Inhibition of carbonic anhydrase isoforms I, II, IX and XII with Schiff's bases incorporating iminoureido moieties

Article abstract of DOI:10.3109/14756366.2014.986118

A series of new Schiff's bases was obtained from the sulfanilamide semicarbazone (4-aminosulfonylphenyl semicarbazide) and aromatic/heterocyclic aldehydes. The new compounds were designed to incorporate moieties known to induce effective inhibitory activi

Full text of DOI:10.3109/14756366.2014.986118

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2015 Informa UK Ltd. DOI: 10.3109/14756366.2014.986118
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RESEARCH ARTICLE
Inhibition of carbonic anhydrase isoforms I, II, IX and XII with Schiff’s
bases incorporating iminoureido moieties
Namrata Singasane1, Prashant S. Kharkar2, Mariangela Ceruso3,4, Claudiu T. Supuran3,4, and
Mrunmayee P. Toraskar1
2
1Department of Pharmaceutical Chemistry, Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai, India, Department of Pharmaceutical
3
Chemistry, SPP School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai, India, Laboratorio di Chimica Bioinorganica,
4
Universita degli Studi di Firenze, Sesto Fiorentino (Firenze), Italy, and Neurofarba Department, Sezione di Scienze Farmaceutiche, Sesto Fiorentino
`
(Firenze), Italy
Abstract
Keywords
A
series of new Schiff’s bases was obtained from the sulfanilamide semicarbazone
Carbonic anhydrase, cytosolic/tumor-
associated enzymes, isoform-selective
inhibitor, Schiff’s base, sulfonamide
(4-aminosulfonylphenyl semicarbazide) and aromatic/heterocyclic aldehydes. The new com-
pounds were designed to incorporate moieties known to induce effective inhibitory activity
against carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in crucial physiologic or
pathologic processes such as the cytosolic CA I and II or the transmembrane, tumor-associated
CA IX and XII: the compounds were medium potency – weak CA I inhibitors, highly effective,
low nanomolar CA II inhibitors, but few of them inhibited effectively CA IX and XII. This may
probably due to the long spacer between the sulfamoylphenyl and imine fragments of the
molecules, which probably induces a highly flexible conformation of the inhibitor bound to the
active site of the enzyme, with destabilizing effects for the adduct. The detailed structure
activity relationship for this class of inhibitors is discussed.
History
Received 9 September 2014
Accepted 3 November 2014
Published online 6 March 2015
Introduction
secreted in saliva and milk (CA VI). Three of these proteins are
acatalytic (CA VIII, X and XI)1–3. CA I and CA II are the two
major CA isozymes present at high concentrations in the cytosol
of red blood cells of most vertebrates, and CA II is also one of the
most active of all CAs, together with the tumor-associated,
transmembrane isoforms CA IX1–3. CA XII, another transmem-
brane isoform, as hCA IX, is also present in some tumors, but
Schiff’s bases incorporating sulfonamide functionalities attached
to aromatic/heterocyclic moieties of the general formula
Ar–CH ¼ N-linker-A-SO2NH2, have been investigated extensively
as carbonic anhydrase (CA, EC 4.2.1.1)1–15 inhibitors (CAIs
starting with the 1990s when the first such compounds were
reported by one of these groups16–27. Some of these derivatives
were among the first reported sulfonamides showing good
selectivity ratios for the inhibition of some human (h) CA
more diffuse in normal tissues compared to hCA IX39–47
.
The human (h) such enzyme, hCAs are therapeutic targets
with the potential to be inhibited or activated, and these
phenomena elicit a number of pharmacologic effects28–47. For
example, inhibitors targeting hCA II/IV/XII are used for the
treatment of glaucoma, those targeting hCA VII/XIV for epilepsy
management, whereas some hCA II/IV inhibitors are used as
diuretics28–33. hCA IX/XII inhibitors show applications as diag-
nostic tools for the imaging of hypoxic tumors, and several such
agents are in early phase clinical development for the treatment of
hypoxic tumors non-responsive to the classical chemo- or radio-
therapy1,39–47. Inhibition of hCA IX/XII leads to the impairment
of the growth of the primary tumor and metastases, and also to the
depletion of the cancer stem cell population, all these phenomena
being useful in the management of hypoxic tumors, for which few
therapeutic options are available nowadays1–3. For such reasons,
the design of new, potent and isoform-selective inhibitors of
various CAs, may lead to clinical applications for treating a
multitude of diseases1–3. The Schiff’s base containing sulfona-
mide inhibitors are in fact just one of the inhibitor classes
investigated for such purposes16–27. In addition to this, a number
isoforms, such as hCA I, II or IV16,17
.
The CA superfamily of enzymes, which act as catalysts for
CO2 hydration to bicarbonate and protons, comprises a large
number of genetic families (six, i.e. the a-, b-, g-, d-, z- and
Z-CAs)28–33. Furthermore, numerous isoforms were discovered so
far in most investigated organisms1–8,34–38. For example, 16
isozymes were described in mammals. Some of them are
cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others
are membrane bound (CA IV, CA IX, CA XII, CA XIV and CA
XV), two are mitochondrial (CA VA and CA VB) and one is
Address for correspondence: Mrunmayee P. Toraskar, Department of
Pharmaceutical Chemistry, Bharati Vidyapeeth’s College of Pharmacy,
Navi Mumbai, India. Tel: +39-055-4573005. Fax: +39-055-4573385.
E-mail: rupalitoraskar@yahoo.com
`
Claudiu T. Supuran, Laboratorio di Chimica Bioinorganica, Universita
degli Studi di Firenze, Sesto Fiorentino (Firenze), Italy. E-mail:
claudiu.supuran@unifi.it

