Inhibition studies on a panel of human carbonic anhydrases with N1-substituted secondary sulfonamides incorporating thiazolinone or imidazolone-indole tails

Article abstract of DOI:10.1080/14756366.2018.1446432

Being the primary sulfonamide among the most efficient zinc binding group (ZBG) to design inhibitors for the metallo-enzymes carbonic anhydrases (CA, EC 4.2.1.1), herein, we propose an investigation on four physiologically important human (h) CAs (hCA I, II, IV, and IX) with N¹-substituted secondary sulfonamides incorporating thiazolinone or imidazolone-indole tails. The effect of the functionalisation of the sulfonamide group with five different substitution patterns, namely acetyl, pyridine, thiazole, pyrimidine, and carbamimidoyl, was evaluated in relation to the inhibition profile of the corresponding primary sulfonamide analogues. With most of these latter being nanomolar inhibitors of all four considered isoforms, a totally counterproductive effect on the inhibition potency can be ascribed to N¹-functionalisations of the ZBG primary sulfonamide structure with pyridine, thiazole, and pyrimidine moieties. On the other hand, incorporation of less hindered groups, such as sulfonylacetamides and sulfonylguanidines, maintained a certain degree of activity dependent on the tailing moiety, with K_Is spanning in the low micromolar range.

Full text of DOI:10.1080/14756366.2018.1446432

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Inhibition studies on a panel of human carbonic
anhydrases with N1-substituted secondary
sulfonamides incorporating thiazolinone or
imidazolone-indole tails
Fadi M. Awadallah, Silvia Bua, Walaa R. Mahmoud, Hossam H. Nada, Alessio
Nocentini & Claudiu T. Supuran
To cite this article: Fadi M. Awadallah, Silvia Bua, Walaa R. Mahmoud, Hossam H. Nada,
Alessio Nocentini & Claudiu T. Supuran (2018) Inhibition studies on a panel of human carbonic
anhydrases with N1-substituted secondary sulfonamides incorporating thiazolinone or imidazolone-
indole tails, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 629-638, DOI:
10.1080/14756366.2018.1446432
To link to this article: https://doi.org/10.1080/14756366.2018.1446432
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 14 Mar 2018.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 629–638
https://doi.org/10.1080/14756366.2018.1446432
RESEARCH PAPER
Inhibition studies on a panel of human carbonic anhydrases with N1-substituted
secondary sulfonamides incorporating thiazolinone or imidazolone-indole tails
Fadi M. Awadallaha, Silvia Buab, Walaa R. Mahmouda, Hossam H. Nadac, Alessio Nocentinib and
Claudiu T. Supuranb
b
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt; Department NEUROFARBA – Pharmaceutical and
c
Nutraceutical Section, University of Firenze, Firenze, Italy; Pharmaceutical Chemistry Department, Faculty of Pharmacy, Badr University,
Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 6 February 2018
Accepted 26 February 2018
Being the primary sulfonamide among the most efficient zinc binding group (ZBG) to design inhibitors for
the metallo-enzymes carbonic anhydrases (CA, EC 4.2.1.1), herein, we propose an investigation on four
physiologically important human (h) CAs (hCA I, II, IV, and IX) with N1-substituted secondary sulfonamides
incorporating thiazolinone or imidazolone-indole tails. The effect of the functionalisation of the sulfona-
mide group with five different substitution patterns, namely acetyl, pyridine, thiazole, pyrimidine, and car-
bamimidoyl, was evaluated in relation to the inhibition profile of the corresponding primary sulfonamide
analogues. With most of these latter being nanomolar inhibitors of all four considered isoforms, a totally
counterproductive effect on the inhibition potency can be ascribed to N1-functionalisations of the ZBG pri-
mary sulfonamide structure with pyridine, thiazole, and pyrimidine moieties. On the other hand, incorpor-
ation of less hindered groups, such as sulfonylacetamides and sulfonylguanidines, maintained a certain
degree of activity dependent on the tailing moiety, with KIs spanning in the low micromolar range.
KEYWORDS
Carbonic anhydrase;
sulfonamides; inhibitor;
indole; zinc binding-group
1. Introduction
ZBG (ring approach)3,5 or appending different tails to the aromatic
ring just mentioned (tail approach)3,5–7
.
Primary sulfonamide is the most efficient zinc binding group (ZBG)
to design inhibitors for the metallo-enzymes carbonic anhydrases
(CA, EC 4.2.1.1), being its structural features ideal for the binding
to the Zn2þ ion present at the bottom of the active site cavity
and the residues nearby1–3. The negatively charged nitrogen of
SO2NHꢀ coordinates the positively charged metal ion replacing
the physiological zinc-bound nucleophile, with the proton on the
coordinated nitrogen atom being at H-bond distance to Thr199
OG1 atom, which act as acceptor (Figure 1)1.
The previously accepted idea8 that only primary sulfonamides
may act as effective inhibitors has been shown to be incorrect,
but only few modifications were carried out on the sulfonamide
moiety1a, in comparison to the high number of tails appended at
the aromatic ring incorporating this ZGB6,3,9,10. The rational of this
choice could be that of not losing the ideal features of primary
sulfonamides, i.e. the negative charge on nitrogen and the pres-
ence of a proton on it, which are both important for the inhibition
mechanism1–3. Conversely, it should be taken into account that
N1-substitution with proper functional moieties could also elicit
novel inhibition mechanisms1.
Benzenesulfonamides constitute the most common and best
characterised class of CA inhibitors (CAIs)3. The presence of an aro-
matic/heteroaromatic scaffold bearing the sulfonamide group fur-
ther stabilise the enzyme-ligand complex, by several Van der
Waals interactions taking place with residues Gln92, Val121,
Secondary sulfonamides maintain the possibility to coordinate
the Zn ion in the deprotonated form, as confirmed by X-ray crys-
tallography. Di Fiore et al. demonstrated that N-hydroxy and N-
methoxy sulfonamides are able to bind to the Zn(II) ion in the
deprotonated form11. An analogous inhibition mechanism has
been identified for the cyclic secondary sulfonamide saccharin and
its derivatives12. Likewise, we previously reported that the incorp-
oration of a nitro-group at the N1-atom of aromatic sulfonamide
moieties does not deprive them of CA inhibitory efficacy, although
the precise inhibitory mode has not been clarified yet13.
Phe131, Leu198, and Thr200 (hCA II binding site (Figure 1))1,3
.
All CAs found in humans belong to the a-class (a-CA) and are
characterised as sixteen isoforms, which differ by molecular fea-
tures, oligomeric arrangement, cellular localisation, distribution in
organs and tissues, expression levels, and kinetic properties1,2
.
Abnormal levels or activities of most sixteen hCA isoforms have
been often associated with different human diseases, making
these isozymes of great interest for the design of inhibitors which
selectively target specific isoforms, in order to reduce the overall
side effects exhibited by most non-isoform selective CAIs clinically
used up to now1–4. Most efforts to design isoform selective CAIs
Tertiary sulfonamides lose their acid character, paving the way
to a totally new inhibition mechanism. Carradori et al.14,15 recently
synthesised different series of tertiary sulfonamides, namely pro-
have been pursued by modulating the ring directly linked to the benecid and saccharin derivatives, obtaining interesting and
CONTACT Fadi M. Awadallah
Street, Cairo 11562, Egypt; Claudiu T. Supuran
fadi.awadallah@pharma.cu.edu.eg
Pharmaceutical Chemistry department, Faculty of Pharmacy, Cairo University, Kasr El-Eini
claudiu.supuran@unifi.it Department NEUROFARBA – Pharmaceutical and Nutraceutical Section, University of
Firenze, via Ugo Schiff 6, Firenze I-50019, Italy
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

630
F. M. AWADALLAH ET AL.
carried out at the Regional Center for Mycology and
Biotechnology, Al-Azhar University, Egypt. Reaction were routinely
monitored by Thin Layer Chromatography (TLC) on silica gel pre-
coated F254 Merck plates. Unless otherwise noted, all solvents and
reagents were commercially available and used without further
purification. Compounds 222, 4a–f23,24, 6a–f23,24, 7a25, 8a,b26,27
and 9a28 were prepared according to the reported procedures.
