Carbonic anhydrase inhibitors: Benzenesulfonamides incorporating cyanoacrylamide moieties are low nanomolar/subnanomolar inhibitors of the tumor-associated isoforms IX and XII

Article abstract of DOI:10.1016/j.bmc.2012.12.004

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues) have been obtained by reaction of sulfanilamide with ethylcyanoacetate followed by condensation with aromatic/heterocyclic aldehydes, isothiocyanates or diazonium salts. The new compounds have been investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4. 2.1.1), and more specifically against the cytosolic human (h) isoforms hCA I and II, as well as the transmembrane, tumor-associated ones CA IX and XII, which are validated antitumor targets. Most of the new benzenesulfonamides were low nanomolar or subnanomolar CA IX/XII inhibitors whereas they were less effective as inhibitors of CA I and II. The structure-activity relationship for this class of effective CA inhibitors is also discussed. Generally, electron donating groups in the starting aldehyde reagent favored CA IX and XII inhibition, whereas halogeno, methoxy and dimethylamino moieties led to very potent CA XII inhibitors.

Full text of DOI:10.1016/j.bmc.2012.12.004

Bioorganic & Medicinal Chemistry 21 (2013) 1396–1403
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors: Benzenesulfonamides incorporating
cyanoacrylamide moieties are low nanomolar/subnanomolar
inhibitors of the tumor-associated isoforms IX and XII
b
Ahmed M. Alafeefy a, , Semra Isik , Hatem A. Abdel-Aziz c,g, Abdelkader E. Ashour d, Daniela Vullo b,
⇑
Nabila A. Al-Jaber e, Claudiu T. Supuran b,f,
⇑
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, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia
d Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia
e Women Students-Medical Studies and Sciences Sections, Chemistry Department, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabia
f Università degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, 50019 Sesto Fiorentino (Florence), Italy
g Department of Applied Organic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 2 January 2013
A
series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues)
have been obtained by reaction of sulfanilamide with ethylcyanoacetate followed by condensation
with aromatic/heterocyclic aldehydes, isothiocyanates or diazonium salts. The new compounds have
been investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4. 2.1.1), and more
specifically against the cytosolic human (h) isoforms hCA I and II, as well as the transmembrane,
tumor-associated ones CA IX and XII, which are validated antitumor targets. Most of the new ben-
zenesulfonamides were low nanomolar or subnanomolar CA IX/XII inhibitors whereas they were less
effective as inhibitors of CA I and II. The structure–activity relationship for this class of effective CA
inhibitors is also discussed. Generally, electron donating groups in the starting aldehyde reagent
favored CA IX and XII inhibition, whereas halogeno, methoxy and dimethylamino moieties led to very
potent CA XII inhibitors.
Keywords:
Sulfonamide
Carbonic anhydrase
Cyanoacrylamide
Isoform-selective inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
HO
H
N
1. Introduction
Cl
O
O
H
N
O
S
O
H2N S
O
S
NH
O
H2N
O
Unsubstituted aromatic sulfonamides were known to inhibit
the metaloenzyme carbonic anhydrase (CA, EC 4.2.1.1) since the
beginning of research in this field, in the 40s.1 Starting with the
1950-s, potent CA inhibitors (CAIs) belonging to the heterocyclic
sulfonamide class (such as acetazolamide AAZ) have been devel-
oped that led to the benzothiadiazine and high-ceiling diuretics
(hydrochlorothiazide HCT, furosemide FUR, etc.), widely used
drugs even nowadays, as well as to the systemic antiglaucoma
drugs (acetazolamide AAZ, methazolamide MZA, ethoxzolamide
EZA and dichlorophenamide DCP, to mention only the first gener-
ation such compounds).2–5
NH
O
S
S
O
H2N
N
O
O
N
AAZ
O
Cl
HCT
FUR
Cl
Cl
O
S
N
O
S
N
H2N
H2N
S NH2
O
N
N
S
O
O
O
DCP
MZA
EZA
Mann and Keilin reported already in 1940 that sulfanilamide SA
acts as a potent and specific CAI,1 and this prompted much re-
search in the field of designing such inhibitors based on the ben-
zenesulfonamide scaffold.6 However, most of the first generation
drugs mentioned above, as well as the second generation
sulfonamide CAIs, dorzolamide DZA and brinzolamide BRZ, were
heterocyclic sulfonamides. The interest in aromatic, benzenesul-
fonamide-based compounds has reemerged only recently, by our
⇑
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 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.004

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403
1397
report that 1,3,5-triazinyl-substituted benzenesulfonamides act as
highly potent, and isoform-selective CAIs targeting two of the most
investigated mammalian CAs,7 that is, the tumor-associated iso-
forms CA IX and XII, which have been shown to be anticancer drug
targets.8–12
On the other hand, compound 1 couples readily with diazotized
aniline in ethanol, in the presence of sodium acetate at 0–5 °C, to
afford the hydrazone 20 (Scheme 2). Furthermore, derivative 1
was reacted with phenyl isothiocyanate in dimethylformamide,
in the presence of potassium hydroxide, yielding the thioacetani-
lide 21 (Scheme 2). Treatment of the latter compound with the
hydrazonoyl chloride in refluxing ethanol and in the presence of
a catalytic amount of triethylamine afforded the corresponding
1,3,4-thiadiazole derivatives 22 (Scheme 2). The IR spectrum of
compound 22 revealed the appearance of two carbonyl absorption
bands around 1685 cmÀ1 in addition to a nitrile absorption band at
around 2225 cmÀ1. The mass spectrum of compound 22 showed a
peak corresponding to its molecular ion. These results indicate that
the reaction of the thioacetanilide 3 with hydrazonyl chloride pro-
ceeded, via loss of hydrogen chloride to form a non-isolable inter-
mediate which undergoes cyclization by elimination of an aniline
molecule to form the 1,3,4-thiadiazole derivatives 22.17–19
O
N
O
O O
S
NH2
S O
O
S
NH2
S O
O
S
S
H3CO
HN
HN
C2H5
BRZ
C2H5
DZA
Indeed, some ureido-substituted benzenesulfonamides devel-
oped in one of our laboratories using the 1,3,5-triazinyl derivatives
mentioned above as lead molecules, were then used in the proof-
of-concept study to show that CA IX/XII inhibition has profound
antitumor effects in vivo, both for the inhibition of the growth of
primary tumors and metastases.13–15 Some of these derivatives
(as well as the corresponding sulfamates) are being evaluated in
preclinical models as potentially new classes of anticancer drugs/
diagnostic tools for imaging hypoxic tumors.8a
Based on this interesting recent data, we report here a new class
of highly effective CAIs incorporating the classical benzenesulfon-
amide scaffold as well as cyanoacrylamide moieties substituted
with aromatic groups, a scaffold never considered earlier for
designing CAIs. The new class of CAIs described here showed excel-
lent activity against the tumor-associated human (h) isoforms hCA
IX and XII, as well as some selectivity for inhibiting the transmem-
brane isoforms versus the cytosolic, offtarget ones CA I and II.
