SLC-0111 enaminone analogs, 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides, as novel selective subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform IX

Article abstract of DOI:10.1016/j.bioorg.2018.11.014

SLC-0111, an ureido substituted benzenesulfonamide, is a selective carbonic anhydrase (CA, EC 4.2.1.1) IX inhibitor that is currently in Phase I/II clinical trials for the treatment of advanced hypoxic tumors complicated with metastases. Herein we report

Full text of DOI:10.1016/j.bioorg.2018.11.014

Accepted Manuscript
SLC-0111 enaminone analogs, 3/4-(3-aryl-3-oxopropenyl) aminobenzenesul-
fonamides, as novel selective subnanomolar inhibitors of the tumor-associated
carbonic anhydrase isoform IX
Wagdy M. Eldehna, Mahmoud F. Abo-Ashour, Emanuela Berrino, Daniela
Vullo, Hazem A. Ghabbour, Sara T. Al-Rashood, Ghada S. Hassan, Hamad M.
Alkahtani, Abdulrahman A. Almehizia, Amal Alharbi, Hatem A. Abdel-Aziz,
Claudiu T. Supuran
PII:
S0045-2068(18)30936-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.014
YBIOO 2622
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 August 2018
6 November 2018
13 November 2018
Please cite this article as: W.M. Eldehna, M.F. Abo-Ashour, E. Berrino, D. Vullo, H.A. Ghabbour, S.T. Al-Rashood,
G.S. Hassan, H.M. Alkahtani, A.A. Almehizia, A. Alharbi, H.A. Abdel-Aziz, C.T. Supuran, SLC-0111 enaminone
analogs, 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides, as novel selective subnanomolar inhibitors of the
tumor-associated carbonic anhydrase isoform IX, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.11.014
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SLC-0111 enaminone analogs, 3/4-(3-aryl-3-oxopropenyl)
aminobenzenesulfonamides, as novel selective subnanomolar inhibitors
of the tumor-associated carbonic anhydrase isoform IX
Wagdy M. Eldehnaa,*, Mahmoud F. Abo-Ashourb, Emanuela Berrinoc, Daniela Vulloc,
Hazem A. Ghabbourd, Sara T. Al-Rashoode, Ghada S. Hassand, Hamad M. Alkahtanie,
Abdulrahman A. Almehiziae, Amal Alharbie, Hatem A. Abdel-Azizf, Claudiu T.
Supuranc,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, Egypt.
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
P.O. Box 11829, Badr City, Cairo, Egypt.
cDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences,
University of Florence, Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy.
dDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura
35516, Egypt.
eDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.
Box 2457, Riyadh 11451, Saudi Arabia.
fDepartment of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622,
Egypt.
Abstract
SLC-0111, an ureido substituted benzenesulfonamide, is a selective carbonic anhydrase
(CA, EC 4.2.1.1) IX inhibitor that is currently in Phase I/II clinical trials for the treatment
of advanced hypoxic tumors complicated with metastases. Herein we report the synthesis
of two series of 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides 5a-i and 6a-j as
SLC-0111 enaminone congeners. The prepared enaminones were in vitro investigated as
inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II,
IV and IX, using a stopped-flow CO2 hydrase assay. All these isoforms were inhibited by
the enaminones reported here in variable degrees. The target tumor-associated isoform
hCA IX was undeniably the most affected one (KIs: 0.21 – 7.1 nM), with 6- to 21-fold
enhanced activity than SLC-0111 (KI = 45 nM). All the prepared enaminones displayed
interesting selectivity towards hCA IX over hCA I (SI: 32 – >35714), hCA II (SI: 2 –
1689) and hCA IV (SI: 11 – >45454). Of particular interest, bioisosteric replacement of
1

phenyl tail with the bulkier 2-naphthyl tail, sulfonamide 6h, achieved the higher II/IX
selectivity herein reported with SI of 1689.
____________
Keywords: Benzenesulfonamide; Carbonic anhydrase; Enaminones; SLC-0111; Selective
CA IX inhibitors.
 Corresponding authors. Tel.: +2-1068837640; e-mail: wagdy2000@gmail.com (WME);
Tel./fax:+39-055-4573729; e-mail: claudiu.supuran@unifi.it (CTS)
2

1-Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are prevalent metalloenzymes in all life
kingdoms. In humans, the CA-catalyzed reaction encompasses three simple chemical
−
entities, CO2, HCO3 , and H+ essential in several physiological and pathological
processes, such as the pH and CO2 homeostasis, bone resorption, electrolyte secretion,
respiration, biosynthetic reactions (like lipogenesis and gluconeogenesis) and
tumorigenicity, to name few [1-3].
The human (h) CA isoforms displayed diverse tissue distribution and location
patterns; thus cytosolic (I, II, III, VII, and XIII), membrane-bound (IV, IX, XII, and
XIV), secreted (VI) and mitochondrial (VA and VB) forms have been described in
mammals. These CA isozymes are important therapeutic targets for a wide range of
disorders such as glaucoma (hCA II, IV and XII), edema (hCA II, IV, XIV), and tumors
(hCA IX and hCA XII) [3-5]. In particular, hCA IX is an extracellular transmembrane
tumor-related protein that overexpressed in a wide variety of human malignancies and
appears to be tightly regulated by microenvironmental hypoxia. During the last years, this
CA isozyme had been stood out as an innovative anticancer drug target and recognized as
a marker of tumor hypoxia and prognostic factor for several cancer types [6-8]. It is
noteworthy, that ubiquitous hCA I and CA II, which are involved in many physiological
processes, are considered to be off-target in the cancer therapies [1], as they are
responsible of most of the side effects of non-selective inhibitors. The “tail approach”
represents the most efficient and exploited tactic to overcome the poor selectivity of
sulfonamide-like CAIs towards the different human isozymes [9-11].
Adopting “tail approach” has led to the development of SLC-0111 (I) (Fig. 1). SLC-
0111, an ureido substituted benzenesulfonamide, is a selective carbonic anhydrase IX
inhibitor that is currently in Phase I/II clinical trials for the treatment of advanced
hypoxic tumors complicated with metastases [12-14]. Being the first-in-class CAI, SLC-
0111 has been used as a lead molecule for designing other compounds with a selective
CA IX inhibitory activity [15-24].
A. Angeli et al. [15] reported the synthesis of novel SLC-0111 congeners via
adopting a divalent bioisosteric replacement approach of the ureido oxygen on SLC-0111
with a sulfur or selenium atom, compounds II and III (Fig. I). Such replacement
3

determined dramatic enhancements of the inhibition potencies towards the hCA I and II
isoforms (Fig. I). While, the thioureido derivative II resulted in only a slight
improvement of the inhibition potency towards hCA IX (KI = 45.0 and 32.1 nM for SLC-
0111 and II, respectively), the introduction of the selenium, compound III, was found to
be detrimental for the activity (KI = 63.0 nM). Accordingly, the inhibition profiles of both
thioureido II and selenoureido III congeners of SLC-0111 displayed loss of selectivity
for the inhibition of the tumor-associated over the cytosolic CA isoforms. Akocak and co-
workers reported two studies about the development of N,N’-diaryl cyanoguanidines and
1,3-diaryltriazenes as novel SLC-0111 analogues, through bioisosteric replacement of
urea linker in SLC-0111 with cyanoguanidine or 1,3-triazene linker, compounds IV and
V (Fig. I) [16,17]. These structural modifications switched the selectivity of
cyanoguanidines and 1,3-diaryltriazenes counterparts to be highly selective for hCA II
over the tumor-associated hCA IV and IX.
Last year, F. Carta et al. [18] explored the synthesis and biological evaluation of
SLC-0111 regioisomers, compound VI (Fig. I). The study outcomes revealed that
shifting primary sulfonamide (-SO2NH2) functionality from the para-position, SLC-0111,
to meta-position, compound VI, resulted in a significant improvement of the inhibitory
activity against hCA II concurrently with worsening of effectiveness towards hCA IX
which massively spoiled the selectivity ratio hCA II/hCA IX for this regioisomer.
Recently, M. Bozdag et al. [19] reported the design and synthesis of ureido containing
compounds based on the 2-aminophenol-4-sulfonamide as new SLC-0111 congeners
with controlled degrees of tail flexibility, compound VII (Fig. I). The phenolic OH was
mainly intended to form a stable intramolecular five-membered ring with the ureido NH
moiety at 2-position (Fig. I), which thought to induce C2-N’ rotational restriction, that
may determine preferential rotational isomers to interact within the hCAs enzymatic sites
thus resulting in improvement of CAs selectivity. Interestingly, the rotational restriction
within compound VII, resulted in 20-fold enhanced selectivity ratio hCA II/hCA IX in
comparison to SLC-0111 (Fig. I).
4