General procedure for the preparation of compounds 4a-f
A mixture of indole-3-carboxaldehyde 2 (1.45 g, 10 mmol) and the
appropriate sulfonamide 3a–f (10 mmol) were grinded and mixed
together using a mortar and a pestle. The mixture was fused on a
watch glass using drops of glacial acetic acid. The resulted paste
was washed with ethanol/water mixture then crystallised from
ethanol to afford compounds 4a–f.
4-{[(1H-indol-3-yl)methylene]amino}benzenesulfonamide (4a)
Figure 1. Schematic representation of the binding mode of benzenesulfonamide
within the hCA II active site.
Yellowish brown powder, (yield 60%), m.p. 180–182 ꢁC; IR (KBr, ꢀ
cmꢀ1): 3479–3244 (NHs), 1311 and 1149 (SO2); 1H NMR (DMSO-d6,
400 MHz) d ppm: 5.80 (s, 2H, NH2, D2O exchangeable), 6.58 (d, 2H,
Ar–H, J ¼ 8.6), 6.88 (s, 1H, NH, D2O exchangeable), 7.21–7.26 (m,
2H, Ar–H), 7.45 (d, 2H, Ar–H, J ¼ 8.6), 7.51 (d, 1H, Ar–H, J ¼ 7.7),
8.09 (d, 1H, Ar–H, J ¼7.3), 8.28 (s, 1H, Ar–H), 9.93 (s, 1H, Ar–H); MS
m/z: 299.0 [M]þ; Anal. Calcd. for C15H13N3O2S (299.35): C, 60.18; H,
4.38; N, 14.04; Found C, 60.41; H, 4.28; N, 13.89.
selective inhibition profile. However, the inhibition mechanism
with such compounds was not yet been elucidated. A plethora of
series of benzenesulfonamide bearing indole or thiazolidinone
moieties have also been reported so far and demonstrated to pos-
sess an effective inhibitory profile against
a wide panel
of hCAs16–20
.
In an earlier investigation of some of us, a series of benzenesul-
fonamides linked to a chromone nucleus through thiazolinone
and imidazolone spacers demonstrated excellent inhibitory activity
against a panel of hCAs in a nanomolar range21. In continuation of
the previous work, and with the aim to pursue the identification
of new potent and selective CAIs, herein we explore the effect of
the functionalisation of the sulfonamide group on the inhibitory
properties by appending several moieties, namely acetyl, pyridine,
thiazole, pyrimidine, and carbamimidoyl, at the N1-atom. A consist-
ent series of imine-, thiazolinone-, and imidazolone benzenesulfo-
namides appending an indole ring instead of the chromone
nucleus (from our earlier work) was thus obtained. All the synthes-
ised derivatives were tested in vitro in order to evaluate their
inhibition profiles against four physiologically important hCAs, i.e.
hCA I, II, IV, and IX. Comparison of the inhibitory efficacy of pri-
mary sulfonamide derivatives with the N1-substituted analogues
further strengthened the role of the sulfonamide in the pharma-
ceutical field related to CAs.
N-{[4-(((1H-indol-3-
yl)methylene)amino)phenyl]sulfonyl}acetamide (4 b)
Off-white powder, (yield 70%), m.p. 170–172 ꢁC; IR (KBr, ꢀ cmꢀ1):
3379–3200 (NHs), 1635 (C ¼ O), 1334 and 1127 (SO2); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.06 (s, 3H, CH3), 5.38 (s, 2H, 2NH,
D2O exchangeable), 6.42–6.44 (m, 2H, Ar–H), 6.56 (d, 1H, Ar–H),
7.36 (d, 2H, Ar–H, J ¼ 8.8), 7.52 (d, 2H, Ar–H, J ¼ 8.8), 7.61 (d, 1H,
Ar–H, J ¼8.8), 8.46 (s, 1H, Ar–H), 9.90 (s, 1H, Ar–H); 13 C NMR
(DMSO-d6) d ppm: 27.1, 110.0, 112.2, 112.8, 121.0, 122.5, 123.5,
127.8, 128.4, 133.4, 150.6, 173.9 175.3; MS m/z: 341.42 [M]þ; Anal.
Calcd. for C17H15N3O3S (341.38): C, 59.81; H, 4.43; N, 12.31; Found
C, 59.63; H, 4.74; N, 12.52.
4-[((1H-indol-3-yl)methylene)amino)-N-(pyrimidin-2-
yl)]benzenesulfonamide (4c)
Brown powder, (yield 68%), m.p. 217–219 ꢁC; IR (KBr, ꢀ cmꢀ1):
3230–3147 (NHs), 1334 and 1126 (SO2); 1H NMR (DMSO-d6,
400 MHz) d ppm: 5.68 (s, 1H, NH, D2O exchangeable), 6.54 (d, 2H,
Ar–H, J ¼ 8.6), 7.20–7.29 (m, 4H, Ar–H), 7.39 (d, 2H, Ar–H, J ¼ 7.6),
7.51 (d, 1H, Ar–H, J ¼ 7.6), 8.09 (d, 2H, Ar–H, J ¼ 7.6), 8.29 (s, 1H,
Ar–H), 9.86 (s, 1H, Ar–H), 12.13 (s, 1H, NH, D2O exchangeable); 13 C
NMR (DMSO-d6) d ppm: 112.5, 112.8, 115.8, 118.6, 121.2, 122.5,
123.9, 124.5, 125.5, 130.2, 137.4, 138.9, 153.3, 158.6, 159.8, 185.4;
MS m/z: 377.12 [M]þ; Anal. Calcd. for C19H19N5O2S (377.42): C,
60.46; H, 4.01; N, 18.56; Found C, 60.29; H, 3.97; N, 18.64.
Materials and methods
Chemistry
Melting points were recorded on a Stuart SMP10 digital melting
point apparatus and were uncorrected. Infrared (IR) spectra were
recorded as KBr disks using a Shimadzu FT-IR 8400S infrared spec-
trophotometer. Mass spectral data are given as m/z (intensity %).
1H NMR spectra were recorded on either on a Varian Mercury VX-
300 MHz spectrophotometer or Bruker AVANCE III Nano Bay
400 MHz FT-NMR spectrophotometer. 13C NMR spectra were run at
100 MHz in deuterated dimethylsulfoxide (DMSO-d6) on Bruker
AVANCE III Nano Bay 400 MHz FT-NMR spectrophotometer.