3. CA inhibition
Inhibition data of compounds 1–22 reported here and acetazol-
amide AAZ (as a standard drug), against the cytosolic CA isozymes
h (h = human) hCA I and II, as well as the tumor-associated, target
isoforms hCA IX and XII, are shown in Table 1.20
The following structure–activity relationship (SAR) can be ob-
served from data of Table 1:
(i) The cytosolic isoforms hCA I was moderately inhibited by
most sulfonamides reported here, with inhibition constants
in the range of 18.7–116 nM. Two compounds, 16 and 17,
were on the other hand low nanomolar hCA I inhibitors
(KIs of 5.3–6.1 nM). Both of them incorporate a five-mem-
bered heterocyclic ring (furan and thiophene, respectively)
in contrast to all other derivatives which are derivatives of
aromatic, substituted benzaldehydes. Thus, the smaller
five-membered ring is more favorable than the six-mem-
bered one in generating potent hCA I inhibitors. On the other
hand, the quite bulky derivative 22, incorporating a highly
substituted 1,3,4-thiadiazole with bulky (phenyl) substitu-
ents in position 3, was one of the least effective hCA I inhib-
itors reported here. It should be however stressed that all
these compounds are better hCA I inhibitors than the clini-
cally used drug acetazolamide, AAZ, which is a weak hCA I
inihibitor (KI of 250 nM). For the compounds incorporating
substituted benzene rings, 2–14, the hCA I inhibition was
modestly influenced by the substitution pattern, as most of
them showed rather similar inhibition power, with KIs vary-
ing between 25.6–71.0 nM (Table 1). The ortho-methoxy
group, present in 5, led to the least effective hCA I inhibitor,
probably due to a steric hindrance caused by the ortho
substituent.
(ii) Compound 1, the starting material from which the other
derivatives were obtained already behaved as a strong hCA
II inhibitor (KI of 6.1 nM). As this is an ubiquitous, house-
keeping isoform, this may be not a valuable property if com-
pounds targeting other isoforms (e.g., the tumor-associated
ones hCA IX and XII) should be obtained. Thus, it is interest-
ing to note that some of the substitution patterns present in
the new derivatives reported here led to less effective hCA II
inhibitors compared to the parent compound 1. Among such
compounds are 2, 3, 8, 10, 13–15, and 18, which showed
inhibition constants in the range of 14.3–61.0 nM. The
remaining ones were highly effective as hCA II inhibitors,
with KIs varying between 2.7–10.9 nM (the same activity
range as acetazolamide AAZ, the standard drug, see Table
2. Chemistry
The key intermediate in the preparation of the new CAIs reported
here was 2-cyano-N-(4-sulfamoylphenyl)acetamide (1), which was
prepared by the reaction of sulfanilamide with ethyl cyanoacetate
according to the reported literature procedure.16 The cyanoacetyl
derivative 1 was then reacted with a series of aromatic/heterocyclic
aldehydes in refluxing ethanol, in the presence of a catalytic amount
of piperidine, to afford the corresponding acrylamides 2–18 and the
chromene 19, respectively (Scheme 1). The rationale behind the
drug design reported here resides in the fact that it has been shown
earlier2–5 by means of X-ray crystallography of adducts of various
CA isoforms with sulfonamide inhibitors, that the most important
contribution for the isoform selectivity is assured by the ‘tail’
of the inhibitor (Fig. 1), which can participate in favorable/non-
favorable interactions with amino acid residues towards the middle
part of the active site or towards its edge.
Thus, we have explored here scaffolds/tails not investigated ear-
lier as CAIs, which incorporate the classical benzenesulfonamide
group which will bind to the Zn(II) ion from the enzyme active site,
to which the rigid substituted acrylamido scaffold has been at-
tached by exploiting the condensation reaction of the active meth-
ylene (NC–CH2–CONH–) from compound
1 with aromatic/
heterocyclic aldehydes. In this way a rather high degree of chemi-
cal diversity may be achieved easily, and this type of scaffold has
not been investigated earlier in the drug design of CAIs.2–5
The new compounds 2–18 were characterized by routine spec-
tral data which confirmed their structures (see Section 5 for de-
tails). For example, the IR spectra of compounds 2–18 showed
the appearance of NH and NH2 absorption band in the region of
3500–3250 cmÀ1 and the band of nitrile function around
2225 cmÀ1 in addition to the carbonyl band at 1700–1650 cmÀ1
.
In their 1H NMR spectra the @CH proton appeared in the region
of 8.15–8.20 ppm in addition to the D2O exchangeable signals of
NH and NH2 (at around 10.50 and 7.50 ppm), respectively.

1398
A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403
O
O
S
H2N
O
CN
N
H
1
R-CHO
2-OH-C6H4-CHO
O
O
O
O
S
S
H2N
O
NH
H2N
O
CN
N
H
O
N
H
2-18
R
19
R =
OMe
MeO
MeO
Cl
Cl
OMe
MeO
Cl
O2N
MeO
MeO
MeO
2
3
5
4
6
7
8
OMe
OMe
MeO
MeO
HO
HO
HO
Me
Me
N
MeO
HO
MeO
10
14
11
12
13
9
O
O
S
17
O
18
15
16
Scheme 1. Preparation of compounds 2–19 by condensation of cyanoacetamido-sulfanilamide 1 with aldehydes.
Hydrophilic
part of active site
new compounds, such as 3, 5, 7 and 10, were less effective
R
TAIL
as hCA IX inhibitors, with KIs in the range of 21.2–35.6 nM.
