7.69 (m, 9H, ArH), 9.14 (s, D2O exchangeable, 1H, NH), 9.32
(s, D2O exchangeable, 1H, NH); 13C NMR (DMSO-d6)
d 10.5,
(CH3), 24.3, 25.5, 52.4 5(CH2), 116.5, 127.2, 128.9, 130.1,
130.8, 132.0, 132.6, 134.5, 146.3, 152.7 (Ar-C), 163.5 (C@O); MS m/z
(Rel Int.) 442 (M+, 29.0). Anal. (C21H26N6O3S, 442.53) C, H, N.
Calcd/Found: C, 57.00 (57.28); H, 5.92 (6.16); N, 18.99 (19.25);
S, 7.25 (7.39).
4.1.4.2.
nyl)propylidene)hydrazinyl)benzene-sulfonamide (5b).
(62%); mp >300 °C; IR
3380–3251 (2NH + NH2), 1652 (C@O) cmꢀ1
4-(2-(2-(2-Chlorobenzoylhydrazono)-1-(phenylsulfo-
Yield
m
;
1H NMR (DMSO-d6) d 2.51 (s, 3H, CH3), 6.64 (s, D2O exchangeable, 2H,
NH2), 7.02–7.99 (m, 13H, ArH), 9.14 (s, D2O exchangeable, 1H, NH),
9.32 (s, D2O exchangeable, 1H, NH); 13C NMR (DMSO-d6) d 15.0
(CH3), 114.0, 127.4, 128.4, 128.7, 129.4, 132.0, 132.5, 134.0, 136.6,
138.3, 138.7, 145.0, 152.6, 155.1 (Ar-C), 165.4 (C@O). MS m/z (Rel
Int.): 536 (M++2, 10.5), 534 (M+, 28.0). Anal. (C22H20ClN5O5S2,
534.01) C, H, N. Calcd/Found: C, 49.48 (49.58); H, 3.78 (3.91); N,
13.11 (12.86); S, 12.01 (11.83).
4.1.3.2.
yl)propylidene)hydrazinyl)benzene-sulfonamide
(4b). Yield (47%); mp >300 °C; IR 3386–3256 (2NH + NH2),
1665 (C@O) cmꢀ1 1H NMR (DMSO-d6) d 1.54 (m, 6H, 3CH2 of
4-(2-(2-(2-Chlorobenzoylhydrazono)-1-(piperidin-1-
m
;
piperidine), 2.23 (s, 3H, CH3), 3.59 (m, 4H, 2CH2 of piperidine),
6.41 (s, D2O exchangeable, 2H, NH2), 7.04–7.98 (m, 8H, ArH), 9.15
(s, D2O exchangeable, 1H, NH), 9.53 (s, D2O exchangeable, 1H,
NH); 13C NMR (DMSO-d6) d 10.7 (CH3), 24.3, 25.5, 52.1 5(CH2),
116.3, 128.7, 130.2, 130.8, 134.7, 138.4, 146.7, 153.0 (Ar-C), 163.8
(C@O); MS m/z (Rel Int.) 478 (M++2, 13.5), 476 (M+, 41.2). Anal.
(C21H25ClN6O3S, 476.98) C, H, N. Calcd/Found: C, 52.88 (53.03); H,
5.28 (5.43); N, 17.62 (17.75); S, 6.72 (6.93).
4.1.4.3. 4-(2-(2-(2-Methoxybenzoylhydrazono)-1-(phenylsulfo-
nyl)propylidene)hydrazinyl)benzene-sulfonamide
(5c).
Yield (57%); mp >300 °C; IR
m 3391–3263 (2NH + NH2),
1661 (C@O) cmꢀ1
;
1H NMR (DMSO-d6) d 2.22 (s, 3H, CH3), 3.70 (s,
3H, OCH3), 6.77 (s, D2O exchangeable, 2H, NH2), 7.06–7.98 (m,
13H, ArH), 9.15 (s, D2O exchangeable, 1H, NH), 9.34 (s, D2O
exchangeable, 1H, NH); 13C NMR (DMSO-d6) d 14.9 (CH3), 55.5
(OCH3), 113.7, 114.0, 126.5, 127.8, 128.3, 128.7, 129.1, 129.5,
134.1, 139.8, 145.9, 151.6, 155.2, 162.7 (Ar-C), 163.5 (C@O); MS
m/z (Rel Int.) 529 (M+, 27.2). Anal. (C23H23N5O6S2, 529.59) C, H, N.
Calcd/Found: C, 52.16 (51.89); H, 4.38 (4.15); N, 13.22 (13.47); S,
12.11 (12.30).
4.1.3.3.