Chemical shifts are expressed in d values (ppm) using the solvent
peak as internal standard. All coupling constant (J) values are
4-[((1H-indol-3-yl)methylene)amino)-N-(pyridin-2-
yl)]benzenesulfonamide (4d)
Yellowish brown powder, (yield 55%), m.p. 144–146 ꢁC; IR (KBr, ꢀ
given in Hz. The abbreviations used are as follows: s, singlet; d, cmꢀ1): 3417–3207 (NHs), 1323 and 1126 (SO2); 1H NMR (DMSO-d6,
doublet; t, triplet; m, multiplet. Elemental microanalyses were 400 MHz) d ppm: 5.91 (s, 1H, NH, D2O exchangeable), 6.53 (d, 1H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
631
Ar–H, J ¼ 9.6), 6.86 (t, 1H, Ar–H, J ¼ 6.2), 7.04 (d, 2H, Ar–H, J ¼ 8.4), N-{[4-((5-((1H-indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-
717–7.24 (m, 3H, Ar–H), 7.49 (d, 2H, Ar–H, J ¼ 8), 7.62 (t, 1H, Ar–H, yl)amino)phenyl]sulfonyl} acetamide (7 b)
J ¼ 8.8), 8.07 (d, 2H, Ar–H, J ¼ 6.8), 8.26 (s, 1H, Ar–H), 9.91 (s, 1H,
Ar–H), 12.10 (s, 1H, NH, D2O exchangeable); 13 C NMR (DMSO-d6) d
ppm: 112.8, 118.6, 121.2, 122.5, 123.9, 124.5, 129.3, 137.4, 138.9,
153.1, 185.4; Anal. Calcd. for C19H19N5O2S (376.43): C, 63.81; H,
4.28; N, 14.88; Found C, 63.97; H, 4.19; N, 14.52.
Green powder, (yield 55%), m.p. 242–244 ꢁC; IR (KBr, ꢀ cmꢀ1):
3371–3213 (NHs), 1701 and 1658 (2 C ¼ Os), 1311 and 1149 (SO2);
1H NMR (DMSO-d6, 300 MHz) d ppm: 1.94 (s, 3H, CH3), 7.18–7.27
(m, 4H, Ar–H), 7.49 (d, 1H, Ar–H, J ¼ 9), 7.51 (s, 1H, NH, D2O
exchangeable), 7.88 (d, 1H, Ar–H, J ¼ 8.1), 8.07–8.09 (m, 3H, Ar–H),
8.26 (s, 1H, Ar–H), 12.10 (s, 2H, 2NHs, D2O exchangeable); Anal.
Calcd. for C20H16N4O4S2 (440.50): C, 54.53; H, 3.66; N, 12.72; Found
C, 54.62; H, 3.92; N, 12.93.
4-[((1H-indol-3-yl)methylene)amino)-N-(thiazol-2-
yl)]benzenesulfonamide (4e)
Yellowish brown powder, (yield 50%), m.p. 148–150 ꢁC; IR (KBr, ꢀ
1
4-{[5-((1H-indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-
yl)amine]-N-(pyrimidin-2-yl)} benzenesulfonamide (7c)
cmꢀ1): 3367–3209 (NHs), 1334 and 1138 (SO2). H NMR (DMSO-d6,
400 MHz) d ppm: 5.18 (s, 1H, NH, D2O exchangeable), 6.52 (t, 1H,
Ar–H, J ¼ 8), 7.15–7.26 (m, 2H, Ar–H), 7.35 (d, 1H, Ar–H, J ¼ 8.8),
7.38 (d, 2H, Ar–H, J ¼ 9.2), 7.49 (d, 2H, Ar–H, J ¼ 7.6), 8.07 (d, 2H,
Ar–H, J ¼ 7.2), 8.26 (s, 1H, Ar–H), 9.91 (s, 1H, Ar–H), 12.10 (s, 1H,
NH, D2O exchangeable); 13 C NMR (DMSO-d6) d ppm: 112.8, 112.9,
118.6, 121.2, 122.5, 123.9, 124.5, 128.0, 128.1, 137.4, 138.9, 152.6,
185.4; Anal. Calcd. for C18H14N4O2S2 (382.46): C, 56.53; H, 3.69; N,
14.65; Found C, 56.40; H, 3.62; N, 14.37.
Off-white powder, (yield 60%), m.p. 228–230 ꢁC; IR (KBr, ꢀ cmꢀ1):
3317–3199 (NHs), 1681 (C ¼ O), 1328 and 1150 (SO2); 1H NMR
(DMSO-d6, 300 MHz) d ppm: 7.03 (t, 2H, Ar–H, J ¼ 4.8), 7.31–7.78
(m, 3H, Ar–H), 7.87 (d, 1H, J ¼ 9), 7.89 (s, 2H, Ar–H), 8.07 (d, 3H,
Ar–H, J ¼ 8.7), 9.06 (d, 2H, Ar–H, J ¼ 5.1), 10.84 (s, 3H, 3NHs, D2O
exchangeable); Anal. Calcd. for C22H16N6O3S2 (476.53): C, 55.45; H,
3.38; N, 17.64; Found C, 55.37; H, 3.76; N, 17.41.
4-{[5-((1H-indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-
yl)amino]-N-(pyridin-2-yl)] benzenesulfonamide (7d)
4-{[(1H-indol-3-yl)methylene]amino}-N-
carbamimidoylbenzenesulfonamide (4f)
Yellow powder, (yield 50%), m.p. 245–247 ꢁC; IR (KBr, ꢀ cmꢀ1):
3350–3200 (NHs), 1697 (C ¼ O), 1334 and 1138 (SO2); 1H NMR
(DMSO-d6, 300 MHz) d ppm: 7.37–7.45 (m, 5H, 4 Ar–H þ1H, NH,
D2O exchangeable), 7.63–7.45 (m, 4H, 2 Ar–H þ 2H, 2NHs, D2O
exchangeable), 8.13–8.17 (m, 3H, Ar–H), 8.34–8.43 (m, 3H, Ar–H),
8.87 (s, 2H, Ar–H); Anal. Calcd. for C23H17N5O3S2 (475.54): C, 58.09;
H, 3.60; N, 14.73; Found C, 57.96; H, 3.42; N, 14.94.
Brown powder, (yield 68%), m.p. 154–156 ꢁC; IR (KBr, ꢀ cmꢀ1):
3433–3332 (NHs), 1334 and 1126 (SO2); 1H NMR (DMSO-d6,
400 MHz) d ppm: 5.65 (s, 3H, 3NHs, D2O exchangeable), 6.51 (d,
2H, Ar–H, J ¼ 8.8), 7.19–7.24 (m, 2H, Ar–H), 7.35 (d, 1H, Ar–H,
J ¼ 8.4), 7.49 (d, 2H, Ar–H, J ¼ 7.6), 8.06 (d, 1H, Ar–H, J ¼ 8.0), 8.26
(s, 1H, Ar–H), 9.91 (s, 1H, Ar–H), 12.05 (s, 2H, 2NHs, D2O exchange-
able); 13 C NMR (DMSO-d6) d ppm: 112.7, 112.8, 118.6, 121.2, 122.5,
123.9, 124.5, 127.6, 131.2, 137.4, 138.9, 151.8, 159.1, 185.4; Anal.
Calcd. for C16H15N5O2S (341.39): C, 56.29; H, 4.43; N, 20.51; Found
C, 56.01; H, 4.37; N, 20.79.
4-{[(5-((1H-indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-
yl)amino]-N-(thiazol-2-yl)] benzenesulfonamide (7e)
Grey powder, (yield 60%), m.p. 218–220 ꢁC; IR (KBr, ꢀ cmꢀ1):
3327–3207 (NHs), 1687 (C ¼ O), 1317 and 1153 (SO2); 1H NMR
(DMSO-d6, 300 MHz) d ppm: 6.80–6.83 (m, 2H, Ar–H), 7.18–7.27 (m,
4H, Ar–H), 7.48 (t, 1H, Ar–H, J ¼ 7.2), 7.81 (d, 2H, Ar–H, J ¼ 9), 8.07
(d, 1H, Ar–H, J ¼ 7,2), 8.26 (s, 1H, Ar–H), 8.27 (s, 1H, Ar–H), 11.95 (s,
1H, NH, D2O exchangeable), 12.10 (s, 2H, 2NHs, D2O exchange-
able); Anal. Calcd. for C21H15N5O3S3 (481.57): C, 52.38; H, 3.14; N,
14.54; Found C, 52.53; H, 3.33; N, 14.71.