They incorporate 2,4-dichlorophenyl-, 2-methoxyphenyl,
3,4,5-trimethoxy and 3,4-dimethoxyphenyl moieties in their
molecules. The remaining sulfonamides reported here showed
excellent hCA IX inhibitory power, with KIs in the range of
0.65–12.8 nM (being much more effective than AAZ, which
has a KI of 25 nM). The most effective, subnanomolar hCA IX
inhibitors (compounds 6, 8, 12 and 13) incorporated the fol-
lowing substitution patterns: 4-methoxyphenyl-, 2,4,5-trime-
thoxyphenyl-, 3,4-dihydroxyphenyl- and 4-hydroxyphenyl. It
may be observed again (as for hCA II, discussed above) that for
some of the most effective and the most ineffective hCA IX
inhibitors there are only small structural differences between
the scaffolds, (residing in the nature of groups substituting the
moiety originating from the starting aldehyde reagent). Thus,
the fine-tuning of the inhibitory activity is dependent of very
small structural changes in the scaffold of these sulfonamide
derivatives.
O
Glu106
SCAFFOLD
R'
N
O
H
O
O
ZBG
H
Hydrophobic part
of active site
N
Thr199
Zn2+
His119
His94
His96
Figure 1. Drug design strategy based on the ‘tail-approach’: the zinc-binding group
(ZBG) of the inhibitor molecule directly coordinates the Zn(II) ion from the enzyme
active site (also coordinated by three His residues), being in this case a SO2NHÀ
moiety. The scaffold of the inhibitor is tailored in such a way as to interact both
with the hydrophilic and the hydrophobic halves of the active site. The tails (one or
more of them; two are shown in this schematic representation) of the cyano-
acrylamide type have never been investigated up until now for the design of CAIs.
(iv) The second tumor-associated isoform, hCA XII, was highly
inhibited by the compounds investigated here, with most
of them being subnanomolar (derivatives 1–9, 13, 14 and
22) or low nanomolar (compounds 10–12, 15–18 and 20)
CAIs. The less effective hCA XII inhibitors were 19 and 21
(KIs of 11.7–24.3 nM) which were anyhow quite effective
CAIs. Thus, a rather large number of substitution patterns
from these derivatives lead to highly effective hCA XII inhib-
itors. Further studies (X-ray crystallography) are currently
being done in order to clarify the exact mechanism of action
and structure–activity correlation for this class of effective
sulfonamide CAIs.
1). It is thus clear that small modifications in the scaffold of
the aldehyde from which the CAIs were obtained, leads to
important differences in the hCA II inhibitory potency (e.g.,
compare the regiomers 7, 8 and 9, which differ in activity
by a factor of 6, comparing the most active with the least
active one).
(iii) The tumor associated target isoform hCA IX was highly inhib-
ited by most sulfonamides reported here, generally in the low
nanomolar to subnanomolar range (Table 1). Thus, several

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403
1399
O
O
S
H2N
O
Ph-N2Cl
1- Ph-NCS
2- HCl
CN
N
H
O
O
1
O
O
S
H2N
O
S
H2N
O
CN
N
H
CN
SH
N
H
HN
N
NH
Ph
20
Ph
21
O
O
O
N
Ph
S
Me
N
H
H2N
O
Cl
CN
Ph
N
H
S
N
N
Me
O
22
Scheme 2. Preparation of compounds 20–22.
Table 1
Table 2
Inhibition data of the newly synthesized compounds 1–22 against isoforms hCA I, II,
Selectivity ratios for the inhibition of hCA IX over hCA II and hCA XII over hCA II for
the compounds 1–22 reported in the paper (and acetazolamide AAZ as standard drug)
IX and XII, by a stopped-flow CO2 hydrase assay20
Compound
KIa (nM)
hCA IX
Compound
Selectivity ratio
hCA I
hCA II
hCA XII
hCA IX/hCA II
hCA XII/hCA II
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
AAZ
52.4
41.3
47.1
40.0
71.0
39.6
38.8
42.4
25.6
31.3
32.9
52.4
58.9
47.3
18.7
6.1
6.1
5.2
8.9
0.67
0.86
0.72
0.66
0.60
0.57
0.59
0.67
0.53
2.3
5.2
2.3
0.65
0.93
2.4
6.3
6.4
8.9
24.3
3.5
11.7
0.64
5.7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
AAZ
1.17
6.85
0.92
5.80
0.21
10.4
0.36
39.8
0.85
0.87
1.11
6.46
50.4
5.70
6.38
1.28
0.73
1.50
0.85
0.89
1.12
23.9
0.48
9.10
70.9
45.7
8.18
11.5
15.8
12.7
57.0
12.6
8.08
1.15
1.82
52.0
20.8
13.0
0.87
0.42
1.61
0.44
2.42
0.55
12.3
2.10
61.0
32.9
5.4
6.9
9.0
7.5
38.2
6.7
18.6
6.0
4.2
35.6
0.93
32.6
0.86
20.5
0.96
7.8
21.2
5.4
0.65
0.67
3.4
4.9
4.3
3.7
9.5
12.8
9.5
5.8
0.33
25
33.8
19.4
31.3
5.5
5.3
2.7
24.7
32.9
34.0
67.6
116
250
14.3
10.9
8.5
6.5
7.9
12
Inhibition with acetazolamide AAZ, a standard sulfonamide CAI, is also provided for
comparison.
a
Mean from 3 different assays. Errors were in the range of 10% of the reported
better for the selective inhibition of hCA XII over hCA I and II
(hCA XII/II selectivity ratio of 15.8). Compound 13 is even
more selective (than 6) as an inhibitor of the tumor-ssociated
over the cytosolic isoforms, with a selectivity ratio for inhib-
iting hCA IX over hCA II of 50.4, and for inhibiting hCA IX over
hCA I of 87.9. The same parameters for inhibiting hCA XII over
hCA II are of 52.0, and for inhibiting hCA XII over hCA I of 90.6,
respectively. Thus, the new class of sulfonamides reported
here may indeed lead to isoform-selective CAIs targeting
the tumor-associated CAs.
values (data not shown).