Fig. 1. Structures of some reported SLC-0111 analogs I-VII and the target enaminones 5a-i
and 6a-j.
In view of the facts mentioned above and as part of our ongoing effort towards
developing effective CAIs [25-29], herein we report two series of 3/4-(3-aryl-3-
oxopropenyl) aminobenzenesulfonamides (5a-i and 6a-j) as SLC-0111 enaminone
analogs (Fig. 1), with the prime aim of developing potent and selective hCA IX
inhibitors. Firstly, the bioisosteric replacement approach was utilized to replace the urea
linker within SLC-0111 with an enaminone one, followed by introduction of two
structural modifications. The first modification focuses on the replacement on 4-
fluorophenyl tail of SLC-0111 with different phenyl, 4-substituted-phenyl, the bulky 2-
naphthyl, and the small 5-membered heterocycles (2-furyl and 2-thienyl). The second
modification includes the development of the regioisomers, through moving the
sulfonamide (-SO2NH2) functionality from the para-position to the meta-position. All the
newly synthesized enaminones were examined in vitro for their inhibitory activity
towards a panel of hCA I, II, IV and IX isoforms, using stopped-flow CO2 hydrase assay.
5

2. Results and Discussion
2.1. Chemistry
The synthetic strategy for the development of the target sulfonamides 5a-i and 6a-j
was depicted in Scheme 1. Different acetophenones 1a-j were refluxed with DMF-DMA
2 in xylene to afford enaminones 3a-j which reacted with m- and p-
aminobenzenesulfonamides 4a,b in refluxing glacial acetic acid to furnish the target
sulfonamides 5a-i and 6a-j, respectively.
The structures of target sulfonamides 5a-i and 6a-j were confirmed under the basis of
their spectral data and elemental analyses. Their IR spectra showed the absorption bands
due to the (NH, NH2) and (C=O) groups in the regions 3120-3400 cm-1 and 1720-1735
cm-1, respectively, besides two bands of (SO2) at 1150-1160 cm-1 and 1300-1320 cm-1.
Furthermore, 1H NMR spectra of sulfonamides 5a-i and 6a-j revealed presence of E/Z
geometric conjugation forms for HaC=CHb. The existing of E-form confirmed by the
coupling constant of the olefinic hydrogens Ha and Hb where JHa-Hb = 12.0-12.8 Hz which
calculated from the down-field doublet signal of E-Ha at δ 6.37-6.69 ppm (Fig. 2).
Similarly, the existing of Z-form proved by the coupling constant of the olefinic
hydrogens Ha and Hb (J = 7.2-8.4 Hz) which elucidated from the up-field doublets of Z-
1
Ha at δ 6.00-6.40 ppm. Additionally, H NMR spectra revealed two sets of doublet,
integrated for a total one proton, each belonging to the NH group of the E and Z forms
which observed at δ 10.24-10.59 and 11.72-12.18 ppm, respectively (Fig. 2). The
downfield lines of -NH protons were assigned to Z form and up-field lines of the same
group to E form. The coupling values of these doublets are in the range of 12.0-12.8 Hz
which provides the existing of NH of sulfonamides 5a-i and 6a-j in trans direction for Hb
around HN-CHb bond in both forms E and Z (Fig. 3). The down-field appearance of Z
NH attributed to its intra-molecular hydrogen bond with the oxygen of C=O group.
Interestingly, x-ray single crystal analyses of 5a showed the Z configuration around
HaC=CHb with trans HN-CHb in solid state and it is also revealed the intra-molecular
hydrogen bond between NH and C=O groups [30].
6

Scheme 1. Synthesis of target enaminones 5a-i and 6a-j; Reagents and conditions: (i)
Dry xylene / reflux 8 h, (ii) Glacial acetic acid / reflux 4 h.
Moreover, their 1H NMR spectra confirmed the presence of singlet signal of NH2 of -
SO2NH2 group around δ 7.20-7.37 ppm, also Compounds 5d and 6e confirmed with
presence of single peak of -CH3 at δ 2.38 ppm and compounds 5e and 6f revealed by
presence of single peak of -OCH3 at δ 3.81-3.84 ppm.
13
In addition, C NMR spectra reveled the presence of E and Z forms as (-CO-CH=CH-)
present in two chemical shift range 94.49-95.36 ppm and 98.77-100.00 ppm for Z and E
forms, respectively, while C=O, presnt in range 177.10-188.08 ppm and 179.55-190.42 ppm.
7

On the other hand, the chemical shift of OCH3 group of compounds 5e, 6f and CH3 group of
compounds 5d, 6e appeared at 55.87, 55.90 ppm and 21.54, 21.55 ppm, respectivly, for E/Z
forms.
1
Figure 2. H NMR of compound 5a which showed the existing of E/Z configurations around
HaC=CHb with trans HN-CHb in DMSO.
8

Figure 3. The existing of compounds 5a as Z configuration around HaC=CH with E HN-CH in
solid state (X-ray single crystal analyses) and as Z/E HaC=CHb, E HN-CHb configurations in
DMSO (1H NMR).
2.2. Single crystal X-ray crystallographic analysis
Single crystal of enaminone 5a was obtained through the slow evaporation of its ethanol
solution. A suitable crystal was selected and analyzed on a Bruker APEX-II CMOS
diffractometer. The data was collected at 293(2) K.
Figure 4. An ORTEP diagram of final X-ray structure of enaminone 5a. Displacement ellipsoids
are plotted at the 40% probability level for non-H atoms.
The structure of enaminone 5a (Fig. 4) was unambiguously determined by X-ray
crystallographic analysis. The molecular structure of 5a (C15H14N2O3S) was crystallized
9

in the monoclinic, space group P21, a = 5.2360 (5) Å, b = 9.4977 (8) Å, c = 14.1960 (14)
Å, β = 98.628 (5) °, V = 697.98 (11) Å3, Z = 2. The crystallographic data and refinement
information are summarized in Table 1. The dihedral angle between the mean planes of
the two phenyl rings is 4.78, which makes the whole structure nearly flat. In the crystal
packing structure, molecules are linked via three intermolecular hydrogen bonds along
the b axis between N2—H1N2···O3i, N2—H1N···O2ii and C5—H5A···O1iii. Symmetry
codes: (i) x+1, y, z; (ii) −x+1, y−1/2, −z+2; (iii) −x+2, y+1/2, −z+1.
Table 1. Crystallographic data and refinements for enaminone 5a
Crystal data
Chemical formula
Mr
5a
C15H14N2O3S
302.34
Crystal system, space group
Temperature (K)
a, b, c (Å)
Monoclinic, P21
293
5.2360 (5), 9.4977 (8), 14.1960 (14)
β (°)
V (Å3)
Z
98.628 (5)
697.98 (11)
2
Radiation type
µ (mm−1)
Mo Kα
0.24
Crystal size (mm)
Data collection
Diffractometer
Absorption correction
0.21 × 0.12 × 0.05
Bruker APEX-II D8 venture diffractometer
Multi-scan
SADABS Bruker 2014
0.951, 0.987
Tmin, Tmax
No. of measured, independent and observed [I
> 2σ(I)] reflections
Rint
27807, 5834, 2695
0.145
Refinement
R[F2 > 2σ( F2)], wR( F2), S
No. of reflections
No. of parameters
No. of restraints
0.076, 0.189, 0.98
5834
202
1
H-atom treatment
H atoms treated by a mixture of
independent and constrained refinement
0.56, −0.34
Δρmax, Δρmin (e Å−3)
Flack parameter
0.14 (11)
10

2.3. Biological Evaluation
2.3.1. Carbonic anhydrase inhibition
All synthesized enaminones 5a-i and 6a-j were tested in vitro for their inhibitory
properties against physiologically relevant hCA isoforms, hCA I, II (cytosolic) and hCA
IV (transmembrane) as well as the tumor-associated hCA IX (transmembrane), by means
of a stopped-flow carbon dioxide hydration assay [31]. Their inhibitory activities were
compared to SLC-0111 and the clinically used standard carbonic anhydrase inhibitor
acetazolamide (AAZ). The following structure–activity relationship (SAR) could be
deduced from the inhibition data displayed in Table 2:
(i) The ubiquitous cytosolic isoform hCA I was inhibited by most of the prepared
enaminones reported here with inhibition constants (KIs) ranging in the low nanomolar -
high micromolar range, in detail, between 8.5 nM and 9.09 μM, except compounds 6g
and 6h which did not inhibit hCA I up to 10 μM. It is noteworthy that shifting the
sulphonamide group from para-position (as compound 6a; KI = 68.8 nM) to the meta-
position (as compound 5a; KIs = 626.7 nM) resulted in a sharp decrease in the inhibitory
activity against hCA I, except enaminones 5d, 5g and 5h. Whereas, bioisosteric
replacement of the phenyl ring (compound 6a; KI = 68.8 nM) with 2-furyl or 2-thienyl
moieties led to the most potent hCA I inhibitors in this study with remarkable increase in
activity (compounds 6i and 6j; KIs = 8.5 and 9.3 nM, respectively).
(ii) The physiologically dominant isoform hCA II was efficiently inhibited by most
sulfonamides prepared in this study (KIs in the range of 0.48-472.8 nM) (Table 2). It is
worth highlighting that the para-substituted enaminones 6a-f, 6i and 6j showed a
generally improved inhibitory profile against hCA II in comparison to their meta-
substituted analogues 5a-e, 5h and 5i. In particular para sulphonamides 6b, 6e, 6i and 6j
stood out as the best hCA II inhibitors with subnanomolar potencies (KIs = 0.48, 0.53,
0.84 and 0.89 nM, respectively).
Noteworthy, the SAR outcomes highlighted that substitution of the pendant phenyl
tail with larger lipophilic groups (Cl, Br, or NO2) or its replacement with the bulkier 2-
naphthyl tail elicited a worsening of effectiveness against hCA II both for the meta-
substituted enaminones 5b, c, e-g (KI: 29.2 – 344.4 nM) and the para-substituted
11