General procedure for the preparation of compounds 7a–f
A mixture of indole-3-carboxaldehyde 2 (1.45 g, 10 mmol), the
appropriate thiazolinone derivative 6a–f (10 mmol) and fused
sodium acetate (0.82 g, 10 mmol) in glacial acetic acid (50 ml) was
refluxed for 12 h. The separated solid was filtered off while hot,
washed with hot water and crystallised from DMF/H2O.
4-{[5-((1H-indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-
yl]amino}benzenesulfonamide (7a)
4-{[5-((1H-indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-
yl]amino}-N-carbamimidoyl benzenesulfonamide (7f)
Yellowish brown powder, (yield 60%), m.p. 230–232 ꢁC; IR (KBr, ꢀ Brown powder, (yield 65%), m.p. 227–229 ꢁC; IR (KBr, ꢀ cmꢀ1):
cmꢀ1): 3360–3199 (NHs), 1674 (C ¼ O), 1334 and 1151 (SO2); 1H 3433–3209 (NHs), 1627 (C ¼ O), 1334 and 1126 (SO2); 1H NMR
NMR (DMSO-d6, 300 MHz) d ppm: 7.14–7.25 (m, 4H, Ar–H), 7.34 (s, (DMSO-d6, 300 MHz) d ppm: 6.88 (s, 1H, Ar–H), 7.16–7.27 (m, 4H,
1H, NH, D2O exchangeable), 7.48 (d, 2H, Ar–H, J ¼ 7.5), 7.61 (s, 1H, Ar–H), 7.48 (t, 1H, Ar–H, J ¼ 6.3), 7.89 (d, 2H, Ar–H, J ¼ 8.7), 8.08 (d,
Ar–H), 7.48 (d, 2H, Ar–H, J ¼ 8.4), 7.94 (s, 1H, Ar–H), 11.89 (s, 2H, 1H, Ar–H, J ¼ 7.2), 8.27 (s, 1H, Ar–H), 11.91 (s, 3H, NHs, D2O
NH2, D2O exchangeable), 12.23 (s, 1H, NH, D2O exchangeable); 13 C exchangeable), 12.11 (s, 3H, NHs, D2O exchangeable); 13 C NMR
NMR (DMSO-d6) d ppm: 115.1, 116.4, 119.8, 120.4, 124.7, 125.9, (DMSO-d6) d ppm: 112.8, 114.4, 118.6, 118.7, 121.2, 122.5, 123.0,
126.9, 127.6, 129.6, 133.8, 137.8, 142.7, 144.0, 159.1, 169.7, 170.2; 123.9, 124.5, 127.2, 128.6, 136.7, 137.5, 138.9, 140.9, 170.9, 185.4;
Anal. Calcd. for C18H14N4O3S2 (398.46): C, 54.26; H, 3.54; N, 14.06; Anal. Calcd. for C19H16N6O3S2 (440.50): C, 51.81; H, 3.66; N, 19.08;
Found C, 54.01; H, 3.75; N, 13.89.
Found C, 52.13; H, 3.90; N, 19.23.

632
F. M. AWADALLAH ET AL.
4-[(1-Acetyl-1H-indol-3-yl)methylene]-2–(4-
methoxyphenyl)oxazol-5(4H)-one (9 b)
1H NMR (DMSO-d6, 400 MHz) d ppm: 1.96 (s, 3H, CH3), 7.06 (t, 1H,
Ar–H, J ¼ 4.2), 7.37–7.48 (m, 3H, Ar–H), 7.59 (t, 2H, Ar–H, J ¼ 7.3),
7.65 (d, 2H, Ar–H, J ¼ 6.8), 7.92 (d, 2H, Ar–H, J ¼ 7.8), 8.00 (s, 1H,
Ar–H), 8.11 (d, 2H, Ar–H, J ¼ 7.2), 8.23 (s, 1H, Ar–H), 8.39 (d, 2H,
Ar–H, J ¼ 8), 8.54 (d, 2H, Ar–H, J ¼ 4.6), 11.91 (s, 1H, NH, D2O
exchangeable); 13 C NMR (DMSO-d6) d ppm: 24.1, 115.0, 116.3,
119.7, 119.9, 124.2, 125.8, 127.7, 128.3, 128.5, 128.8, 129.0, 129.7,
131.2, 132.3, 133.8, 134.9, 144.4, 158,7, 164.8, 164.8, 169.7; Anal.
Calcd. for C30H22N6O4S (562.60): C, 64.05; H, 3.94; N, 14.94; Found;
C, 64.39; H, 4.20; N, 15.21.
A mixture of indole-3-carboxaldehyde 2 (1.45 g, 10 mmol), N-(4-
methoxybenzoyl)glycine 8 b (2.09 gm, 10 mmol) and fused sodium
acetate (0.5 g, 6 mmol) in acetic anhydride (20 ml) was heated in a
boiling water bath for 5 h. The mixture was cooled and ethanol
(20 ml) was added slowly and allowed to stand overnight in the
refrigerator. The crystalline product was filtered off, washed with
hot water and recrystallised from benzene to give compound 9 b.
Buff powder, (yield 60%), m.p. 149–151 ꢁC; IR (KBr, ꢀ cmꢀ1):
1743 and 1689 (2 C ¼ Os); 1H NMR (DMSO-d6, 400 MHz) d ppm:
1.91 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 7.17 (d, 2H, Ar–H, J ¼ 8),
7.39–7.46 (m, 2H, Ar–H), 7.50 (s, 1H, Ar–H), 8.07 (d, 2H, Ar–H,
J ¼ 8), 8.35 (d, 2H, Ar–H, J ¼ 8), 8.79 (s, 1H, Ar–H); Anal. Calcd. for
C21H16N2O4 (360.36): C, 69.99; H, 4.48; N, 7.77; Found C, 70.23; H,
4.65; N, 7.91.
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-y])-N-(pyridin-2-
yl)benzenesulfonamidee (10d)
Yellow powder, (yield 60%), m.p. 248–250 ꢁC; IR (KBr, ꢀ cmꢀ1):
3325 (NH), 1730 and 1631 (2 C ¼ Os), 1327 and 1136 (SO2); 1H
NMR (DMSO-d6, 300 MHz) d ppm: 2.05 (s, 3H, CH3), 6.86 (t, 1H,
Ar–H, J ¼ 6.3), 7.11 (d, 1H, Ar–H, J ¼ 8.7), 7.18–7.28 (m, 5H, Ar–H),
7.50 (d, 1H, Ar–H, J ¼ 6.6), 7.65–7.69 (m, 4H, Ar–H), 7.70 (s, 1H,
Ar–H), 7.79 (d, 2H, Ar–H, J ¼ 8.7), 8.01 (s, 1H, Ar–H), 8.08 (d, 2H,
Ar–H, J ¼ 7.2), 8.28 (d, 1H, Ar–H, J ¼ 6.6), 12.10 (s, 1H, NH, D2O
exchangeable); Anal. Calcd. for C31H23N5O4S (561.61): C, 66.30; H,
4.13; N, 12.47; Found C, 66.23; H, 4.38; N, 12.68.
General procedure for the preparation of compounds 10a–l
An equimolar amount of compound 9a,b (10 mmol) and the
appropriate sulfonamide 3a–f (10 mmol) in glacial acetic acid
(20 ml) containing freshly fused sodium acetate (0.03 g, 0.36 mmol)
was heated in a boiling water bath with constant stirring for
10–14 h. The separated solid was filtered off, washed with water,
and crystallised from DMF/water to give compounds 10a–l.