(v) Selectivity for inhibiting the tumor-associated isoforms (hCA
IX and XII) over the widespread cytosolic ones (hCA I and II) is
a critical issue when designing CAIs.2,3 As seen from data of
Table 2, where these ratios are provided, many of the com-
pounds reported here showed relatively strong inhibitory
properties against all four CAs investigated (e.g., compounds
16 and 17 showed highly similar inhibitory properties
against hCA I, II, IX and XII, with KIs in the range of 2.7–
6.4 nM, Tables 1 and 2). This is not highly desirable when
only the tumor-associated isoforms would be targeted. How-
ever, some of the compounds reported here do show indeed
such an inhibition profile. For example 6 has a selectivity
ratio for inhibiting hCA IX over hCA II of 10.5, and for
inhibiting hCA IX over hCA I of 46. These parameters are even
4. Conclusions
We report
a
series of benzenesulfonamides incorporating
cyanoacrylamide moieties (tyrphostine analogues), which have
been obtained by reaction of sulfanilamide with ethylcyanoacetate,

1400
A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403
followed by condensation of the active methylene from the cyano-
acetamide group with aromatic/heterocyclic aldehydes, diazonium
salts or isothiocyanates. Most of the new benzenesulfonamides ob-
tained and evaluated in this study were low nanomolar or subn-
anomolar CA IX/XII inhibitors whereas they were less effective as
inhibitors of CA I and II, but substantially inhibited also those iso-
forms. Some CA IX/XII-selective inhibitors were also detected in
this study. Further studies should be done to explore in detail
the exact mechanism of action and to broaden the structure–activ-
ity relationship for this interesting class of sulfonamide CAIs.
5.2.4. 2-Cyano-3-(2,4-dichlorophenyl)-N-(4-sulfamoylphenyl)
acrylamide (3)
Compound 3: Yield: 80%, mp: 235–37 °C. IR,
m
(cmÀ1): 3410,
3328, 3257 (SO2NH2 and NH), 3049 (Ar-CH), 2944 (CH), 2222
(CN), 1666 (C@O). 1H NMR (DMSOd6): d 7.01 (d, 1H, J = 6.4 Hz,
Ar-H), 7.13 (d, 1H, J = 6.4 Hz, Ar-H), 7.25(s, 1H, Ar-H), 7.87 (d,
J = 7.5 Hz, 2H, H-3 and H-5, PhSO2), 7.91 (d, J = 7.5 Hz, 2H, H-2
and H-6, PhSO2), 8.21 (s, 2H, SO2NH2), 8.33 (s, 1H, @CH), 10.46
(s, 1H, N–H, exchange). 13C NMR: d 106.50 (C-CN), 116.43 (CN),
122.03, 126.90, 127.82, 128.92, 129.64, 131.41, 132.35, 133.24,
135.30, 139.15, 153.42 (Ar-C), 164.34 (C@O). MS m/z (Rel. Int.):
398 (M++2, 26), 396 (M+, 8.5). Anal. (C16H11Cl2N3O3S) C, H, N.
5. Experimental protocols
5.1. General
5.2.5. 2-Cyano-3-(4-nitrophenyl)-N-(4-sulfamoylphenyl)
acrylamide (4)
Compound 4: Yield: 49%, mp: 218–220 °C. IR,
m
(cmÀ1): 3415,
Melting points (°C, uncorrected) were determined in open cap-
illaries on a Gallenkamp melting point apparatus (Sanyo Gallenk-
amp, 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 re-
corded on Bruker AC-300 Ultra Shield NMR spectrometer (Bruker,
Flawil, Switzerland, d ppm) at 300 MHz for 1H and 75 MHz for
13C, using TMS as internal standard and peak multiplicities are de-
signed 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 anal-
yses 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.
3324, 3257 (SO2NH2 and NH), 3075 (Ar-CH), 2981 (CH), 2230
(CN), 1684 (C@O). 1H NMR (DMSOd6): d 7.59 (d, J = 7.5 Hz, 2H, H-
2 and H-6, PhNO2), 7.66 (s, 2H, SO2NH2), 7.88 (d, J = 7.6 Hz, 2H,
H-3 and H-5, PhSO2), 7.92 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhSO2),
8.12 (d, J = 8.0 Hz, 2H, H-3 and H-5, PhNO2), 8.31 (s, 1H, @CH),
10.27 (s, 1H, N–H, exchange). 13C NMR: d 106.3 (C-CN), 115.5,
116.4 (CN), 121.4, 127.5, 127.64, 129.3, 135.7, 139.5, 153.8, 158.4
(Ar-C), 164.6 (C@O). MS m/z (Rel. Int.): 374 (M++2, 20). Anal.
(C16H12N4O5S) C, H, N.
5.2.6. 2-Cyano-3-(2-methoxyphenyl)-N-(4-sulfamoylphenyl)
acrylamide (5)
Compound 5: Yield: 52%, mp: 208–10 °C. IR,
m
(cmÀ1): 3412,
3331, 3253 (SO2NH2 and NH), 3067 (Ar-CH), 2984 (CH), 2257
(CN), 1680 (C@O). 1H NMR (DMSOd6): d 3.73 (s, 3H, OCH3), 7.05–
7.22 (m, 4H, Ar-H), 7.45 (s, 2H, SO2NH2), 7.77 (d, J = 7.5 Hz, 2H,
H-3 and H-5, PhSO2), 7.92 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhSO2),
8.35 (s, 1H, @CH), 10.40 (s, 1H, N–H, exchange). 13C NMR: d 56.3
(OCH3), 106.5 (C-CN), 114.7, 115.4, 116.7 (CN), 120.8, 121.6,
127.5, 129.2, 135.7, 139.5, 153.9, 157.5 (Ar-C), 164.0 (C@O). MS
m/z (Rel. Int.): 357 (M+, 36). Anal. (C17H15N3O4S) C, H, N.
5.2. Chemistry
5.2.1. 2-Cyano-N-(4-sulfamoylphenyl)acetamide (1)
This compound was prepared according to reported literature
procedures.19 It had the same physico-chemical parameters (1H,
13C NMR spectra, as well as MS, as reported in Ref. 19, data not
shown).