enaminones 6c, d, f-h (KIs: 6.3 – 472.8 nM) in comparison to unsubstituted counterparts
5a and 6a (KIs = 21.7 and 5.9 nM, respectively). In addition, bioisosteric replacement of
the phenyl tail for the meta-substituted sulphonamide 5a (KIs = 21.7 nM) with 2-furyl or
2-thienyl tail moieties led to about two- and four-fold diminished hCA II inhibition
efficacy (enaminones 5h and 5i; KIs = 34.6 and 76.6 nM, respectively). Conversely,
adopting this replacement for the para-substituted derivative 5a (KIs = 5.9 nM) resulted
in a seven-fold efficacy enhancement (enaminones 6i and 6j; KIs = 0.84 and 0.89 nM,
respectively).
(iii) The in vitro kinetic data reported in Table 2 showed that inhibition of trans-
membrane isoform hCA IV by the prepared enaminones was ranged from weak to potent
inhibition, with KI values ranging from 6.5 to 6140.8 nM, except compound 6a (KI >
10000 nM). Interestingly, enaminones 5h, 5i, 6i and 6j, possessing 2-furyl/thienyl tail,
displayed potent inhibitory activity against hCA IV (KIs = 64.5, 8.7, 22.40 and 6.5 nM,
respectively), hinting that replacement of the phenyl tail with the 2-furyl or 2-thienyl tails
is advantageous for inhibitory activity toward hCA IV. Moreover, sulphonamides 5f and
6g, possessing 4-nitrophenyl tail, beside 6h effectively inhibited hCA IV with inhibition
constants equals 80.70, 56.30 and 64.2 nM, respectively.
(iv) The tumor-associated isoform hCA IX was potently inhibited by all enaminones
reported here. The hCA IX inhibition profile was found to be rather flat, since the
measured KIs ranged between 0.21 and 1.5 nM, aside from enaminones 5b and 5c whose
efficacy raised at slightly higher concentrations (KIs = 7.1 and 7.0 nM, respectively).
Superiorly, all the investigated enaminones emerged as subnanomolar hCA IX inhibitors
(KIs: 0.21 – 0.31 nM), except sulfonamides 5b, 5c and 5g displayed single-digit
nanomolar inhibitory activity towards hCA IX (KIs: 1.5 – 7.1 nM). All the examined
enaminones 5a-i and 6a-j were 6 – 21 times more potent than SLC-0111 (KI = 45 nM). It
is worth noting that a decrease of potency (2.7-33 folds) was obtained upon shifting the
sulphonamide group from para-position to the meta-position.
12

Table 2. Inhibition data of human CA isoforms hCA I, II, IV and IX with SLC-0111 and
enaminones 5a-i and 6a-j, determined by stopped-flow CO2 hydrase assay, using
acetazolamide (AAZ) as a standard drug.
KI (nM)*
Comp.
Ar
C6H5
hCA I
626.7
2985
3803
693
hCA II
21.70
29.20
183.20
6.70
hCA IV
1355.80
4391.50
6140.80
2616.80
5266.70
80.70
hCA IX
0.90
7.10
7.00
0.82
0.96
0.90
1.50
0.73
0.77
0.22
0.27
0.22
0.21
0.21
0.23
0.31
0.28
0.27
0.22
45.0
25.80
5a
5b
5c
5d
5e
5f
4-ClC6H4
4-BrC6H4
4-CH3C6H4
4-CH3OC6H4
4-NO2C6H4
naphthalen-2-yl
furan-2-yl
916
265.50
190.40
344.40
34.60
76.60
5.90
9095
8143
155.6
566.5
68.80
72.40
323.10
7888
30.80
70.90
<10000
<10000
8.50
5g
5h
5i
793.0
64.5
thiophen-2-yl
C6H5
8.7
6a
6b
6c
6d
6e
6f
<10000
772.40
874.10
893.40
798.60
589.0
4-FC6H4
0.48
4-ClC6H4
8.70
4-BrC6H4
88.90
0.53
4-CH3C6H4
4-CH3OC6H4
4-NO2C6H4
naphthalen-2-yl
furan-2-yl
6.30
6g
6h
6i
464.40
472.80
0.84
56.30
64.20
22.40
6j
thiophen-2-yl
9.30
0.89
6.50
SLC-0111 -
AAZ
5080
250
960.0
12.10
286.0
-
74.0
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
 5-10 % of the reported values).
13

(v) As a result of the inhibitory trends extrapolated from the data in Table 2,
interesting selectivity index (SI) arose for all enaminones herein reported (Table 3).
Interestingly, all the examined sulfonamides 5a-i and 6a-j displayed interesting
selectivity towards hCA IX over hCA I (SI: 32 – >35714), hCA II (SI: 2 – 1689) and
hCA IV (SI: 11 – >45454). Of particular interest, bioisosteric replacement of phenyl tail
with the the bulkier 2-naphthyl tail, sulfonamide 6h, achieved the higher II/IX selectivity
herein reported with SI of 1689 (Table 3).
As discussed above, the Z configuration around HaC=CHb resulted in an intra-molecular
hydrogen bond between NH and C=O groups, affording stable intra-molecular five-
membered ring. It’s suggested that this intra-molecular ring furnished a rotational restriction
with a controlled degree for flexibility of the tail, which in turn led to a preferential
interaction within the hCAs active sites resulting in an improvement for CAs selectivity.
Table 3. Selectivity ratios for the inhibition of hCA IX over hCA I, II and hCA IV for
enaminones 5a-i and 6a-j reported in the paper, SLC-0111 and acetazolamide.
Selectivity ratio
Cpd.
5a
I/IX
II/IX
IV/IX
Cpd.
6a
6b
6c
I/IX
313
II/IX
27
IV/IX
<45454
2861
3973
4254
3803
2561
182
696
24.1
1506
268
2
420
543
846
959
10106
5429
213
736
9.7
4
619
877
3191
5486
90
1469
37561
147
40
5b
5c
26
6d
6e
423
3
8
5d
5e
308
27
277
212
230
47
6f
<32258
<35714
32
5f
6g
6h
6i
1498
1689
3
529
88
229
5g
5h
5i
83
100
0.5
11
42
4
30
6j
AAZ
2.9
SLC-0111 112.9
21.3
6.4
14

3. Conclusion
In summary, this study reports the design and synthesis of two series of 3/4-(3-aryl-3-
oxopropenyl) aminobenzenesulfonamides 5a-i and 6a-j as SLC-0111 enaminone
congeners. The synthesized derivatives incorporated an enaminone moiety, instead of the
ureido moiety in SLC-0111, as a linker between the sulfonamide hCA inhibitor (CAI)
warhead and the molecular tail. Furthermore two structural modifications were
introduced. The first modification focuses on replacement on 4-fluorophenyl tail of SLC-
0111 with different phenyl, 4-substituted-phenyl, the bulky 2-naphthyl, and the small 5-
membered heterocycles (2-furyl and 2-thienyl). The second modification includes
development of the regioisomers, through moving the sulfonamide (-SO2NH2)
functionality from the para-position to the meta-position. The prepared enaminones were
in vitro investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1) isoforms hCA I, II, IV and IX, using a stopped-flow CO2 hydrase assay. All the
examined isoforms were inhibited by the sulfonamides reported here in variable degrees;
hCA I was inhibited with KIs in the range of 8.5-9095 nM, hCA II in the range of 0.48-
472.8 nM; hCA IV in the range of 6.5-6140.8 nM, whereas hCA IX in the range of 0.21 -
7.1 nM. Superiorly, all the investigated enaminones emerged as subnanomolar hCA IX
inhibitors (KIs: 0.21 – 0.31 nM), except sulfonamides 5b, 5c and 5g displayed single-digit
nanomolar inhibitory activity towards hCA IX (KIs: 1.5 – 7.1 nM). All sulfonamides 5a-i
and 6a-j displayed interesting selectivity towards hCA IX over hCA I (SI: 32 – >35714),
hCA II (SI: 2 – 1689) and hCA IV (SI: 11 – >45454). Of particular interest, bioisosteric
replacement of phenyl tail with the the bulkier 2-naphthyl tail, sulfonamide 6h, achieved
the higher II/IX selectivity herein reported with SI of 1689.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured with a Stuart melting point apparatus and were
uncorrected. Infrared spectra were recorded on Schimadzu FT-IR 8400S
spectrophotometer. The NMR spectra were recorded by Bruker spectrophotometer at 400
15