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl]-N-(thiazol-2-
yl)benzenesulfonamide (10e)
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl]benzenesulfonamide (10a)
Green powder, (yield 65%), m.p. 265–267 ꢁC; IR (KBr, ꢀ cmꢀ1): 3251
(NH), 1703 and 1627 (2 C ¼ Os), 1328 and 1147 (SO2); 1H NMR
(DMSO-d6, 300 MHz) d ppm: 1.89 (s, 3H, CH3), 6.80–6.87 (m, 2H,
Ar–H), 7.23–7.37 (m, 2H, Ar–H), 7.42 (t, 1H, Ar–H, J ¼ 6.9), 7.53 (t,
2H, Ar–H, J ¼ 7.3), 7.77 (d, 2H, Ar–H, J ¼ 8.7), 7.85–7.80 (m, 2H,
Ar–H), 7.92 (s, 1H, Ar–H), 8.05 (d, 2H, J ¼ 6.9), 8.16 (s, 1H, Ar–H),
8.33 (d, 2H, Ar–H, J ¼ 8.1), 12.72 (s, 1H, NH, D2O exchangeable);
13 C NMR (DMSO-d6) d ppm: 24.3, 115.1, 116.4, 120.3, 123.9, 125.8,
126.8, 127.7, 128.9, 129.0, 129.7, 131.2, 133.9, 134.9, 137.2, 139.0,
142.2, 144.5, 158.6, 159.2, 168.9, 170.1; Anal. Calcd. for
C29H21N5O4S2 (567.64): C, 61.36; H, 3.73; N, 12.34; Found C, 61.08;
H, 3.44; N, 12.59.
Grey powder, (yield 60%), m.p. 240–242 ꢁC; IR (KBr, ꢀ cmꢀ1): 3371,
3305 (NH2), 1708 and 1650 (2 C ¼ Os), 1338 and 1157 (SO2); 1H
NMR (DMSO-d6, 300 MHz) d ppm: 1.90 (s, 3H, CH3), 7.26 (s, 2H, NH2,
D2O exchangeable), 7.31–7.42 (m, 2H, Ar–H), 7.45 (s, 1H, Ar–H),
7.51–7.61 (m, 3H, Ar–H), 7.79 (d, 1H, Ar–H, J ¼ 9), 7.87 (d, 2H, Ar–H,
J ¼ 6.9), 7.93 (d, 1H, Ar–H, J ¼ 9), 8.06 (d, 2H, Ar–H, J ¼ 7.2), 8.16 (s,
1H, Ar–H), 8.33 (d, 2H, Ar–H, J ¼ 6.9); 13 C NMR (DMSO-d6) d ppm:
24.1, 115.1, 116.4, 119.9, 120.4, 124.7, 125.9, 126.9, 127.6, 128.6,
128.9, 129.1, 131.2 132.3, 133.6, 137.5 138.9, 142.7 144.0, 168.4,
169.7, 170.2; Anal. Calcd. for C26H20N4O4S (484.53): C, 64.45; H, 4.16;
N, 11.56; Found C, 64.18; H, 4.19; N, 11.48.
N-[(4–(4-((1-acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl)phenyl)sulfonyl]acetamide (10 b)
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl]-N-
carbamimidoylbenzenesulfonamide (10f)
Brown powder, (yield 55%), m.p. 237–239 ꢁC; IR (KBr, ꢀ cmꢀ1):
3390 (NH), 1730, 1701 and 1635 (3 C ¼ Os), 1327 and 1165 (SO2);
1H NMR (DMSO-d6, 300 MHz) d ppm: 1.91 (s, 3H, CH3), 1.92 (s, 3H,
CH3), 7.15–7.26 (m, 2H, Ar–H), 7.31–7.73 (m, 3H, Ar–H), 7.88 (d, 2H,
Ar–H, J ¼ 8.7), 7.93–7.99 (m, 2H, Ar–H), 8.05 (d, 2H, Ar–H, J ¼ 6.9),
8.18 (s, 1H, Ar–H), 8.36 (d, 2H, Ar–H, J ¼ 6.9), 8.81 (s, 1H, Ar–H),
12.51 (s, 1H, NH, D2O exchangeable); 13 C NMR (DMSO-d6) d ppm:
23.6, 24.1, 115.0, 116.3, 119.9, 124.2, 124.7, 125.9, 127.7, 128.3,
128.5, 128.8, 129.1, 129.3, 129.6, 131.1, 132.0, 132.3, 133.8, 135.0,
144.4, 164.9, 169.1, 169.7; Anal. Calcd. for C28H22N4O5S (526.56): C,
63.87; H, 4.21; N, 10.64; Found C, 63.81; H, 4.27; N, 10.97.
Brown powder, (yield 65%), m.p. 252–254 ꢁC; IR (KBr, ꢀ cmꢀ1):
3217 (NH), 1708 and 1635 (2 C ¼ Os), 1315 and 1138 (SO2); 1H
NMR (DMSO-d6, 300 MHz) d ppm: 1.90 (s, 3H, CH3), 7.14–7.25 (m,
4H, Ar–H), 7.34 (s, 2H, NH2, D2O exchangeable), 7.61 (s, 1H, Ar–H),
7.47–7.61 (m, 5H, Ar–H), 7.83–7.94 (m, 5H, Ar–H), 11.89 (s, 2H,
2NHs, D2O exchangeable); 13 C NMR (DMSO-d6) d ppm: 24.4, 108.5,
115.0, 116.6, 119.8, 120.4, 124.6, 125.8, 127.2, 127.6, 128.5, 129.0,
129.7, 131.2, 132.3, 134.9, 135.7, 136.8, 142.3, 143.0, 164.9, 168.9,
169.7; Anal. Calcd. for C27H22N6O4S (526.57): C, 61.59; H, 4.21; N,
15.96; Found C, 61.31; H, 4.50; 16.04.
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl]-N-(pyrimidin-2-
yl)benzenesulfonamide (10c)
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-2–(4-methoxyphenyl)-5-
oxo-4,5-dihydro-1H-imidazol-1-yl]benzenesulfonamide (10 g)
Brownish red powder, (yield 55%), m.p. 248–250 ꢁC; IR (KBr, ꢀ Off-white powder, (yield 70%), m.p. 226–228 ꢁC; IR (KBr, ꢀ cmꢀ1):
cmꢀ1): 3383 (NH), 1689 and 1643 (2 C ¼ Os), 1323 and 1153 (SO2); 3479, 3375 (NH2), 1627 and 1597 (2 C ¼ Os), 1315 and 1145 (SO2);

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633
1H NMR (DMSO-d6, 400 MHz) d ppm: 2.08 (s, 3H, CH3) , 3.80 (s, 3H, OCH3), 5.80 (d, 1H, Ar–H, J ¼ 4.8), 6.49–6.55 (m, 3H, Ar–H), 6.72 (d,
OCH3), 5.77 (s, 2H, NH2, D2O exchangeable), 7.5–6.85 (m, 3H, 1H, Ar–H, J ¼ 4.4), 6.95–7.00 (m, 2H, Ar–H), 7.18 (d, 1H, Ar–H,
Ar–H), 6.85 (s, 1H, Ar–H), 6.99 (d, 1H, J ¼ 7.2), 7.17–7.26 J ¼ 6.4), 7.35 (d, 1H, Ar–H, J ¼ 8.4), 7.37 (s, 1H, Ar–H), 7.41 (d, 2H,
Ar–H, J ¼ 8.8), 7.73 (s, 1H, Ar–H), 7.55 (s, 1H, Ar–H), 7.87 (d, 2H,
Ar–H, J ¼ 8.8), 11.15 (s, 1H, NH, D2O exchangeable); Anal. Calcd. for
C30H23N5O5S2 (597.66): C, 60.29; H, 3.88; N, 11.72; Found C, 60.13;
H, 4.20; N, 11.70.