5.2.7. 2-Cyano-3-(4-methoxyphenyl)-N-(4-sulfamoylphenyl)
acrylamide (6)
Compound 6: Yield: 47%, mp: 215–17 °C. IR,
m
(cmÀ1): 3414,
3329, 3254 (SO2NH2 and NH), 3068 (Ar-CH), 2987 (CH), 2231
(CN), 1683 (C@O). 1H NMR (DMSOd6): d 3.74 (s, 3H, OCH3), 7.12
(d, J = 7.5 Hz, 2H, H-2 and H-6, PhOCH3), 7.41 (s, 2H, SO2NH2),
7.68 (d, J = 7.6 Hz, 2H, H-3 and H-5, PhSO2), 7.79 (d, J = 7.5 Hz,
2H, H-2 and H-6, PhSO2), 8.02 (d, J = 8.0 Hz, 2H, H-3 and H-5,
PhOCH3), 8.31 (s, 1H, @CH), 10.46 (s, 1H, N–H, exchange). 13C
NMR: d 56.3 (OCH3), 106.7 (C-CN), 115.2, 116.5 (CN), 121.3,
127.6, 129.3, 135.7, 139.5, 153.9, 158.5 (Ar-C), 164.3 (C@O). MS
m/z (Rel. Int.): 357 (M+, 36). Anal. (C17H15N3O4S) C, H, N.
5.2.2. Synthesis of compounds 2–19
To
a mixture of 2-cyano-N-(4-sulfamoylphenyl)acetamide
(0.239 g, 1 mmol) and the appropriate aldehyde (1 mmol) in etha-
nol (25 ml), few drops of piperidine were added. The reaction mix-
ture was refluxed for 4 h, and then cooled to room temperature.
The precipitate was filtered, dried and washed with ethanol. The
crude product was recrystallized from EtOH/DMF to afford com-
pounds 2–19, respectively, in 45–80% yield.
5.2.8. 2-Cyano-N-(4-sulfamoylphenyl)-3-(3,4,5-trimethoxyphenyl)
5.2.3. 3-(2-Chlorophenyl)-2-cyano-N-(4-sulfamoylphenyl)
acrylamide (7)
m
(cmÀ1): 3398,
acrylamide (2)
Compound 7: Yield: 50%, mp: 225–27 °C. IR,
Compound 2: Yield: 81%, mp: 221–23 °C. IR,
m
(cmÀ1): 3412,
3324, 3250 (SO2NH2 and NH), 3073 (Ar-CH), 2955 (CH), 2233
(CN), 1689 (C@O). 1H NMR (DMSOd6): d 3.74 (s, 9H, 3OCH3), 6.27
(s, 2H, H-2 and H-6, Ar-H), 7.41 (s, 2H, SO2NH2), 7.82 (d,
J = 8.0 Hz, 2H, H-3 and H-5, HNPhSO2), 7.90 (d, J = 7.6 Hz, 2H, H-2
and H-6, HNPhSO2), 8.32 (s, 1H, @CH), 10.48 (s, 1H, N–H, ex-
change). 13C NMR: d 56.5 (2OCH3), 56.6 (OCH3), 102.9, 106.7 (C-
CN), 111.4, 113.2, 116.2 (CN), 121.4, 127.3, 135.6, 139.8, 143.5,
150.2, 151.6, 153.9 (Ar-C), 165.0 (C@O). MS m/z (Rel. Int.): 417
(M+, 37). Anal. (C19H19N3O6S) C, H, N.
3331, 3253 (SO2NH2 and NH), 3045 (Ar-CH), 2941 (CH), 2225
(CN), 1668 (C@O). 1H NMR (DMSOd6): d 7.01–7.23 (m, 4H, Ar-H),
7.42 (s, 2H, SO2NH2), 7.73 (d, J = 7.5 Hz, 2H, H-3 and H-5, PhSO2),
7.85 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhSO2), 8.33 (s, 1H, @CH),
10.46 (s, 1H, N–H, exchange). 13C NMR: d 106.50 (C-CN), 116.43
(CN), 122.03, 126.90, 127.82, 128.92, 129.64, 131.41, 133.24,
135.30, 139.15, 146.0, 153.42 (Ar-C), 164.34 (C@O). MS m/z (Rel.
Int.): 363 (M++2, 17), 361 (M+, 5.7). Anal. (C16H12ClN3O3S) C, H, N.

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403
1401
5.2.9. 2-Cyano-N-(4-sulfamoylphenyl)-3-(2,4,5-trimethoxyphenyl)
164.21 (C@O). MS m/z (Rel. Int.): 359 (M+, 65). Anal. (C16H13N3O5S)
acrylamide (8)
C, H, N.
Compound 8: Yield: 46%, mp: 226–28 °C. IR,
m
(cmÀ1): 3414,
3330, 3253 (SO2NH2 and NH), 3075 (Ar-CH), 2956 (CH), 2236
(CN), 1685 (C@O). 1H NMR (DMSOd6): d 3.74 (s, 3H, OCH3), 3.80
(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.19 (s, 1H, Ar-H), 6.60 (s, 1H,
Ar-H), 7.41 (s, 2H, SO2NH2), 7.76 (d, J = 8.0 Hz, 2H, H-3 and H-5,
HNPhSO2), 7.89 (d, J = 7.6 Hz, 2H, H-2 and H-6, HNPhSO2), 8.32 (s,
1H, @CH), 10.48 (s, 1H, N–H, exchange). 13C NMR: d 56.33
(2OCH3), 56.65 (OCH3), 101.90, 106.37 (C-CN), 109.14, 113.20,
116.23 (CN), 121.40, 127.35, 135.65, 139.58, 143.52, 150.22,
151.46, 153.99 (Ar-C), 165.0 (C@O). MS m/z (Rel. Int.): 417 (M+,
67). Anal. (C19H19N3O6S) C, H, N.
5.2.14. 2-Cyano-3-(4-hydroxyphenyl)-N-(4-sulfamoylphenyl)
acrylamide (13)
Compound 13: Yield: 42%, mp: 219–21 °C. IR,
m
(cmÀ1): 3426
(OH), 3345, 3241 (SO2NH2 and NH), 3041 (Ar-CH), 2938 (CH),
2227 (CN), 1672 (C@O). 1H NMR (DMSOd6): d 6.68 (d, J = 7.0 Hz,
2H, Ar-H), 7.11 (d, J = 6.6 Hz, 2H, Ar-H), 7.60 (s, 2H, SO2NH2), 7.87
(d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2), 7.92 (d, J = 7.5 Hz, 2H,
H-2 and H-6, HNPhSO2), 8.25 (s, 1H, @CH), 8.54 (s, 1H, OH),
10.30 (s, 1H, N–H, exchange). 13C NMR: d 106.3 (C-CN), 115.5,
116.4 (CN), 122.4, 127.0, 127.6, 134.5, 138.7, 153.4, 156.7 (Ar-C),
164.2 (C@O). MS m/z (Rel. Int.): 359 (M+, 65). Anal. (C16H13N3O4S)
C, H, N.