13
MHz. C NMR spectra were run at 100 MHz in deuterated dimethylsulfoxide (DMSO-
d6). Chemical shifts (δH) are reported relative to TMS as internal standard. All coupling
constant (J) values are given in hertz. Chemical shifts (δC) are reported relative to
DMSO-d6 as internal standards. High-resolution mass spectra were recorded using a
Bruker MicroTOF spectrometer. Elemental analyses were carried out at the Regional
Center for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Unless
otherwise noted, all solvents and reagents were commercially available and used without
further purification.
4.1.2. General procedure for preparation of enaminone intermediates 3a-j.
Compounds 3a-j were prepared as reported [32, 33]
4.1.3. General procedure for preparation of target sulfonamides 5a-i and 6a-j.
The appropriate enaminone intermediate 3a-j (1 mmol) was added to a hot stirred
solution of p- or m-aminobenzenesulfonamide 4a, b (0.17 gm, 1 mmol) in glacial acetic
acid (15 mL). The reaction mixture was heated under reflux for 4 h. The formed solid
upon cooling was filtered off, washed with cold methanol, dried and recrystallized from
dioxane to afford the target enaminones 5a-s.
4.1.3.1. 3-((3-Oxo-3-phenylprop-1-en-1-yl)amino)benzenesulfonamide (5a).
Yellow crystals (yield 65 %), m.p. 175-177 °C; IR (KBr, ν cm-1): 3122-3395 broad band
(NH, NH2), 1728 (C=O), 1300, 1155 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.23
(d, 2/3H, Ar-H, J= 8 Hz), 6.53 (d, 1/3H, Ar-H, J= 12.8 Hz), 7.37-7.66 (m, 8H, 6H of Ar-
H and 2H of NH2, D2O exchangeable), 7.78 (s, 1H, Ar-H), 7.91-8.01 (m, 2.73H, Ar-H),
8.12 (t, 1/3H, Ar-H, J= 12.4 Hz), 10.38 (d, ½ H, NH exchanged with D2O, J= 12.4 Hz),
13
12.12 (d, ½ H, NH exchanged with D2O, J= 12.4 Hz); CNMR (DMSO-d6,100 MHz)
ppm: 94.78, 99.21 (-CO-CH=CH-, cis and trans conformers), 112.58, 113.68, 119.24,
119.42, 119.83, 120.45, 127.73 (2C), 127.77 (2C), 129.04 (2C), 129.11 (2C), 130.85
(2C), 132.22, 132.46, 138.90, 139.66, 141.13, 142.09, 144.09, 145.70, 145.97, 146.05,
188.08, 190.42 (C=O, cis and trans conformers); MS m/z [%]: 302 [M+, 13.99], 301
16

[100]; Anal. Calcd. for C15H14N2O3S (302.35): C, 59.59; H, 4.67; N, 9.27; found C,
59.72; H, 4.70; N, 9.40.
4.1.3.2. 3-((3-(4-Chlorophenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (5b).
Yellow crystals (yield 74 %), m.p. 156-157 °C; IR (KBr, ν cm-1): 3122-3392 broad band
(NH, NH2), 1724 (C=O), 1300, 1160 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.23
(d, 1/3H, Ar-H, J= 8 Hz), 6.53 (d, 2/3H, Ar-H, J= 12 Hz), 7.37-7.66 (m, 7H, 5 H of Ar-H
and 2H of NH2, D2O exchangeable), 7.76 (s, 1H, Ar-H), 7.90-8.02 (m, 2.73H, Ar-H),
8.12 (t, 1/3H, Ar-H, J= 12.4 Hz), 10.44 (d, ½ H, NH exchanged with D2O, J= 12 Hz),
13
12.09 (d, ½ H, NH exchanged with D2O, J= 12 Hz); CNMR (DMSO-d6,100 MHz)
ppm: 94.49, 98.77 (-CO-CH=CH-, cis and trans conformers), 112.66, 113.77, 119.32,
119.56, 119.97, 120.59, 129.13, 129.20, 129.64, 129.67, 130.87, 136.99, 137.31, 137.54,
138.29, 140.99, 141.92, 144.58, 145.93, 146.01, 146.23 (Aromatic carbons), 186.74,
188.91 (C=O, cis and trans conformers); HRMS (ESI) for C15H13ClN2O3S, calcd
337.04082, found 337.04088 [M+H]+; Anal. Calcd. for C15H14ClN2O3S (337.04): C,
53.49; H, 3.89; N, 8.32; found C, 53.65; H, 3.79; N, 8.50.
4.1.3.3. 3-((3-(4-Bromophenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (5c).
Beige crystals (yield 73 %), m.p. 211-213 °C; IR (KBr, ν cm-1): 3125-3390 broad band
(NH, NH2), 1720 (C=O), 1307, 1154 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.20
(d, ½ H, Ar-H, J= 7.6 Hz), 6.52 (d, ½ H, Ar-H, J= 12.4 Hz), 7.04 (d, 2H, Ar-H, J= 7.2
Hz), 7.36-7.56 (m, 5H, 3H of Ar-H and 2H of NH2, D2O exchangeable), 7.64 (s, ½ H, Ar-
H), 7.73 (s, ½ H, Ar-H), 7.84-7.99 (m, 2.5H, Ar-H), 8.16 (br s, ½ H, Ar-H), 10.27 (d, ½
H, NH exchanged with D2O, J= 10 Hz), 12.09 (d, ½ H, NH exchanged with D2O, J= 11.6
Hz); MS m/z [%]: 383 [M++2, 50.95], 381 [M+, 56.26], 332 [100]; Anal. Calcd. for
C15H13BrN2O3S (381.24): C, 47.26; H, 3.44; N, 7.35; found C, 47.32; H, 3.61; N, 7.49.
4.1.3.4. 3-((3-Oxo-3-(p-tolyl)prop-1-en-1-yl)amino)benzenesulfonamide (5d).
Yellow crystals (yield 76 %), m.p. 270-271 °C; IR (KBr, ν cm-1): 3127-3396 broad band
(NH, NH2), 1725 (C=O), 1302, 1151 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 2.38
(s, 3H, CH3), 6.21 (d, 1/3H, Ar-H, J= 8 Hz), 6.50 (d, 2/3H, Ar-H, J= 12 Hz), 7.31-7.63
17

(m, 7.5 H, 5.5 H of Ar-H and 2H of NH2, D2O exchangeable), 7.74 (s, ½ H, Ar-H), 7.80
(d, 1 H, Ar-H, J= 8 Hz), 7.88-7.93 (m, 1.5H, Ar-H), 8.13 (t, ½ H, Ar-H, J= 12 Hz), 10.33
(d, ½ H, NH exchanged with D2O, J= 12 Hz), 12.09 (d, ½ H, NH exchanged with D2O,
J= 12 Hz); 13CNMR (DMSO-d6,100 MHz) ppm: 2 (21.55) (CH3, cis and trans
conformers), 94.71, 99.15 (-CO-CH=CH-, cis and trans conformers), 112.39, 113.49,
119.14, 119.26, 119.73, 120.28, 127.84, 127.88, 129.60, 129.69, 130.85, 136.27, 136.99,
141.17, 142.14, 142.34, 142.66, 143.70, 145.34, 145.93, 146.01 (Aromatic carbons),
187.64, 190.17 (C=O, cis and trans conformers); HRMS (ESI) for C16H16N2O3S, calcd
317.09544, found 317.09525 [M+H]+; Anal. Calcd. for C16H17N2O3S (317.09): C, 60.74;
H, 5.10; N, 8.85; found C, 61.01; H, 5.03; N, 8.97.
4.1.3.5. 3-((3-(4-Methoxyphenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (5e).
Yellow crystals (yield 63 %), m.p. 200-201 °C; IR (KBr, ν cm-1): 3121-3392 broad band
(NH, NH2), 1723 (C=O), 1304, 1155 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 3.84
(s, 3H, OCH3), 6.20 (d, ½ H, Ar-H, J= 7.2 Hz), 6.52 (d, ½ H, Ar-H, J= 12.4 Hz), 7.04 (d,
2 H, Ar-H, J= 7.6 Hz), 7.36-7.56 (m, 5H, 3H of Ar-H and 2H of NH2, D2O
exchangeable), 7.64 (s, ½ H, Ar-H), 7.73 (s, ½ H, Ar-H), 7.84-7.99 (m, 2.5H, Ar-H), 8.12
(t, ½ H, Ar-H, J= 12 Hz), 10.27 (d, ½ H, NH exchanged with D2O, J= 12 Hz), 12.08 (d,
½ H, NH exchanged with D2O, J= 11.6 Hz); 13CNMR (DMSO-d6,100 MHz) ppm:
55.87, 55.90 (OCH3), 94.65, 99.06 (-CO-CH=CH-, cis and trans conformers), 112.35,
113.40, 114.25 (2C), 114.34 (2C), 119.07, 119.15, 119.57, 120.14, 129.90 (2C), 130.81
(2C), 130.84 (2C), 131.58, 132.24, 141.27, 142.26, 143.28, 144.84, 145.96, 146.04,
162.65, 162.85, 186.72, 189.49 (C=O, cis and trans conformers); MS m/z [%]: 331 [M+,
61.03], 332 [100]; Anal. Calcd. for C16H16N2O4S (332.37): C, 57.82; H, 4.85; N, 8.43;
found C, 57.82; H, 4.79; N, 8.50.
4.1.3.6. 3-((3-(4-Nitrophenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (5f).
Yellow crystals (yield 68 %), m.p. 195-197 °C; IR (KBr, ν cm-1): 3126-3397 broad band
(NH, NH2), 1728 (C=O), 1309, 1150 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.26
(d, 1/3H, Ar-H, J= 7.6 Hz), 6.48 (d, 2/3H, Ar-H, J= 12.4 Hz), 7.37-7.68 (m, 5.33 H, 3.33
H of Ar-H and 2H of NH2, D2O exchangeable), 7.81 (s, 2/3 H, Ar-H), 8.02-8.34 (m, 5H,
18