(m, 3H, Ar–H), 7.42 (d, 1H, Ar–H, J ¼ 6.8), 7.86 (d, 2H, Ar–H, J ¼ 7.2),
8.07 (d, 2H, Ar–H, J ¼ 8.4), 8.26 (s, 1H, Ar–H); 13 C NMR (DMSO-d6) d
ppm: 24.5, 55.7, 112.8, 113.9, 114.2, 118.9, 124.0, 127.0, 127.3,
127.8, 129.3, 133.0, 138.4, 140.9, 142.7, 152.4, 161.8, 162.9, 165.6,
169.5, 173.1, 173.9; Anal. Calcd. for C27H22N4O5S (514.55): C, 63.02;
H, 4.31; N, 10.89; Found C, 63.09; H, 4.64; N, 11.07.
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-2–(4-methoxyphenyl)-5-
oxo-4,5-dihydro-1H-imidazol-1-yl]-N-
carbamimidoylbenzenesulfonamide (10 l)
N-[(4–(4-((1-acetyl-1H-indol-3-yl)methylene)-2–(4-
methoxyphenyl)-5-oxo-4,5-dihydro-1H-imidazol-1-
yl)phenyl)sulfonyl]acetamide (10 h)
Brown powder, (yield 70%), m.p. 234–236 ꢁC; IR (KBr, ꢀ cmꢀ1):
Brown powder, (yield 70%), m.p. 222–224 ꢁC; IR (KBr, ꢀ cmꢀ1):
3433–3213 (NHs), 1635 and 1608 (2 C ¼ Os), 1300 and 1126 (SO2);
1H NMR (DMSO-d6, 400 MHZ) d ppm: 1.90 (s, 3H, CH3), 3.80 (s, 3H,
OCH3), 5.68 (s, 3H, NHs, D2O exchangeable), 6.54 (d, 2H, Ar–H,
J ¼ 8.6), 7.02 (d, 1H, Ar–H, J ¼ 8.8), 7.20–7.28 (m, 2H, Ar–H), 7.39 (d,
2H, Ar–H, J ¼ 8.6), 7.51 (d, 2H, Ar–H, J ¼ 7.6), 7.67 (s, 1H, Ar–H),
7.90 (d, 1H, Ar–H, J ¼ 8.8), 8.10 (d, 2H, Ar–H, J ¼ 7.2), 8.29 (s, 1H,
Ar–H), 12.13 (s, 1H, NH, D2O exchangeable); Anal. Calcd. for
C28H24N6O5S (556.59): C, 60.42; H, 4.35; N, 15.10; Found C, 60.72;
H, 4.62; N, 15.39.
1
3367 (NH), 1685 (br.) and 1604 (3 C ¼ Os), 1330 and 1168 (SO2); H
NMR (DMSO-d6, 400 MHz) d ppm: 2.02 (s, 3H, CH3), 2.06 (s, 3H,
CH3), 3.80 (s, 3H, OCH3), 7.21–7.33 (m, 3H, Ar–H), 7.42–7.44 (m, 2H,
Ar–H), 7.51 (d, 1H, Ar–H, J ¼ 8.8), 7.61 (d, 2H, Ar–H, J ¼ 8.8), 7.72 (s,
1H, Ar–H), 7.84 (s, 1H, Ar–H), 7.85 (d, 2H, Ar–H, J ¼ 7.2), 7.89 (d, 2H,
Ar–H, J ¼ 6.8), 10.36 (s, 1H, NH, D2O exchangeable); 13 C NMR
(DMSO-d6) d ppm: 22.0, 24.5, 55.7, 112.9, 113.5, 113.8, 118.2, 118.5,
118.9, 121.2, 122.5, 123.8, 125.9, 127.1, 128.0, 131.3, 131.6, 138.4,
142.6, 162.5, 168.4, 169.5, 173.1, 185.5; Anal. Calcd. for
C29H24N4O6S (556.59): C, 62.58; H, 4.35; N, 10.07; Found C, 62.24;
H, 4.67; N, 10.03.
Carbonic anhydrase inhibition assay
An applied photophysics stopped-flow instrument has been used
for assaying the CA catalysed CO2 hydration activity29. 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
7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the
ionic strength), following the initial rates of the CA-catalysed CO2
hydration reaction for a period of 10–100 s. The CO2 concentra-
tions ranged from 1.7 to 17 mM for the determination of the kin-
etic parameters and inhibition constants. For each inhibitor at
least six traces of the initial 5–10% of the reaction have been used
for determining the initial rate. The uncatalysed rates were deter-
mine d in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (0.1 mM) were prepared
in distilled-deionised water and dilutions up to 0.01 nM were done
thereafter with the assay buffer. Inhibitor and enzyme solutions
were preincubated together for 15 min at room temperature prior
to assay, in order to allow for the formation of the E–I complex.
The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3 and the Cheng–Prusoff equation, as
reported earlier30–33, and represent the mean from at least three
different determinations. All CA isoforms were recombinant ones
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-2–(4-methoxyphenyl)-5-
oxo-4,5-dihydro-1H-imidazol-1-yl]-N-(pyrimidin-2-
yl)benzenesulfonamide (10i)
Off-white powder, (yield 65%), m.p. 178–180 ꢁC; IR (KBr, ꢀ cmꢀ1):
3356 (NH), 1651 and 1597 (2 C ¼ Os), 1327 and 1153 (SO2); 1H
NMR (DMSO-d6, 400 MHz) d ppm: 2.07 (s, 3H, CH3), 3.81 (s, 3H,
OCH3), 6.00 (s, 1H, NH, D2O exchangeable), 6.56 (d, 2H, Ar–H,
J ¼ 8.8), 6.99–7.03 (m, 3H, Ar–H), 7.61 (d, 2H, Ar–H, J ¼ 8.7), 7.73 (d,
2H, Ar–H, J ¼ 8.8), 7.84 (d, 2H, Ar–H, J ¼ 8.8), 7.89 (d, 2H, Ar–H,
J ¼ 8.8), 7.95 (s, 1H, Ar–H), 8.25 (s, 1H, Ar–H), 8.47 (d, 2H, Ar–H,
J ¼ 8.8); Anal. Calcd. for C31H24N6O5S (592.62): C, 62.83; H, 4.08; N,
14.18; Found; C, 63.17; H, 4.40; N, 14.56.
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-2–(4-methoxyphenyl)-5-
oxo-4,5-dihydro-1H-imidazol-1-yl]-N-(pyridin-2-
yl)benzenesulfonamide (10j)
Dark brown powder, (yield 75%), m.p. 198–200 ꢁC; IR (KBr, ꢀ
cmꢀ1): 3244 (NH), 1660 and 1635 (2 C ¼ Os), 1320 and 1126 (SO2);
1H NMR (DMSO-d6, 400 MHz) d ppm: 2.04 (s, 3H, CH3), 3.80 (s, 3H,
OCH3), 6.51–6.54 (m, 3H, Ar–H), 6.58–6.98 (m, 3H, Ar–H), 7.04 (t,
1H, Ar–H, J ¼10.6), 7.11–7.26 (m, 2H, Ar–H), 7.50 (d, 2H, Ar–H,
J ¼ 8.8), 7.71–7.64 (m, 4H, Ar–H), 8.07 (d, 2H, Ar–H, J ¼ 8.4), 8.25 (s,
1H, Ar–H), 11.01 (s, 1H, NH, D2O exchangeable); Anal. Calcd. for
C32H24N5O5S (591.64): C, 64.96; H, 4.26; N, 11.84; Found C, 65.19;
H, 4.62; N, 11.88.
obtained in-house as reported earlier34–36
.