5.2.10. 2-Cyano-N-(4-sulfamoylphenyl)-3-(2,4,6-trimethoxyphenyl)
acrylamide (9)
Compound 9: Yield: 43%, mp: 226–28 °C. IR,
m
(cmÀ1): 3399,
5.2.15. 2-Cyano-3-(4-(dimethylamino)phenyl)-N-(4-sulfamoyl-
3324, 3253 (SO2NH2 and NH), 3074 (Ar-CH), 2953 (CH), 2236
(CN), 1686 (C@O). 1H NMR (DMSOd6): d 3.72 (s, 9H, 3OCH3), 5.97
(s, 2H, Ar-H), 7.81 (s, 2H, SO2NH2), 7.88 (d, J = 8.0 Hz, 2H, H-3 and
H-5, HNPhSO2), 7.93 (d, J = 7.6 Hz, 2H, H-2 and H-6, HNPhSO2),
8.33 (s, 1H, @CH), 10.27(s, 1H, N–H, exchange). 13C NMR: d 56.2
(2OCH3), 56.4 (OCH3), 102.5, 106.3 (C-CN), 112.1, 113.6, 116.3
(CN), 121.0, 127.4, 135.8, 139.8, 143.6, 150.5, 151.4, 154.0 (Ar-C),
165.3 (C@O). MS m/z (Rel. Int.): 417 (M+, 60). Anal. (C19H19N3O6S)
C, H, N.
phenyl)acrylamide (14)
Compound 14: Yield: 49%, mp: 212–14 °C. IR,
m
(cmÀ1): 3305,
3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN), 1674
(C@O). 1H NMR (DMSOd6): d 6.56 (d, J = 7.0 Hz, 2H, Ar-H), 7.12 (d,
J = 6.6 Hz, 2H, Ar-H), 7.66 (s, 2H, SO2NH2), 7.89 (d, J = 8.5 Hz, 2H,
H-3 and H-5, HNPhSO2), 7.93 (d, J = 7.5 Hz, 2H, H-2 and H-6,
HNPhSO2), 8.34 (s, 1H, @CH), 10.30 (s, 1H, N–H, exchange). 13C
NMR: d 41.5 (2CH3), 107.1 (C-CN), 114.9, 116.4 (CN), 122.4, 125.0,
127.2, 127.5, 135.5, 139.3, 149.0, 153.4, (Ar-C), 163.8 (C@O). MS
m/z (Rel. Int.): 370 (M+, 36). Anal. (C18H18N4O3S) C, H, N.
5.2.11. 2-Cyano-3-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenyl)
acrylamide (10)
5.2.16. 2-Cyano-5-phenyl-N-(4-sulfamoylphenyl)penta-2,4-
Compound 10: Yield: 46%, mp: 205–07 °C. IR,
m
(cmÀ1): 3421,
dienamide (15)
3342, 3255 (SO2NH2 and NH), 3075 (Ar-CH), 2953 (CH), 2243
(CN), 1682 (C@O). 1H NMR (DMSOd6): d 3.81 (s, 3H, OCH3), 3.86
(s, 3H, OCH3), 6.78 (d, J = 7.0 Hz, 1H, Ar-H), 6.96 (d, J = 7.0 Hz, 1H,
Ar-H), 7.41 (s, 2H, SO2NH2), 7.77 (d, J = 8.5 Hz, 2H, H-3 and H-5,
HNPhSO2), 7.99 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.22 (s,
1H, @CH), 10.43 (s, 1H, N–H, exchange). 13C NMR: d 56.33
(2OCH3), 56.45 (OCH3), 107.08 (C-CN), 112.20, 116.23 (CN),
117.0, 120.14, 122.10, 127.65, 128.87, 135.95, 139.14, 148.75,
149.54, 153.96 (Ar-C), 165.10 (C@O). MS m/z (Rel. Int.): 387 (M+,
65). Anal. (C18H17N3O5S) C, H, N.
Compound 15: Yield: 72%, mp: 187–89 °C. IR, m
(cmÀ1): 3352,
3318, 3261 (NH2 and NH), 3075 (Ar-CH), 2229 (CN), 1685 (C@O).
1H NMR (DMSOd6): d 6.66 (d, J = 7.0 Hz, 1H, C@H), 6.90 (m, 1H,
C@H), 7.12 (m, 1H, Ar-H), 7.21 (m, 2H, Ar-H), 7.36 (d, J = 7.0 Hz,
2H, C@H), 7.53 (s, 2H, SO2NH2), 7.90 (d, J = 7.0 Hz, 2H, Ar-H), 7.92
(d, J = 7.0 Hz, 2H, Ar-H), 8.0 (d, J = 7.0 Hz, 1H, C@H), 8.45 (s, 1H,
N–H, exchange). 13C NMR: d 93.0 (C-CN), 115.3, 116.2 (C„N),
120.1, 121.2, 125.0, 125.9, 127.2, 128.0, 128.5, 131.26, 133.8,
135.1, 135.3, 139.5 (Ar-C), 163.5 (C@O). MS m/z (Rel. Int.): 353
(M+, 5.5). Anal. (C18H15N3O3S) C, H, N.
5.2.12. 2-Cyano-3-(4-hydroxy-3-methoxyphenyl)-N-(4-sulfamoyl-
5.2.17. 2-Cyano-3-(furan-2-yl)-N-(4-sulfamoylphenyl)
phenyl)acrylamide (11)
acrylamide (16)
Compound 11: Yield: 48%, mp: 211–13 °C. IR,
m
(cmÀ1): 3429,
Compound 16: Yield: 46%, mp: 201–203 °C. IR, m
(cmÀ1): 3305,
3345, 3234 (SO2NH2 and NH), 3044 (Ar-CH), 2935 (CH), 2222
(CN), 1677 (C@O). 1H NMR (DMSOd6): d 3.65 (s, 3H, OCH3), 6.46
(d, J = 7.0 Hz, 1H, Ar-H), 6.60 (s, 1H, Ar-H), 6.65 (d, J = 7.0 Hz, 1H,
Ar-H), 7.80 (s, 2H, SO2NH2), 7.90 (d, J = 8.5 Hz, 2H, H-3 and H-5,
HNPhSO2), 7.94 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.18 (s,
1H, @CH), 8.56 (s, 1H, OH, exchange.), 10.36 (s, 1H, N–H, exchange).