4-(2-(2-(2-Methoxybenzoylhydrazono)-1-(piperidin-1-
Yield
3390–3262 (2NH + NH2), 1671
yl)propylidene)hydrazinyl)benzene-sulfonamide (4c).
(42%); mp 266–267 °C; IR
m
(C@O) cmꢀ1; 1H NMR (DMSO-d6) d 1.55 (m, 6H, 3CH2 of piperidine),
2.34 (s, 3H, CH3), 3.61 (m, 4H, 2CH2 of piperidine), 3.85 (s, 3H, OCH3),
6.61 (s, D2O exchangeable, 2H, NH2), 7.05–7.86 (m, 8H, ArH), 9.11 (s,
D2O exchangeable, 1H, NH), 9.51 (s, D2O exchangeable, 1H, NH); 13
C
NMR (DMSO-d6) d 10.0 (CH3), 24.2, 25.6, 52.3 5(CH2), 56.1 (OCH3),
114.5, 116.7, 125.5, 128.3, 130.5, 131.0, 134.5, 146.0, 152.7, 162.5
(Ar-C), 164.1 (C@O); MS m/z (Rel Int.) 472 (M+, 26.5). Anal. (C22H28-
N6O4S, 472.56) C, H, N. Calcd/Found: C, 55.92 (56.07); H, 5.97 (6.22);
N, 17.78 (17.56); S, 6.79 (6.48).
4.1.4.4.
nyl)propylidene)hydrazinyl)benzene-sulfonamide (5d).
(62%); mp >300 °C; IR
3384–3261 (2NH + NH2), 1657 (C@O) cmꢀ1
4-(2-(2-(2-Chlorobenzoylhydrazono)-1-(phenylsulfo-
Yield
m
;
1H NMR (DMSO-d6) d 2.27 (s, 3H, CH3), 6.68 (s, D2O exchangeable, 2H,
NH2), 7.07–8.49 (m, 12H, ArH), 9.11 (s, D2O exchangeable, 1H, NH),
9.34 (s, D2O exchangeable, 1H, NH); 13C NMR (DMSO-d6) d 10.6
(CH3), 115.3, 128.0, 128.4, 128.9, 129.5, 129.8, 133.6, 135.4, 137.2,
137.6, 139.4, 146.5, 153.1, 155.6 (Ar-C), 166.1 (C@O). MS m/z (Rel
Int.): 505 (M+, 35.0). Anal. (C20H19N5O5S3, 505.59) C, H, N. Calcd/
Found: C, 47.51 (47.66); H, 3.79 (4.01); N, 13.85 (14.11); S, 19.03
(18.79).
4.1.3.4. 4-(2-(1-(Piperidin-1-yl)-2-(2-(thine-2-carbonyl)hydrazon-
o)propylidene)hydrazinyl)-benzenesulfonamide (4d).
Yield
(45%); mp 235–237 °C; IR 3382–3251 (2NH + NH2), 1661
m
1
(C@O) cmꢀ1; H NMR (DMSO-d6) d 1.55 (m, 6H, 3CH2 of piperidine),
2.40 (s, 3H, CH3), 3.60 (m, 4H, 2CH2 of piperidine), 6.60 (s, D2O
exchangeable, 2H, NH2), 7.02–8.15 (m, 7H, ArH), 9.14 (s, D2O
exchangeable, 1H, NH), 9.31 (s, D2O exchangeable, 1H, NH); 13C
NMR (DMSO-d6) d 10.5 (CH3), 24.3, 25.2, 52.1 5(CH2), 116.5, 129.5,
130.2, 130.4, 130.9, 131.6, 134.5, 137.8, 146.4, 153.6 (Ar-C), 168.5
(C@O); MS m/z (Rel Int.) 448 (M+, 41.5). Anal. (C19H24N6O3S2,
448.56) C, H, N. Calcd/Found: C, 50.87 (51.02); H, 5.39 (5.64); N,
18.74 (18.93); S, 14.30 (14.67).
4.1.5. Synthesis of 4-(2-(6-methyl-3-substituted-7H-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazin-7-ylidene)hydrazinyl)benzen-
esulfonamide 7a, b
To a solution of compound 1 (0.276 g, 1 mmol) in ethanol
(30 mL), the appropriate 4-amino-5-(methyl/phenyl)-4H-[1,2,4]-
triazole-3-thione 6a, b (1 mmol) was added. The reaction mixture
was heated under reflux for 10 h. The precipitated solid was fil-
tered, washed with ethanol and dried. Crystallization from
DMF/H2O yielded the corresponding compounds 7a, b,
respectively.