Results and discussion
Chemistry
The target compounds 4a–f, 7a–f, and 10a–l were synthesised as
illustrated in Schemes 1–3. The key stating material, indole-3-car-
boxaldehyde 2, was prepared via Vilsemier-Hack formylation reac-
tion, as reported in literature22. IR spectrum of compound 2
revealed the presence of (NH) stretching band at 3363 cmꢀ1 along
with a (C ¼ O) stretching band at 1633 cmꢀ1. In addition, 1H NMR
showed a singlet signal at 9.94 ppm attributed to the proton of
the (CH ¼ O) group and a D2O exchangeable signal of the (NH)
4-[4-((1-Acetyl-1H-indol-3-yl)methylene)-2–(4-methoxyphenyl)-5-
oxo-4,5-dihydro-1H-imidazol-1-yl]-N-(thiazol-2-
yl)benzenesulfonamide (10k)
Light brown powder, (yield 65%), m.p. 206–208 ꢁC; IR (KBr, ꢀ proton at 12.15 ppm. Schiff’s bases 4a–f were obtained via the
cmꢀ1): 3294 (NH), 1670 and 1639 (2 C ¼ Os), 1330 and 1145 (SO2); condensation of the aldehyde compound 2 with different sulfona-
1H NMR (DMSO-d6, 400 MHz) d ppm: 2.06 (s, 3H, CH3), 3.80 (s, 3H, mide derivatives 3a–f. The reported method for the preparation of

634
F. M. AWADALLAH ET AL.
Scheme 1. Synthesis of compounds 4a–f. Reagents and reaction conditions: (i) Phosphorous oxychloride, DMF, 5 ꢁC; (ii) glacial acetic acid.
Scheme 2. Synthesis of compounds 7a–f. Reagents and reaction conditions: (i) Chloroacetyl chloride, DMF, rt; (ii) Ammonium thiocyanate, absolute alcohol; (iii) Indole-
3-carboxaldehyde 2, fused sodium acetate, glacial acetic acid, reflux.
these compounds failed to give the expected compounds, and in to obtain the thiazolinone compounds 6a–f23,24. Knoevenagel con-
some derivatives a very poor yield was obtained37. Searching for densation of the aldehyde 2 with the active methylene group in
an alternative method, Naqvi et al.38 reported an energy efficient compounds 6a–f afforded the newly synthesised compounds
greener methodology for the preparation of Schiff’s bases involv- 7a–f. IR spectra of compounds 7a–f were consistent with their
ing a mechano-chemical solvent-free procedure. Applying this new proposed structures where broad stretching bands appeared at
methodology afforded compounds 4a–f in good yields and high 3433–3199 cmꢀ1 corresponding to the (NH) groups. The (C ¼ O)
purity in a short reaction time. IR spectra of compounds 4a–f band of the thiazolinone ring appeared at 1697–1627 cmꢀ1 with
were characterised by the disappearance of the (C ¼ O) stretching an additional (C ¼ O) band at 1701 cmꢀ1 in compound 7 b. The
band of the aldehyde 2 and broadening of the (NH) stretching two (SO2) stretching bands of all derivatives of the series were dis-
band at 3350–3200 cmꢀ1 due to the additional (NH) groups. Also, played at 1334–1311 and 1151–1126 cmꢀ1. Furthermore, com-
two characteristic stretching bands corresponding to the (SO2) pounds 7a–f showed characteristic 1H NMR signal in the region
1
group were identified at 1334–1311 and 1149–1126 cmꢀ1. H NMR 7.78–8.87 ppm corresponding to the alkene (CH¼) proton accom-
of compounds 4a–f displayed the presence of sharp singlet signal panied with the absence of the singlet signal corresponding to
at 9.86–9.93 ppm attributed to the azomethine proton (CH ¼ N) in the aliphatic (CH2) protons with the usual pattern of the protons
addition to the (NH) singlet signals, all disappeared by D2O of both indole and benzene sulfonamide rings. The three protons
exchange. As for compound 4 b; a singlet signal at 2.06 ppm inte- of the acetamide group of compound 7 b appeared as singlet sig-
grated for the three protons of the (CH3) group in 1H NMR spec- nal at 1.94 ppm. 13C NMR spectra of compounds 7a and 7f were
trum and a signal at 27.1 ppm in 13C NMR spectrum confirmed its consistent with their carbon skeleton.
structure. In addition, 13C NMR of compounds 4c–f adopted the
expected pattern of their carbon content. Mass spectra of com-
Scheme 3 illustrated the synthesis of the imidazolinone deriva-
tives 10a–l. The oxazolone intermediates 9a28 and 9 b were syn-
thesised by Erlenmeyer reaction of hippuric acid or 4-methoxy
pounds 4a–c gave m/z at 299.0, 341.42 and 377.42 corresponding
hippuric acid with indole-3-carboxaldehyde 2 in acetic anhydride
to their molecular weights, respectively.
The thiazolinone derivatives 7a–f were synthesised according and fused sodium acetate. Interestingly, the IR spectra of com-
to Scheme 2. The appropriate sulfonamide derivatives 3a–f were pounds 9a,b explored two (C ¼ O) stretching bands for each at
reacted with chloroacetyl chloride at r.t. in DMF to give the inter- 1634, 1689 and 1788, 1743 cmꢀ1, respectively, assigned for the N-
mediate chloroacetyl derivatives 5a–f which were then intramo- acetyl and oxazolone carbonyl groups, respectively. This confirmed
lecularly cyclised by reflux with ammonium thiocyanate in ethanol, that the (NH) group of the indole ring was acetylated under the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
635
Scheme 3. Synthesis of compounds 10a–l. Reagents and reaction conditions: (i) Glycine, 10% sodium hydroxide, ice bath, (0ꢁC); (ii) Indole-3-carboxaldehyde 2, acetic
anhydride, fused sodium acetate, (100 ꢁC); (iii) The appropriate sulfonamide 3a–f, glacial acetic acid, fused sodium acetate, (100 ꢁC).
1
reaction conditions. H NMR of both compounds revealed a singlet
signal at 1.90 ppm assigned for the (CH3) protons of the acetyl
moiety. Subsequent reaction of compounds 9a,b with the appro-
priate sulfonamide derivatives 3a–f afforded the expected imida-
zolinone derivatives 10a–l through a mechanism involving an
open intermediate. The IR spectra of compounds 10a–l showed
two (C ¼ O) bands in the range of 1730–1635 cmꢀ1 and
1701–1597 cmꢀ1 and two characteristic bands for the (SO2) group
Table 1. Inhibition data of human CA isoforms hCA I, II, IV and IX with com-
pounds 4a–f, 7a–f and 10a–l reported here and the standard sulfonamide
inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.