13C NMR: d 56.5 (OCH3), 106.6 (C-CN), 114.0, 116.4 (CN), 118.1,
121.1, 122.3, 127.6, 128.8, 135.8, 139.3, 146.7, 147.7, 153.6 (Ar-
C), 164.2 (C@O). MS m/z (Rel. Int.): 373 (M+, 35). Anal.
(C17H15N3O5S) C, H, N.
3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),
1674 (C@O). 1H NMR (DMSOd6): d 6.62 (t, J = 6.3 Hz, 1H, Ar-H),
7.10 (d, J = 6.9 Hz, 1H, Ar-H), 7.80 (d, J = 6.5 Hz, 1H, Ar-H), 7.86 (s,
2H, SO2NH2), 7.90 (d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2), 7.94
(d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.33 (s, 1H, @CH),
10.23 (s, 1H, N–H, exchange). 13C NMR: d 111.1 (C-CN), 112.5,
114.0, 116.4 (CN), 121.5, 127.5, 135.7, 139.4, 147.0, 152.0, 154.7,
(Ar-C), 164.2 (C@O). MS m/z (Rel. Int.): 317 (M+, 34). Anal.
(C14H11N3O4S) C, H, N.
5.2.18. Cyano-N-(4-sulfamoylphenyl)-3-(thiophen-2-yl)
5.2.13. 2-Cyano-3-(3,4-dihydroxyphenyl)-N-(4-sulfamoylphenyl)
acrylamide (17)
acrylamide (12)
Compound 17: Yield: 51%, mp: 186–88 °C. IR, m
(cmÀ1): 3305,
Compound 12: Yield: 41%, mp: 235–37 °C. IR,
m
(cmÀ1): 3428
3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),
1674 (C@O). 1H NMR (DMSOd6): d 7.32 (t, J = 6.3 Hz, 1H, Ar-H),
7.60 (d, J = 6.9 Hz, 1H, Ar-H), 7.63 (d, J = 6.5 Hz, 1H, Ar-H), 7.85 (s,
2H, SO2NH2), 7.91 (d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2), 7.95
(d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.32 (s, 1H, @CH),
10.20 (s, 1H, N–H, exchange). 13C NMR: d 112.0 (C-CN), 116.4
(CN), 121.5, 127.5, 128.6, 130.8, 135.7, 139.4, 147.0, 152.0, 154.7,
(Ar-C), 164.2 (C@O). MS m/z (Rel. Int.): 333 (M+, 26). Anal.
(C14H11N3O3S2) C, H, N.
(2OH), 3348, 3239 (SO2NH2 and NH), 3046 (Ar-CH), 2939 (CH),
2223 (CN), 1677 (C@O). 1H NMR (DMSOd6): d 6.58 (d, J = 7.0 Hz,
1H, Ar-H), 7.09 (s, 1H, Ar-H), 7.41 (s, 2H, SO2NH2), 7.60 (d,
J = 8.5 Hz, 1H, Ar-H), 7.77 (d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2),
7.99 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.18 (s, 1H, @CH),
9.98 (s, 2H, 2OH), 10.39 (s, 1H, N–H, exchange). 13C NMR: d
106.32 (C-CN), 113.22, 116.24 (CN), 117.11, 120.13, 122.04,
127.46, 129.88, 135.85, 139.37, 146.75, 147.74, 153.76 (Ar-C),

1402
A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403
5.2.19. (3-(Benzo[d][1,3]dioxol-5-yl)-2-cyano-N-(4-sulfamoyl-
change). 13C NMR: d 86.43 (C-CN), 115.38, 116.75 (CN), 119.10,
122.25, 127.95, 130.26, 136.14, 139.85, 144.56 (Ar-C), 160.54
(@CH), 163.57 (C@O). MS m/z (Rel. Int.): 374 (M+, 5.5). Anal.
(C16H14N4O3S2) C, H, N.
phenyl)acrylamide (18)
Compound 18: Yield: 50%, mp: 195–97 °C. IR,
m
(cmÀ1): 3305,
3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),
1674 (C@O). 1H NMR (DMSOd6): d 5.92 (s, 2H, CH2), 6.60 (d,
J = 6.7 Hz, 1H, Ar-H), 6.77 (d, J = 6.5 Hz, 1H, Ar-H), 6.90 (s, 1H, Ar-
H), 7.85 (s, 2H, SO2NH2), 7.91 (d, J = 8.5 Hz, 2H, H-3 and H-5,
HNPhSO2), 7.96 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.34 (s,
1H, @CH), 10.20 (s, 1H, N–H, exchange). 13C NMR: d 100.9 (CH2),
107.3 (C-CN), 112.1, 115.0, 116.1 (CN), 122.0, 127.6, 128.5, 135.4,
139.6, 147.8, 149.0, 152.8 (Ar-C), 163.5 (C@O). MS m/z (Rel. Int.):
371 (M+, 41). Anal. (C17H13N3O5S) C, H, N.
5.2.23. 2-(5-Acetyl-3-phenyl-1,3,4-thiadiazol-2(3H)-ylidene)-2-
cyano-N-(4-sulfamoylphenyl)acetamide (22)
To a solution of thioanilide 21 (0.37 g, 1 mmol) dissolved in eth-
anol (20 mL), (Z)-2-oxo-N’-phenylpropanehydrazonoyl chloride
(22) (0196 g, 1 mmol), and of triethylamine (0.5 mL) were added.
The mixture was refluxed for 6 h, then allowed to cool. The formed
solid was filtered off, washed with ethanol and recrystallized from
EtOH/DMF to afford 1,3,4-thiadiazole derivative 22 in 46% yield.