Ar-H), 10.59 (d, ½ H, NH exchanged with D2O, J= 12.4 Hz), 12.16 (d, ½ H, NH
13
exchanged with D2O, J= 12.4 Hz); CNMR (DMSO-d6100 MHz) ppm: 94.87, 99.07 (-
CO-CH=CH-, cis and trans conformers), 113.07, 114.19, 119.53, 119.97, 120.28, 121.05,
123.83, 124.25 (2C), 129.02 (2C), 129.06 (2C), 130.88 (2C), 140.77, 141.68, 144.00,
144.94, 145.60, 145.97, 146.05, 147.29, 149.50, 149.65, 186.45, 188.02 (C=O, cis and
trans conformers); MS m/z [%]: 347 [M+, 96.23], 346[100]; Anal. Calcd. for
C15H13N3O5S (347.35): C, 51.87; H, 3.77; N, 12.10; found C, 52.03; H, 3.78; N, 12.17.
4.1.3.7. 3-((3-(Naphthalen-2-yl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (5g).
Yellow crystals (yield 64 %), m.p. 183-185 °C; IR (KBr, ν cm-1): 3120-3391 broad band
(NH, NH2), 1722 (C=O), 1303, 1154 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.24
(d, ½ H, Ar-H, J= 8 Hz), 6.69 (d, ½ H, Ar-H, J= 12 Hz), 7.37-7.45 (m, 3 H, 1 H of Ar-H
and 2H of NH2, D2O exchangeable), 7.51-7.67 (m, 4.5 H, Ar-H), 7.78 (s, ½ H, Ar-H),
7.79-8.15 (m, 4.5 H, Ar-H), 8.22 (t, ½ H, Ar-H, J= 12 Hz), 8.52 (s, ½ H, Ar-H), 8.66 (s,
½ H, Ar-H), 10.44 (d, ½ H, NH exchanged with D2O, J= 12 Hz), 12.18 (d, ½ H, NH
13
exchanged with D2O, J= 12 Hz); CNMR (DMSO-d6 100 MHz) ppm: 95.00, 99.41 (-
CO-CH=CH-, cis and trans conformers), 112.54, 113.62, 119.25, 119.40, 119.88, 120.44,
124.18, 124.52, 127.18, 127.21, 128.07, 128.39, 128.47, 128.54, 128.63, 128.69, 128.72,
129.82, 129.87, 130.88, 132.81, 132.86, 134.96, 135.03, 136.18, 136.98, 141.13, 142.09,
144.05, 145.67, 145.95, 146.03 (Aromatic carbons) 187.91, 190.24 (C=O, cis and trans
conformers); HRMS (ESI) for C19H17N2O3S, calcd 353.09544, found 353.09537 [M+H]+;
Anal. Calcd. for C19H16N2O3S (353.09): C, 64.76; H, 4.58; N, 7.95; found C, 64.54; H,
4.67; N, 8.10.
4.1.3.8. 3-((3-(Furan-2-yl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (5h).
Yellow crystals (yield 70 %), m.p. 195-197 °C; IR (KBr, ν cm-1): 3125-3396 broad band
(NH, NH2), 1727 (C=O), 1308, 1159 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.00
(d, 1/3H, Ar-H, J= 7.6 Hz), 6.37 (d, 2/3H, Ar-H, J= 12.8 Hz), 6.69 (s, 1H, Ar-H), 7.22 (s,
1H, Ar-H), 7.33-7.56 (m, 5 H, 3 H of Ar-H and 2H of NH2, D2O exchangeable), 7.64 (s,
2/3H, Ar-H), 7.72 (s, 1/3 H, Ar-H), 7.86-7.94 (m, 1.33 H, Ar-H), 8.12 (t, 2/3 H, Ar-H, J=
12.8 Hz), 10.35 (d, ½ H, NH exchanged with D2O, J= 12.8 Hz), 11.77 (d, ½ H, NH
19

exchanged with D2O, J= 12.4 Hz); 13CNMR (DMSO-d6 100 MHz)  ppm: 94.81, 99.00 (-
CO-CH=CH-, cis and trans conformers), 112.53, 112.82, 112.96, 113.53, 115.29, 115.81,
119.24, 119.42, 119.67, 120.38, 130.83 (2C), 141.08, 142.00, 143.15, 145.34, 145.95,
146.02, 146.55, 147.04, 153.41, 154.25, 177.11, 179.55 (C=O, cis and trans conformers);
MS m/z [%]: 292 [M+, 41.49], 184.49 [100]; Anal. Calcd. for C13H12N2O4S (292.31): C,
53.42; H, 4.14; N, 9.58; found C, 53.08; H, 4.22; N, 9.65.
4.1.3.9. 3-((3-Oxo-3-(thiophen-2-yl)prop-1-en-1-yl)amino)benzenesulfonamide (5i).
Yellow crystals (yield 65 %), m.p. 208-210 °C; IR (KBr, ν cm-1): 3120-3391 broad band
(NH, NH2), 1722 (C=O), 1303, 1154 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.12
(d, 1/3H, Ar-H, J= 8 Hz), 6.41 (d, 2/3H, Ar-H, J= 12.0 Hz), 7.21 (m, 1H, Ar-H), 7.35-
7.43 (m, 4 H, 2 H of Ar-H and 2H of NH2, D2O exchangeable), 7.49-7.56 (m, 1.5 H, Ar-
H), 7.63 (s, ½ H, Ar-H), 7.73 (s, 1/3 H, Ar-H), 7.77 (br s, 2/3 H, Ar-H), 7.85-7.92 (m,
1.33 H, Ar-H), 8.11 (t, 2/3 H, Ar-H, J= 12 Hz), 10.38 (d, ½ H, NH exchanged with D2O,
J= 12 Hz), 11.77 (d, ½ H, NH exchanged with D2O, J= 12 Hz); 13CNMR (DMSO-d6 100
MHz)  ppm: 94.88, 99.01 (-CO-CH=CH-, cis and trans conformers), 112.47, 113.47,
119.25, 119.39, 119.72, 120.33, 128.98, 129.14, 130.30, 130.78, 130.85, 133.07, 133.68,
141.09, 142.00, 143.34, 145.13, 145.92, 145.99, 146.27, 147.00, 180.80, 183.66 (C=O,
cis and trans conformers); HRMS (ESI) for C13H12N2O3S2, calcd 309.03621, found
309.03589 [M+H]+; Anal. Calcd. for C13H13N2O3S2 (309.03): C, 50.63; H, 3.92; N, 9.08;
found C, 50.89; H, 3.98; N, 9.13.
4.1.3.10. 4-((3-Oxo-3-phenylprop-1-en-1-yl)amino)benzenesulfonamide (6a).
Yellow crystals (yield 70 %), m.p. 223-225 °C (reported: 220-222 °C [34]); IR (KBr, ν
1
cm-1): 3122-3395 broad band (NH, NH2), 1724 (C=O), 1300, 1150 (SO2); H NMR
(DMSO-d6, 400 MHz)  ppm: 6.24 (d, ½ H, Ar-H, J= 8 Hz), 6.54 (d, ½ H, Ar-H, J= 16
Hz), 7.26 (s, 2H, NH2, D2O exchangeable), 7.31 (d, 1H, Ar-H, J= 8.8 Hz), 7.51-7.59 (m,
4H, Ar-H), 7.77 (t, ½ H, Ar-H, J= 8.8 Hz), 7.89 (d, 2H, Ar-H, J= 7.6 Hz), 7.99 (d, 2H,
Ar-H, J= 7.6 Hz), 8.17 (t, ½ H, Ar-H, J= 12.4 Hz), 10.38 (d, ½ H, NH exchanged with
D2O, J= 12 Hz), 12.05 (d, ½ H, NH exchanged with D2O, J= 12 Hz); 13CNMR (DMSO-
d6,100 MHz)  ppm: 94.96, 99.58 (-CO-CH=CH-, cis and trans conformers), 115.58,
20