KIa (nM)
Compound
hCA I
hCA II
hCA IV
hCA IX
4a
4 b
4c
4d
4e
4f
7a
88.5
575.8
>10000
>10000
>10000
>10000
8288.3
76.4
2744.6
>10000
>10000
>10000
>10000
>10000
4429.3
327.4
1615.9
>10000
>10000
>10000
1314.5
308.0
8576.7
>10000
>10000
>10000
7534.5
96.4
at 1338–1300 and 1168–1126 cmꢀ1. H NMR demonstrated a sing-
1
let signal in the range of 1.90–2.08 ppm assigned for the CH3 pro-
tons of the acetyl group, in addition to the (OCH3) group protons
in compounds 10 g–l in the range of 3.80–3.81 ppm. Moreover,
compounds 10 b and 10 h revealed an additional singlet signal at
1.92 and 2.04 ppm, respectively, assigned for (CH3) protons of the
acetamido sulfamoyl moiety. 13C NMR spectra of this series were
in accordance with their carbon skeleton where the (CH3) group
of the indole acetyl moiety appeared in the range of
24.0–24.4 ppm, in addition to the (CH3) signals of the second
acetamide moiety at 23.6 and 22.0 ppm in compounds 10 b and
10 h, respectively. Also, compound 10 h was characterised by
another signal corresponding to the (OCH3) group at 55.7 ppm.
7 b
7c
7d
7e
7f
7978.0
>10000
>10000
>10000
9067.9
96.6
5867.4
>10000
>10000
>10000
5764.1
93.7
5719.1
>10000
>10000
>10000
6898.2
250
>10000
>10000
>10000
>10000
7181.8
56.4
>10000
>10000
>10000
>10000
>10000
262.7
>10000
>10000
>10000
>10000
>10000
2771.2
778.3
>10000
>10000
>10000
1396.7
318.4
1467.2
>10000
>10000
>10000
1636.4
328.0
1400.3
>10000
>10000
>10000
1206.6
25
10a
10 b
10c
10d
10e
10f
10 g
10 h
10i
10j
10k
10 l
AAZ
487.3
>10000
>10000
>10000
3816.2
602.3
548.9
3048.1
>10000
>10000
>10000
>10000
12
>10000
>10000
>10000
>10000
74
Carbonic anhydrase inhibitory activity
The CA inhibitory activities of compounds 4a–f, 7a–f, and 10a–l in
addition to acetazolamide (AAZ) as standard inhibitor, were meas-
ured against hCAI, hCA II, hCA IV and hCA IX by a stopped flow
CO2 hydrase assay29. The rational for the choice of these four iso-
aMean from three different assays, by a stopped flow technique (errors were in
the range of 5–10% of the reported values).
the main off-target isoforms both for the antiglaucoma or anti-
forms is that hCA II and IV are targets for antiglaucoma drugs1–3
,
cancer CAIs therapeutic application1,3
.
whereas hCA IX have been validated as targets for the treatment
The following structure-activity-relationships (SARs) were
and prognosis of hypoxic cancers39,40. Otherwise hCA I is one of obtained from the inhibition data reported in Table 1.

636
F. M. AWADALLAH ET AL.
(i) As expected the inhibitory profile of the tested compounds herein, we propose an investigation on four physiologically
against the four CA isoforms was strictly dependent on the func- important human (h) CAs (I, II, IV, and IX) of N1-substituted sec-
tionalisation mode of the sulfonamide group, with the primary ondary sulfonamides incorporating a thiazolinone or imidazolone-
SO2NH2 bearing derivatives (4a, 7a, 10a, and 10 g) arisen as the
best hCA inhibitors herein reported. Sulfonylacetamido (4 b, 7 b,
10 b, and 10 h) and sulfonylguanidino (4c, 7c, 10c, and 10i) com-
pounds maintained a certain degree of activity, dependent on the
tailing moiety and the considered isozymes. Conversely, a totally
counterproductive effect on the inhibition potency can be
ascribed to all other N1-functionalisations, with all following con-
siderations regarding uniquely the active ZBG bearing-derivatives.
(ii) The cytosolic isoform hCA I was significantly inhibited by all
primary sulfonamide compounds with KIs in the range of
88.5–96.6 nM. It is pertinent to mention that the corresponding
sulfonylguanidine (10f) and sulfonylacetamide (10 h) derivatives
lose to a great extent their efficacy, showing a KI of 5764.1 and of
5719.1 nM, respectively.
(iii) hCA II was the most inhibited isoform, among those consid-
ered here by the primary sulfonamide derivatives (4a, 7a, 10a,
and 10 g) with KIs value 575.8, 76.4, 56.4, 602.3 nM, respectively. In
analogy to the SAR reported for the previous isoform, it is interest-
ing to note that the nature of the tail appended at the benzene-
sulfonamide scaffold greatly affected the inhibition of this isoform
in the sulfonylacetamide subset, being the imidazolones (10 b)
and (10 h) medium nanomolar inhibitors (KI 487.3 and 548.9 nM),
and imine (4 b) and thiazolinone (7 b) devoid of any efficacy.
(iv) Uniquely the primary sulfonamides (4a, 7a, 10a, and 10 g)
exhibited significant activity against the membrane-associated iso-
form IV (KIs spanning between 262.7 and 4429.3 nM), with excep-
tion of sulfonylacetamide (10 h) which showed a micromolar
inhibition with a KI of 3048.1 nM.
(v) The general tendencies described above were also applic-
able to the tumour-associated isoform hCA IX, which was moder-
ately inhibited by the primary sulfonamides (4a, 7a, 10a, and
10 g) with binding affinities ranging between 308.0 and 328.0 nM.
Sulfonylacetamido (4 b, 7 b, 10 b, and 10 h) and sulfonylguanidino
(4c, 7c, 10c, and 10i) compounds were shown to possess compar-
able effects on hCA IX, inhibiting it in the low micromolar range
(KIs of 778.3–1615.9 nM and 1206.6–1636.4 nM).
(vi) The inhibition data reported in Table 1 unexpectedly high-
lighted a total loss of efficacy upon functionalisation of the ZBG pri-
mary sulfonamide structure by pyridine, thiazolinone and pyrimidine
moieties, at least within the present set of imine-, thiazolidinone-
and imidazolone-indole benzenesulfamides. On the other hand,
incorporation of less hindered groups as in case of sulfonylaceta-
mides and sulfonylguanidines maintained a certain degree of activ-
ity dependent on the tailing moiety and the considered isozymes.
(vii) Comparing the results of the current study with the earlier
investigation in which a chromone nucleus was used instead of
the indole ring21, revealed that the chromone derivatives showed
excellent activity profile despite being secondary sulfonamides
and bearing similar N1-substitutions. So, it can be claimed that
activity is not solely linked to the absence or presence of the N1-
substitution, but also affected by other distant fragments in the
molecule (chromone vs. indole). In other words, the presence of
secondary sulfonamide moiety is not to be exclusively accused of
the dramatic decrease in activity of the indole derivatives com-
pared to their chromone counterparts.
indole scaffold. The effect of functionalisation of the sulfonamide
group with five different substitution patterns, namely acetyl, pyri-
dine, thiazole, pyrimidine and carbamimidoyl, was evaluated in
relation to the inhibition profile of the primary sulfonamide ana-
logues. With most of these latter compounds acting as nanomolar
inhibitors of all four considered isoforms, a total loss of inhibitory
efficacy upon functionalisation of the ZBG primary sulfonamide
structure arose in case of incorporation of pyridine, thiazole and
pyrimidine moieties. On the other hand, incorporation of less hin-
dered groups, such as sulfonylacetamide (4 b, 7 b, 10 b, and 10 h)
and sulfonylguanidino (4c, 7c, 10c, and 10i) maintained a certain
degree of activity dependent on the tailing moiety and the consid-
ered isozymes, with KIs spanning in the low micromolar range.
Nevertheless, it is worth to be mentioned that the same function-
alisation of the sulfonamide group but with chromone scaffolds,
led to potent inhibitors, in the nanomolar range as revealed in a
previous report21. This drew our attention to the role of the indole
nucleus as a key fragment responsible for switching the activity of
this type of compounds.
Disclosure statement
The authors declare no conflict of interest.
Funding
This work was in part financed by intramural Grants from the
University of Florence (ex 60%).
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