4.1.4. 4-(2-(2-(2-Aroylhydrazono)-1-(piperidin-1-yl)propylidene)-
hydrazinyl)benzenesulfonamides 5a–d
To a solution of compounds 3a–d (1 mmol) in absolute ethanol
(50 mL), sodium benzenesulphinate dihydrate (0.4 g, 2 mmol) was

146
A. M. Alafeefy et al. / Bioorg. Med. Chem. 22 (2014) 141–147
4.1.5.1. 4-(2-(3,6-Dimethyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thi-
adiazin-7-ylidene)hydrazinyl)-benzenesulfonamide
426.47) C, H, N. Calcd/Found: C, 53.51 (53.27); H, 3.31 (3.16); N,
13.14 (12.88); S, 15.04 (14.81).
(7a).
Yield (63%); mp 252–54 °C; IR
m
3385–3268
(NH + NH2) cmꢀ1
;
1H NMR (DMSO-d6) d 2.09 (s, 3H, CH3), 2.47 (s,
4.1.6.4.
ylidene)-2-cyano-N-(4-sulfamoylphenyl)acetamide (10d).
(52%); mp >300 °C; IR 3387–3252 (NH + 2NH2), 2223 (C„N), 1736,
1718 (2C@O) cmꢀ1 1H NMR (DMSO-d6) d 2.16 (s, 3H, CH3), 6.67 (s,
D2O exchangeable, 2H, NH2), 6.79 (d, J = 8.5 Hz, 2H, PhSO2), 7.69 (d,
J = 8.5 Hz, 4H, PhSO2), 7.95 (d, J = 7.5 Hz, 2H, PhSO2), 9.21 (s, D2O
exchangeable, 1H, N-H). 13C NMR: d 24.5 (CH3), 86.9 (C-CN), 115.6
(CN), 116.8, 118.3, 129.5, 130.1, 131.2, 136.3, 141.0, 148.4, 149.5,
159.8 (Ar-C), 164.8, 185.3 (2C@O). MS m/z (Rel Int.): 520 (M+, 38.0). Anal.
(C19H16N6O6S3, 420.56)) C, H, N. Calcd/Found: C, 43.84 (44.01); H, 3.10
(2.92); N, 16.14 (15.89); S, 18.48 (18.71).
2-(5-Acetyl-3-(4-sulfamoylphenyl)-1,3,4-thiadiazol-2(3H)-
3H, CH3), 6.65 (s, 2H, NH2, exchangeable), 6.91 (d, J = 8.5 Hz, 2H,
H-3 and H-5, PhSO2), 7.66 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhSO2),
9.20 (s, 1H, N-H, exchangeable); 13C NMR (DMSO-d6) d 12.8
(CH3), 14.7 (CH3), 90.6, 115.8, 118.5, 126.5, 128.1, 128.4, 128.9,
130.0, 134.6, 144.1, 148.9, 153.1 (Ar-C), 174.9 (C@O). MS m/z (Rel
Int.): 351 (M+, 29.0). Anal. (C12H13N7O2S2, 351.41) C, H, N. Calcd/
Found: C, 41.01 (40.84); H, 3.73 (3.92); N, 27.90 (27.65); S, 18.25
(17.95).
Yield
m
;
4.1.5.2. 4-(2-(6-Methyl-3-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thi-
adiazin-7-ylidene)hydrazinyl)-benzenesulfonamide (7b).
Yield
(68%); mp 236–38 °C; IR
m
3386–3253 (NH + NH2) cmꢀ1
;
1H NMR
4.2. CA activity and inhibition measurements
(DMSO-d6) d 2.06 (s, 3H, CH3), 6.65 (s, 2H, NH2, exchange.), 6.95 (d,
J = 8.5 Hz, 2H, H-3 and H-5, PhSO2), 7.13-7.57 (m, 5H, Ar-H), 7.66 (d,
J = 7.5 Hz, 2H, H-2 and H-6, PhSO2), 9.20 (s, 1H, N-H, exchange.). 13C
NMR: d 14.1 (CH3), 116.2, 127.7, 129.3, 130.1, 130.5, 130.8, 131.6,
146.5, 151.3, 154.0, 156.6, 158.3 (Ar-C). MS m/z (Rel Int.): 413 (M+,
27.6). Anal. (C17H15N7O2S2, 413.48) C, H, N. Calcd/Found: C, 49.38
(49.62); H, 3.66 (3.83); N, 23.71 (24.03); S, 15.51 (15.74).