115.87, 115.97, 116.09, 116.19, 116.53, 127.91, 128.10, 130.48, 130.53, 130.57, 130.62,
135.42, 136.14, 137.64, 138.76, 143.34,143.99, 144.29, 145.59, 163.46, 163.67, 165.94
(Aromatic carbons), 186.67,189.04 (C=O, cis and trans conformers); MS m/z [%]: 302
[M+, 31.74], 301 [100]; Anal. Calcd. for C15H14N2O3S (302.35): C, 59.59; H, 4.67; N,
9.27; found C, 59.81; H, 4.73; N, 9.44.
4.1.3.11. 4-((3-(4-Fluorophenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (6b).
Pale yellow crystals (yield 65 %), m.p. 225-227 °C; IR (KBr, ν cm-1): 3120-3390 broad
1
band (NH, NH2), 1720, (C=O), 1310, 1155 (SO2); H NMR (DMSO-d6, 400 MHz) 
ppm: 6.23 (d, ½ H, Ar-H, J= 8 Hz), 6.50 (d, ½ H, Ar-H, J= 12 Hz), 7.27-7.36 (m, 5H, 3H
of Ar-H and 2H of NH2, D2O exchangeable), 7.51 (d, 1H, Ar-H, J= 8 Hz), 7.75 (t, 2H,
Ar-H, J= 8 Hz), 7.95-7.99 (m, 1.5H, Ar-H), 8.06-8.10 (m, 1H, Ar-H), 8.17 (d, ½ H, Ar-
H, J= 12 Hz), 12.0 (d, 1H, NH exchanged with D2O, J= 12 Hz); HRMS (ESI) for
C15H13FN2O3S, calcd 321.07037, found 321.07037 [M+H]+; Anal. Calcd. for
C15H14FN2O3S (320.34): C, 56.24; H, 4.09; N, 8.75; found C, 56.41; H, 4.18; N, 8.97.
4.1.3.12. 4-((3-(4-Chlorophenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (6c).
Reddish yellow crystals (yield 80 %), m.p. 257-259 °C; IR (KBr, ν cm-1): 3122-3395
broad band (NH, NH2), 1735 (C=O), 1320, 1160 (SO2); 1H NMR (DMSO-d6, 400 MHz)
 ppm: 6.21 (d, ½ H, Ar-H, J= 8 Hz), 6.49 (d, ½ H, Ar-H, J= 12.8 Hz), 7.27 (s, 2H, NH2,
D2O exchangeable), 7.31 (d, 1H, Ar-H, J= 10.8 Hz), 7.51 (d, 1H, Ar-H, J= 8.4 Hz), 7.56
(d, 2H, Ar-H, J= 8 Hz), 7.77 (t, ½ H, Ar-H, J= 8.8 Hz), 7.89 (d, 2H, Ar-H, J= 8 Hz), 7.99
(d, 2H, Ar-H, J= 8 Hz), 8.18 (t, ½ H, Ar-H, J= 12.8 Hz), 10.43 (d, ½ H, NH exchanged
with D2O, J= 12.8 Hz), 12.02 (d, ½ H, NH exchanged with D2O, J= 12.4 Hz); MS m/z
[%]: 338 [M++2, 24.15], 336 [M+, 58.57], 335 [100]; Anal. Calcd. for C15H13ClN2O3S
(336.79): C, 53.49; H, 3.89; N, 8.32; found C, 53.72; H, 3.95; N, 8.45.
4.1.3.13. 4-((3-(4-Bromophenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (6d).
White crystals (yield 74 %), m.p. 268-270 °C; IR (KBr, ν cm-1): 3130-3400 broad band
(NH, NH2), 1730 (C=O), 1300, 1160 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.22
(d, ½ H, Ar-H, J= 8 Hz), 6.48 (d, ½ H, Ar-H, J= 12 Hz), 7.62-7.33 (m, 3H, 1H of Ar-H
21

and 2H of NH2, D2O exchangeable), 7.52 (d, 1H, Ar-H, J= 8 Hz), 7.72-7.84 (m, 5H, Ar-
H), 7.93 (d, 1H, Ar-H, J= 8 Hz), 7.98-8.03 (m, ½ H, Ar-H), 8.19 (d, ½ H, Ar-H, J= 12
Hz), 10.47 (s, ½ H, NH exchanged with D2O), 12.02 (d, ½ H, NH exchanged with D2O,
13
J= 12 Hz); CNMR (DMSO-d6,100 MHz) ppm: 94.90, 99.52, 115.68, 116.64, 126.08,
126.49, 127.90, 128.09, 129.84, 129.87, 132.10, 132.17, 137.75, 137.83, 138.61, 138.89,
143.26, 144.20, 144.33, 145.94, 187.01 (Aromatic carbons), 189.19 (C=O); HRMS (ESI)
for C15H13BrN2O3S, calcd 380.99030, found 380.99020 [M+H]+; Anal. Calcd. for
C15H14BrN2O3S (380.99): C, 47.26; H, 3.44; N, 7.35; found C, 47.08; H, 3.41; N, 7.45.
4.1.3.14. 4-((3-Oxo-3-(p-tolyl)prop-1-en-1-yl)amino)benzenesulfonamide (6e).
Buff crystals (yield 65 %), m.p. 240-242 °C; IR (KBr, ν cm-1); 3125-3395 broad band
(NH, NH2), 1728 (C=O), 1315, 1150 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 2.38
(s, 3H, CH3), 6.21 (d, ½ H, Ar-H, J= 8.4 Hz), 6.53 (d, ½ H, Ar-H, J= 12.4 Hz), 7.25 (s,
2H, NH2, D2O exchangeable), 7.29-7.33 (m, 3H, Ar-H), 7.49 (d, 1H, Ar-H, J= 8.8 Hz),
7.76-7.82 (m, 2.5 H, Ar-H), 7.89-7.96 (m, 2 H, Ar-H), 8.14 (t, ½ H, Ar-H, J= 12.8 Hz),
10.32 (d, ½ H, NH exchanged with D2O, J= 12.8 Hz), 12.03 (d, ½ H, NH exchanged with
D2O, J= 12 Hz); 13CNMR (DMSO-d6,100 MHz) ppm: 21.54, 21.55 (CH3), 95.22,
100.00 (-CO-CH=CH-, cis and trans conformers), 115.44 (2C), 116.35 (2C), 127.87 (2C),
127.91 (2C), 127.95 (2C), 128.13 (2C), 129.61 (2C), 129.70 (2C), 136.26, 137.00,
137.48, 138.60, 142.38, 142.74, 143.39, 143.47, 144.46, 144.98, 187.76, 190.32 (C=O,
cis and trans conformers); MS m/z [%]: 316 [M+, 28.81], 301 [100]; Anal. Calcd. for
C16H16N2O3S (316.38): C, 60.74; H, 5.10; N, 8.85; found C, 60.98; H, 5.13; N, 8.99.
4.1.3.15. 4-((3-(4-Methoxyphenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (6f).
Yellow crystals (yield 68 %), m.p. 250-252 °C; IR (KBr, ν cm-1): 3127-3395 broad band
(NH, NH2), 1724 (C=O), 1310, 1155 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 3.81
(s, 3H, OCH3), 6.18 (d, ½ H, Ar-H, J = 8 Hz), 6.50 (d, ½ H, Ar-H, J = 16 Hz), 7.01 (d,
2H, Ar-H, J= 8 Hz), 7.20 (s, 2H, NH2, D2O exchangeable), 7.24 (d, 2H, Ar-H, J = 12
Hz), 7.44 (d, 1H, Ar-H, J = 8 Hz), 7.71 (t, 1.5 H, Ar-H, J = 8 Hz), 7.85-7.90 (m, 1H, Ar-
H), 7.95 (d, 1H, Ar-H, J = 8 Hz), 8.08 (t, ½ H, Ar-H, J = 12 Hz), 10.24 (d, ½ H, NH
exchanged with D2O, J = 12 Hz), 11.98 (d, ½ H, NH exchanged with D2O, J = 12 Hz);
22