Compound 22: Yield: 46%, mp: 204–06 °C. IR,
m
(cmÀ1): 3305,
5.2.20. 2-Imino-N-(4-sulfamoylphenyl)-2H-chromene-3-
carboxamide (19)
3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),
1674 (C@O). 1H NMR (DMSOd6): d 2.27 (s, 3H, CH3), 6.46 (d,
J = 6.5 Hz, 2H, Ar-H), 6.63 (t, J = 6.5 Hz, 1H, Ar-H), 6.98 (t, J = 6.7 Hz
, 2H, Ar-H), 7.88 (s, 2H, SO2NH2), 7.91 (d, J = 8.5 Hz, 2H, H-3 and
H-5, HNPhSO2), 7.94 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2),
10.23 (s, 1H, N–H, exchange). 13C NMR: d 24.0 (CH3), 87.6 (C-CN),
116.0, 116.7 (CN), 119.3, 119.7, 121.3, 127.5, 129.3, 135.6, 139.7,
146.5, 154.6, 161.2 (Ar-C), 163.1, 184.7 (C@O). MS m/z (Rel. Int.):
441 (M+, 21). Anal. (C19H15N5O4S2) C, H, N.
Compound 19: Yield: 44%, mp: 222–24 °C. IR,
m
(cmÀ1): 3305,
3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),
1674 (C@O). 1H NMR (DMSOd6): d 5.70 (bs, 1H, =NH, D2O
exchangeable), 6.67 (d, J = 6.4 Hz, 1H, Ar-H), 6.89 (t, J = 6.2 Hz, 1H,
Ar-H), 6.94 (t, J = 6.3 Hz, 1H, Ar-H), 7.14 (d, J = 6.5 Hz, 1H, Ar-H),
7.87 (s, 2H, SO2NH2), 7.92 (d, J = 8.5 Hz, 2H, H-3 and H-5,
HNPhSO2), 7.95 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.35 (s,
1H, @CH), 10.21 (s, 1H, N–H, exchange). 13C NMR: d 115.7 (CH2),
116.3, 119.7, 121.5, 127.3, 127.8, 128.4, 129.2, 135.5, 139.8, 153.9
(Ar-C), 163.1, 164.5. MS m/z (Rel. Int.): 343 (M+, 41). Anal.
(C16H13N3O4S) C, H, N.
5.3. CA inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.20 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 con-
stant the ionic strength), following the initial rates of the CA-cata-
lyzed CO2 hydration reaction for a period of 10–100 s.20 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 inhibitor (10 mM) were pre-
pared in distilled-deionized water and dilutions up to 0.001 nM
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temper-
ature 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, as reported earlier,10–15
and represent the mean from at least three different determina-
tions. All CA isoforms were recombinant ones obtained in house
as reported earlier.10–15 The concentration of the enzymes in the
assays was of 8.3 nM for hCA I, of 5.7 nM for hCA II, of 6.9 nM for
hCA IX and of 7.5 nM for hCA XII, respectively.
5.2.21. 2-Oxo-N’-phenyl-2-(4-
sulfamoylphenylamino)acetohydrazonoyl cyanide (20)
To a stirred solution of 2-cyano-N-(4-sulfamoylphenyl)acetam-
ide (0.239 g, 1 mmol) in ethanol (50 ml) sodium acetate trihydrate
(0.13 g, 1 mmol) was added. After stirring for 15 min, the mixture
was chilled at 0 °C and treated with cold solution of aniline
(0.93 g, 1 mmol) in 6 M hydrochloric acid (1.5 ml) with sodium ni-
trite solution (0.07 g, 1 mmol) in water (3 mL). The addition of the
diazonium salt was stirred for an additional 2 h at 0–5 °C and then
left for 8 h in a refrigerator (4 °C). The resulting solid was collected
by filtration, washed thoroughly with water and dried. The crude
product was crystallized from ethanol to give hydrazone 20 in
55% yield.
Compound 20: Yield: 55%, mp: 226–28 °C. IR,
m
(cmÀ1): 3385,
3230, 3190 (SO2NH2 and NH), 3040 (Ar-CH), 2225 (CN), 1679
(C@O). 1H NMR (CH3OD): d 6.58 (d, J = 6.4 Hz, 2H, ArH), 6.70 (t,
J = 6.2 Hz, 1H, ArH), 7.11 (t, J = 6.8 Hz, 2H, ArH), 7.36 (s, 2H,
SO2NH2), 7.78 (d, J = 8.0 Hz, 2H, H-2 and H-6, NHPhSO2NH2), 7.91
(d, J = 8.0 Hz, 2H, H-3 and H-5, NHPhSO2NH2), 8.35 (s, 1H, N–H, ex-
change.), 10.48 (s, 1H, CON–H, exchange). 13C NMR: d 107.13,
114.25, 116.10, 119.0, 122.4, 128.1, 129.7, 135.7, 137.25, 140.0,
143.9 (Ar-C), 162.7 (C@O). MS m/z (Rel. Int.): 343 (M+, 20). Anal.
(C15H13N5O3S) C, H, N.
5.2.22. 2-Cyano-3-mercapto-3-(phenylamino)-N-(4-sulfamoyl-
phenyl)acrylamide (21)
Acknowledgments
To a stirred solution of KOH (0.56 g 10 mmol) in DMF (20 mL),
compound 1 (2.39 g, 10 mmol) was added. After stirring for
30 min phenyl isothiocyanate (1.35 g, 10 mmol) was added to the
resulting mixture. Stirring was continued for 6 h and then poured
over crushed ice containing HCl. The solid product so formed was
filtered off, washed with water, dried and finally crystallization
from EtOH to afford compound 21 as pale yellow solid in 41% yield.
This research was financed by a 7th FP EU project (Metoxia) and
by a grant from the National Plan of Science, Technology and Inno-
vation (Grant No. 10-MED1188-02 & Grant No. 11-MED-1874-2),
King Saud University, Riyadh, by the Graduate Studies and Scien-
tific Research Agency, Salman bin Abdulaziz University, Grant No.
2.H.33, Alkharj, Saudi Arabia. Semra Isik was supported by a grant
from the Council of Higher Education of Turkey.
Compound 21: Yield: 41%, mp: 196–98 °C. IR,
m
(cmÀ1): 3353,
3330, 3264 (SO2NH2 and NH), 3062 (Ar-CH), 2221 (CN), 1682
(C@O). 1H NMR (DMSOd6): d 5.06 (s, 1H, NH, exchange.), 6.75–
7.05 (m, 5H, Ar-H), 7.33 (s, 2H, SO2NH2), 7.76 (d, J = 8.0 Hz, 2H,
H-2 and H-6, NHPhSO2NH2), 7.90 (d, J = 8.0 Hz, 2H, H-3 and H-5,
NHPhSO2NH2), 10.45 (s, 1H, N–H, exchange.), 11.51 (s, 1H, S-H, ex-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.004.

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403
1403
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