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity. Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 10–20 mM
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 for maintaining con-
stant the ionic strength, following the initial rates of the CA-cata-
lyzed CO2 hydration reaction for a period of 10–100 s.29 The CO2
concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants. For each inhib-
itor, at least six traces of the initial 5–10% of the reaction have been
used for determining the initial velocity. The uncatalyzed rates
were determined in the same manner and subtracted from the to-
tal observed rates. Stock solutions of inhibitors (0.01 mM) were
prepared in distilled-deionized water and dilutions up to 0.01 nM
were done thereafter with distilled-deionized water. Inhibitor
and enzyme solutions were preincubated together for 15 min at
room temperature prior to assay, in order to allow for the forma-
tion of the E-I complex. The inhibition constants were obtained
by non-linear least-squares methods using PRISM 3 and the
Cheng–Prusoff equation, whereas the kinetic parameters for the
uninhibited enzymes from Lineweaver–Burk plots, as reported ear-
lier,22 and represent the mean from at least three different
determinations.
4.1.6. Synthesis of 2,4,5-trisubstituted-(1,3,4)-thiadiazole benzene
sulfonamides 10a–d
To a solution of the appropriate thioanilide derivatives 8a–d
(1 mmol) dissolved in ethanol (20 mL), 2-oxo-N0-phen-
ylpropanehydrazonoyl chloride (1) (0.276 g, 1 mmol), and triethyl-
amine (0.5 mL) were added. The mixture was refluxed for 6 h. The
formed solid was filtered off, washed with ethanol and crystallized
from EtOH/DMF to afford 1,3,4-thiadiazole derivatives 10a–d,
respectively.
4.1.6.1.
diazol-3(2H)-yl)benzenesulfonamide (10a).
mp 235–37 °C; IR 3385, 3377 (NH2), 2227 (C„N), 1770
(C@O) cmꢀ1 1H NMR (DMSO-d6) d 2.45 (s, 3H, CH3), 6.65 (s, D2O
4-(5-Acetyl-2-(cyanophenylmethylene)-1,3,4-thia-
Yield (68%);
m
;
exchangeable, 2H, NH2), 7.3-7.83 (m, 9H, ArH); 13C NMR (DMSO-
d6) d 23.7 (CH3), 87.6, 115.8, 118.5 (CN), 126.5, 128.1, 128.4,
128.9, 130.0, 134.6, 144.1, 148.9, 153.1 (Ar-C), 184.9 (C@O). MS
m/z (Rel Int.): 398 (M+, 20.0). Anal. (C18H14N4O3S2, 398.46) C, H,
N. Calcd/Found: C, 54.26 (53.97); H, 3.54 (3.29); N, 14.06 (13.88);
S, 16.09 (15.79).
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) and by an FP7 EU project—Metoxia
(to CTS).
4.1.6.2.
diazol-2(3H)-ylidene)-2-cyanoacetate (10b).
201–03 °C; IR 3387, 3352 (NH2), 2224 (C„N), 1760, 1741
(2C@O) cmꢀ1 1H NMR (DMSO-d6) d 1.17 (t, J = 3.32 Hz, 3H, CH3),
Ethyl
2-(5-acetyl-3-(4-sulfamoylphenyl)-1,3,4-thia-
Yield (55%); mp
m
;
References and notes
2.05 (s, 3H, CH3), 4.25 (q, J = 2.7 Hz, 2H, CH2), 6.62 (s, D2O exchange-
able, 2H, NH2), 7.22–7.84 (m, 4H, ArH); 13C NMR (DMSO-d6) d 14.3
(CH3), 23.2 (CH3), 61.5 (CH2), 112.5 (@C-CN), 116.6 (CN), 118.2,
128.5, 129.2, 148.9, 152.9, 160.2 (Ar-C), 165.8, 179.3 (2C@O). MS
m/z (Rel Int.): 394 (M+, 19.2). Anal. (C15H14N4O5S2, 394.43) C, H, N.
Calcd/Found: C, 45.68 (45.83); H, 3.58 (3.29); N, 14.20 (13.98); S,
16.26 (15.94).
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4.1.6.3. 4-(5-Acetyl-2-(1-cyano-2-oxo-2-phenylethylidene)-1,3,4-
thiadiazol-3(2H)-yl)benzene-sulfonamide (10c).
mp 243–45 °C; IR 3389, 3275 (NH2), 2225 (C„N), 1766, 1748
(2C@O) cmꢀ1
Yield (60%);
m
;
1H NMR (DMSO-d6) d 2.23 (s, 3H, CH3), 6.58 (s, D2O
exchangeable, 2H, NH2), 6.95–7.82 (m, 9H, ArH); 13C NMR (DMSO-
d6) d 23.7 (CH3), 87.4 (C-CN), 116.6 (CN), 118.9, 129.3, 130.1,
130.7, 131.5, 135.2, 136.6, 147.9, 148.8, 159.5 (Ar-C), 174.9, 182.7
(2C@O). MS m/z (Rel Int.): 426 (M+, 20.0). Anal. (C19H14N4O4S2,

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