MS m/z [%]: 332 [M+, 29.04], 331 [100]; Anal. Calcd. for C16H16N2O4S (332.37): C,
57.82; H, 4.85; N, 8.43; found C, 57.60; H, 4.97; N, 8.50.
4.1.3.16. 4-((3-(4-Nitrophenyl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (6g).
Yellow crystals (yield 70 %), m.p. 235-237 °C; IR (KBr, ν cm-1): 3122-3380 broad band
(NH, NH2), 1720 (C=O), 1300, 1150 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.27
(d, ½ H, Ar-H, J= 8 Hz), 6.49 (d, ½ H, Ar-H, J= 12.8 Hz), 7.27-7.37 (m, 3H, 1H of Ar-H
and 2H of NH2, D2O exchangeable), 7.56 (d, 1H, Ar-H, J= 8.4 Hz), 7.77-7.82 (m, 2H,
Ar-H), 8.06-8.10 (m, 1.5H, Ar-H), 8.19-8.35 (m, 3.5H, Ar-H), 10.59 (d, ½ H, NH
exchanged with D2O, J= 12.8 Hz), 12.08 (d, ½ H, NH exchanged with D2O, J= 12.8 Hz);
13CNMR (DMSO-d6,100 MHz) ppm: 95.29, 99.88, (-CO-CH=CH-, cis and trans
conformers), 115.99 (2C), 117.01 (2C), 123.84 (2C), 124.27 (2C), 127.91 (2C), 128.09
(2C), 129.07 (2C), 129.09 (2C), 138.05, 139.42, 143.03, 143.94, 143.95, 144.91, 145.36,
146.97, 149.52, 149.71, 186.66, 188.20 (C=O, cis and trans conformers); MS m/z [%]:
347 [M+, 33.42], 346 [100]; Anal. Calcd. for C15H13N3O5S (347.35): C, 51.87; H, 3.77;
N, 12.10; found C, 52.04; H, 3.80; N, 12.15.
4.1.3.17. 4-((3-(Naphthalen-2-yl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (6h).
White crystals (yield 73 %), m.p. 271-272 °C; IR (KBr, ν cm-1): 3120-3380 broad band
(NH, NH2), 1721 (C=O), 1300, 1150 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.40
(d, ½ H, Ar-H, J= 8 Hz), 6.68 (d, ½ H, Ar-H, J= 12.8 Hz), 7.22 (s, 2H, NH2, D2O
exchangeable), 7.27 (s, 1H, Ar-H), 7.30 (d, 1H, Ar-H, J= 8 Hz), 7.51 (d, 1H, Ar-H, J= 8
Hz), 7.57-7.64 (m, 2H, Ar-H), 7.73-7.79 (m, 2H, Ar-H), 7.95-8.07 (m, 2.5H, Ar-H), 8.10
(d, 1H, Ar-H, J= 8 Hz), 8.18 (t, ½ H, Ar-H, J= 12 Hz), 8.48 (s, ½ H, Ar-H), 8.64 (s, ½ H,
Ar-H), 10.40 (d, ½ H, NH exchanged with D2O, J= 12 Hz), 12.08 (d, ½ H, NH
exchanged with D2O, J= 12 Hz); MS m/z [%]: 352 [M+, 8.22], 303 [100]; Anal. Calcd.
for C19H16N2O3S (352.41): C, 64.76; H, 4.58; N, 7.95; found C, 64.90; H, 4.57; N, 8.01.
4.1.4.18. 4-((3-(Furan-2-yl)-3-oxoprop-1-en-1-yl)amino)benzenesulfonamide (6i).
Yellow crystals (yield 81 %), m.p. 263-265 °C; IR (KBr, ν cm-1): 3125-3395 broad band
(NH, NH2), 1724 (C=O), 1318, 1150 (SO2); 1H NMR (DMSO-d6, 400 MHz)  ppm: 6.01
23

(d, ½ H, Ar-H, J= 8 Hz), 6.39 (d, ½ H, Ar-H, J= 12.8 Hz), 6.69 (s, 1 H, Ar-H), 7.22-7.36
(m, 4H, 2H of Ar-H and 2H of NH2, D2O exchangeable), 7.46 (d, 1H, Ar-H, J= 8.4 Hz),
7.75-7.79 (m, 2H, Ar-H), 7.89-7.95 (m, 1.5H, Ar-H), 8.13 (t, ½ H, Ar-H, J= 12.8 Hz),
10.36 (d, ½ H, NH exchanged with D2O, J= 12.8 Hz), 11.72 (d, ½ H, NH exchanged with
13
D2O, J= 12 Hz); CNMR (DMSO-d6,100 MHz) ppm: 95.29, 99.74 (-CO-CH=CH-, cis
and trans conformers), 112.84, 113.00, 115.42, 115.53 (2C), 116.02, 116.32 (2C), 127.93
(2C), 128.10 (2C), 137.61, 138.64, 142.86, 143.36, 144.29, 144.99, 146.63, 147.18,
153.36, 154.21, 177.10, 179.58 (C=O, cis and trans conformers); MS m/z [%]: 292.31
[M+, 39.27], 42 [100]; Anal. Calcd. for C13H12N2O4S (292.31): C, 53.42; H, 4.14; N,
9.58; found C, 53.19; H, 4.11; N, 9.45.
4.1.3.19. 4-((3-Oxo-3-(thiophen-2-yl)prop-1-en-1-yl)amino)benzenesulfonamide (6j).
Yellow crystals (yield 80 %), m.p. 255-257 °C (reported: 254.6 °C [35]); IR (KBr, ν cm-
1): 3125-3400 broad band (NH, NH2), 1720 (C=O), 1300, 1150 (SO2); 1H NMR (DMSO-
d6, 400 MHz)  ppm: 6.14 (d, ½ H, Ar-H, J= 8 Hz), 6.44 (d, ½ H, Ar-H, J= 12 Hz), 7.22-
7.32 (m, 4H, 2H of Ar-H and 2H of NH2, D2O exchangeable), 7.48 (d, 1H, Ar-H, J= 8
Hz), 7.75-7.78 (m, 3H, Ar-H), 7.88-7.94 (m, 1.5H, Ar-H), 8.12 (t, ½ H, Ar-H, J= 12 Hz),
10.39 (d, ½ H, NH exchanged with D2O, J= 12 Hz), 11.72 (d, ½ H, NH exchanged with
13
D2O, J= 12 Hz); CNMR (DMSO-d6,100 MHz) ppm: 95.36, 99.76 (-CO-CH=CH-, cis
and trans conformers), 115.52, 116.32, 127.90, 128.09, 129.01, 129.17, 130.39, 130.96,
133.18, 133.86, 137.57, 138.58, 143.04, 143.38, 144.29, 144.79, 146.24, 146.94
(Aromatic carbons), 180.81, 183.76 (C=O, cis and trans conformers); HRMS (ESI) for
C13H12N2O3S2, calcd 309.03621, found 309.03607 [M+H]+; Anal. Calcd. for
C13H13N2O3S2 (309.03): C, 50.63; H, 3.92; N, 9.08; found C, 50.88; H, 4.01; N, 9.14.
4.2. Single crystal X-ray crystallographic analysis
Enaminone 5a was obtained as single crystals by slow evaporation from ethanol
solution of the pure compound at room temperature. Data were collected on a Bruker
APEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Mo Kα
radiation, λ = 0.71073 Å at 293 (2) K. Cell refinement and data reduction were carried
out by Bruker SAINT. SHELXT [36-38] was used to solve structure. The final
24

refinement was carried out by full-matrix least-squares techniques with anisotropic
thermal data for nonhydrogen atoms on ??. CCDC 1856635 contains the supplementary
crystallographic data for this compound can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
4.3. Biological Evaluation
4.3.1. CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity, as reported earlier [31]. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff
equation as reported earlier [39], and represent the mean from at least three different
determinations. The four tested CA isofoms were recombinant ones obtained in-house as
reported earlier [40].
Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship of
Scientific Research at King Saud University for its funding of this research through the
Research Group Project no. RG-1439-65.
References
References
[1] C.T. Supuran, Structure and function of carbonic anhydrases, Biochem. J. 473 (2016)
2023-2032.
[2] C. T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and
activators. Nat. Rev. Drug Discovery 7 (2008) 168-181.
[3] M. Aggarwal, C.D. Boone, B. Kondeti, R. McKenna, Structural annotation of human
carbonic anhydrases, J. Enzyme Inhib. Med. Chem. 28 (2013) 267-277.
[4] C.T. Supuran. How many carbonic anhydrase inhibition mechanisms exist? J Enzyme
Inhib Med Chem. 31 (2016) 345-360.
25

[5] C.T. Supuran, Structure-based drug discovery of carbonic anhydrase inhibitors, J.
Enzyme Inhib. Med. Chem. 27 (2012) 759-772.
[6] G. De Simone, C.T. Supuran, Carbonic anhydrase IX: Biochemical and
crystallographic characterization of a novel antitumor target. Biochim. Biophys. Acta
1804 (2010) 404-409.
[7] C.T. Supuran, Carbonic Anhydrase Inhibition and the Management of Hypoxic
Tumors. Metabolites 7 (2017) 48.
[8] C.T. Supuran, V. Alterio, A. Di Fiore, K. D’Ambrosio, F. Carta, S.M. Monti, G. De
Simone, Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and
cancer stem cells: Three for the price of one. Med. Res. Rev. 2018;in press; doi:
10.1002/med.21497.
[9] P.C. McDonald, J.Y. Winum, C.T. Supuran, S. Dedhar, Recent Developments in
Targeting Carbonic Anhydrase IX for Cancer Therapeutics. Oncotarget 3 (2012) 84-97.
[10] W.M. Eldehna, M.F. Abo-Ashour, A. Nocentini, R.S. El-Haggar, S. Bua, A.
Bonardi, S.T. Al-Rashood, G.S. Hassan, P. Gratteri, H.A. Abdel-Aziz, C.T. Supuran,
Enhancement of the tail hydrophobic interactions within the carbonic anhydrase IX active
site via structural extension: Synthesis of novel N-substituted isatins-SLC-0111 hybrids
as carbonic anhydrase inhibitors and antitumor agents, Eur. J. Med. Chem. (2018)
Accepted manuscript (Doi: 10.1016/j.ejmech.2018.10.068).
[11] H. S. Ibrahim, H.A. Allam, W R. Mahmoud, A. Bonardi, A. Nocentini, P. Gratteri,
E. S. Ibrahim, H. A. Abdel-Aziz, and C. T. Supuran, Dual-tail arylsulfone-based
benzenesulfonamides differently match the hydrophobic and hydrophilic halves of human
carbonic anhydrases active sites: Selective inhibitors for the tumor-associated hCA IX
isoform, Eur. J. Med. Chem. 152 (2018) 1-9.
[12] Y. Lou, P.C. McDonald, A. Oloumi, S. Chia, C. Ostlund, A. Ahmadi, A. Kyle, U.
Auf dem Keller, S. Leung, D. Huntsman, B. Clarke, B.W. Sutherland, D. Waterhouse, M.
Bally, C. Roskelley, C.M. Overall, A. Minchinton, F. Pacchiano, F. Carta, A. Scozzafava,
N. Touisni, J.Y. Winum, C.T. Supuran, S. Dedhar, Targeting tumor hypoxia: suppression
26

of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors, Cancer
Res. 71 (2011) 3364-3376.
[13] F. Pacchiano, M. Aggarwal, B.S. Avvaru, A.H. Robbins, A. Scozzafava, R.
McKenna, C.T. Supuran, Selective hydrophobic pocket binding observed within the
carbonic anhydrase II active site accommodate different 4-substituted-ureido-
benzenesulfonamides and correlate to inhibitor potency, Chem. Commun. (Camb) 46
(2010) 8371–8373.
[14] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar,
C.T. Supuran, Ureido-substituted benzenesulfonamides potently inhibit carbonic
anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis, J.
Med. Chem. 54 (2011) 1896–1902.
[15] A. Angeli, D. Tanini, T.S. Peat, L. Di C. Mannelli, G. Bartolucci, A. Capperucci, C.
Ghelardini, C.T. Supuran, F. Carta, Discovery of new selenoureido analogues of 4-(4-
fluorophenylureido) benzenesulfonamide as carbonic anhydrase inhibitors, ACS Med.
Chem. Lett. 8 (2017) 963-968.
[16] S. Akocak, N. Lolak, S. Bua, I. Turel, C.T. Supuran, Synthesis and biological
evaluation of novel N,N0-diaryl cyanoguanidines acting as potent and selective carbonic
anhydrase II inhibitors, Bioorg. Chem. 77 (2018) 245-251.
[17] N. Lolak, S. Akocak, S. Bua, M. Koca, C.T. Supuran, Design and synthesis of novel
1,3-diaryltriazene-substituted sulfonamides as potent and selective carbonic anhydrase II
inhibitors, Bioorg. Chem. 77 (2018) 542-547.
[18] F. Carta, D. Vullo, S. M. Osman, Z. AlOthman, C.T. Supuran, Synthesis and
carbonic anhydrase inhibition of a series of SLC-0111 analogs, Bioorg. Med. Chem. 25
(2017) 2569-2576.
[19] M. Bozdag, F. Carta, M. Ceruso, M. Ferraroni, P.C. McDonald, S. Dedhar, C.T.
Supuran, Discovery of 4-Hydroxy-3-(3-(phenylureido)benzenesulfonamides As SLC-
0111 Analogues For the Treatment of Hypoxic Tumors Overexpressing Carbonic
27

Anhydrase
IX,
J.
Med.
Chem.,
Accepted
Manuscript,
DOI:
10.1021/acs.jmedchem.8b00770.
[20] R.G. Gieling, M. Babur, L. Mamnani, N. Burrows, B.A. Telfer, F. Carta, J.Y.
Winum, A. Scozzafava, C.T. Supuran, K.J. Williams, Antimetastatic effect of sulfamate
carbonic anhydrase IX inhibitors in breast carcinoma xenografts. J. Med. Chem. 55
(2012) 5591-5600.
[21] C. Asakawa, M. Ogawa, K. Kumata, M. Fujinaga, T. Yamasaki, L. Xie, J. Yui, K.
Kawamura, T. Fukumura, M.R. Zhang, Radiosynthesis of three [11C]ureido-substituted
benzenesulfonamides as PET probes for carbonic anhydrase IX in tumors. Bioorg. Med.
Chem. Lett. 21 (2011) 7017-7020.
[22] J. Pan, J. Lau, F. Mesak, N. Hundal, M. Pourghiasian, Z. Liu, F. Benard, S. Dedhar,
C.T. Supuran, K.S. Lin, Synthesis and evaluation of 18F-labeled carbonic anhydrase IX
inhibitors for imaging with positron emission tomography. J. Enzyme Inhib. Med. Chem.
29 (2014) 249-255.
[23] C. L. Lomelino, B. P. Mahon, R. McKenna, F. Carta, C.T. Supuran, Kinetic and X-
ray crystallographic investigations on carbonic anhydrase isoforms I, II, IX and XII of a
thioureido analog of SLC-0111, Bioorg. Med. Chem. 5 (2016) 976-981.
[24] M.Y. Mboge, B.P. Mahon, N. Lamas, L. Socorro, F. Carta, C.T. Supuran, S.C. Frost,
R. McKenna, Structure activity study of carbonic anhydrase IX: Selective inhibition with
ureidosubstituted benzenesulfonamides, Eur. J. Med. Chem. 132 (2017) 184-191.
[25] W.M. Eldehna, M. Fares, M. Ceruso, H.A. Ghabbour, S.M. Abou-Seri, H.A. Abdel-
Aziz, D.A. El Ella, C.T. Supuran, Amido/ureidosubstituted benzenesulfonamides-isatin
conjugates as low nanomolar/subnanomolar inhibitors of the tumor-associated carbonic
anhydrase isoform XII, Eur. J. Med. Chem. 110 (2016) 259-266.
[26] A.M. Alafeefy, R. Ahmad, M. Abdulla, W.M. Eldehna, A.M.S. Al-Tamimi, H.A.
Abdel-Aziz, O. Al-Obaid, F. Carta, A.A. Al-Kahtani, C.T. Supuran, Development of
certain new 2-substituted-quinazolin-4-yl-aminobenzenesulfonamide as potential
antitumor agents, Eur. J. Med. Chem. 109 (2016) 247-253.
28

[27] W.M. Eldehna, G.H. Al-Ansary, S. Bua, A. Nocentini, P. Gratteri, A. Altoukhy, H.
Ghabbour, H.Y. Ahmed, C.T. Supuran, Novel indolin-2-one-based sulfonamides as
carbonic anhydrase inhibitors: synthesis, in vitro biological evaluation against carbonic
anhydrases isoforms I, II, IV and VII and molecular docking studies, Eur. J. Med. Chem.
127 (2017) 521-530.
[28] W.M. Eldehna, M.F. Abo-Ashour, A. Nocentini, P. Gratteri, H.I. Eissa, M. Fares,
O.E.Ismael, H.A. Ghabbour, M.M. Elaasser, H.A. Abdel-Aziz, C.T. Supuran, Novel 4/3-
((4-oxo-5-(2-oxoindolin-3-ylidene) thiazolidin-2-ylidene) amino) benzenesulfonamides:
Synthesis, carbonic anhydrase inhibitory activity, anticancer activity and molecular
modelling studies, Eur. J. Med. Chem. 139 (2017) 250-262.
[29] M.F. Abo-Ashour, W.M. Eldehna, A. Nocentini, H.S. Ibrahim, S. Bua, S.M. Abou-
Seri, C.T. Supuran, Novel hydrazido benzenesulfonamides-isatin conjugates: Synthesis,
carbonic anhydrase inhibitory activity and molecular modeling studies, Eur. J. Med.
Chem. 157 (2018) 28-36.
[30] H.A. Abdel-Aziz, H.A. Ghabbour, W.M. Eldehna, M.M. Qabeel, H.-K. Fun,
Synthesis, Crystal Structure, and Biological Activity of cis/trans Amide Rotomers of (Z)-
N'-(2-Oxoindolin-3-ylidene)formohydrazide, Journal of Chemistry, 2014, Article ID
760434, 7 pages (2014).
[31] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase I. Stop-
flow kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246 (1971)
2561-2573.
[32] (a) W. M. Eldehna, A. Altoukhy, H. Mahrous, H. A. Abdel-Aziz, Design, synthesis
and QSAR study of certain isatin-pyridine hybrids as potential anti-proliferative agents,
Eur. J. Med. Chem. 90 (2015), 684-694. (b) W. M. Eldehna, M. Fares, M.M. Abdel-Aziz,
H.A. Abdel-Aziz, Design, Synthesis and Antitubercular Activity of Certain Nicotinic
Acid Hydrazides, Molecules 20, (2015) 8800-8815.
[33] (a) D.H. Soliman, W.M. Eldehna, H.A. Ghabbour, M.M. Kabil, M.M. Abdel-Aziz,
H.A. Abdel-Aziz, Novel 6-Phenylnicotinohydrazide Derivatives: Design, Synthesis and
29