2
N. Singasane et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
of Schiff’s base derivatives have been reported to exert notable General procedure for synthesis of N4-(4-aminosulfonylphenyl)-
biological activity (antibacterial, antitubercular, antitumor, antil- N1-(aryl/substituted aryl/heteroaryl) semicarbazones (3a–l)
eishmanial, DNA-binding activities, etc.)27.
In a 250-mL flask, equimolar quantities of 2 (0.05 mol) and
In this study, we report the synthesis of series of 12 N4-(4-
aromatic aldehyde (0.05 mol) were dissolved in EtOH (50 mL)50.
aminosulfonylphenyl)-N1 (aryl/substitutedaryl/heteroaryl) semi-
Glacial HOAc (2–3 mL) was added to adjust the pH to 5–6. The
carbazones derivatives, and investigation their CA inhibitory
reaction mixture was further refluxed for 10–25 h. After comple-
activity against four physiologically relevant human isoforms,
tion of reaction (as seen from TLC), the mixture was concentrated
hCA I, II, IX and XII.
under reduced pressure, poured onto the crushed ice and the
precipitated product was filtered, washed twice with ice-cold
water and recrystallized from EtOH.
Materials and methods
Chemistry
Synthetic and spectral characterization details for compounds
3a–l can be found in Supplementary Material.
All the reagents and solvents were obtained from commercial
suppliers and were used as received unless otherwise indicated.
Solvents were dried, wherever necessary, according to standard
procedures. All reactions were performed under N2 atmosphere,
2-(4-Fluorobenzylidene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3a)
unless otherwise indicated. Analytical silica gel 60 F254-coated Purity (HPLC): 98%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
TLC plates were purchased from Sigma-Aldrich, and were 10.93 (s, 1H, CO–NH), 9.24 (s, 1H, Ar–NH), 7.96 (s, 1H,
visualized with UV light. Infrared (IR) spectra were recorded on N ¼ CH), 7.91–7.94 (m, 1H, C–H aromatic), 7.87–7.84 (d, 2H, C–
Shimadzu – FTIR 8400S instrument (Shimadzu Corp. Tokyo, H aromatic), 7.75–7.72 (d, 2H, C–H aromatic), 7.30–7.24 (m, 3H,
Japan) using KBr pellet technique. 1H NMR spectra were routinely C–H aromatic), 7.22 (s, 2H, SO2NH2); 13C NMR (100 MHz,
obtained with a Varian Mercury Plus 300 MHz NMR (Agilent DMSO-d6): ꢀ ¼ 152.4, 141.7, 137.2, 134.8 (JC–F ¼ 243 Hz), 136.7,
Technologies, Santa Clara, CA) and Me4Si was used as an internal 133.5, 130.2, 128.9, 128, 127.0, 126.5, 118.5. LC-MS (ESI)
standard. 13C NMR spectra were obtained with Bruker Avance II (M + H)+: 337.12; IR (KBr) cmꢂ1: 3375 (NH str of NH2), 3333
400 NMR spectrometer SAIF Punjab University Chandigarh. LC- (N–H str), 3099 (aromatic C–H str), 1687 (C ¼ O str), 1593
MS spectra were recorded on 6110 AA Series Quadrupole LC/MS (C ¼ N str), 1533 (aromatic C ¼ C str), 1411, 1321, 1153 (S ¼ O
system (Agilent Technologies, Santa Clara, CA). Analytical HPLC str), 1095 (C–F str).
analyses ware carried out with a KromasilÕ 100-5C18 column
(150 mm ꢀ 4.6 mm) on PerkinElmer Series 200 HPLC system with 2-(4-Bromobenzylidene)-N-(4-sulfamoylphenyl)
autosampler and PDA detector (PerkinElmer, Inc., Waltham, MA) hydrazinecarboxamide (3b)
using ACN:Water (50:50) mobile phase (isocratic elution). Melting
Purity (HPLC): 97 %; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
points were recorded using a VeegoÕ (VMP)-D capillary melting
11.0 (s, 1H, CO–NH), 9.27 (s, 1H, Ar–NH), 7.94 (s, 1H, N ¼ CH),
point apparatus (Veego Instruments Corp. Mumbai, India) and are
7.87–7.85 (d, 2H, C–H aromatic, J ¼ 7.2 Hz), 7.84–7.82 (d, 2H,
uncorrected.
C–H aromatic, J ¼ 6.6 Hz), 7.76–7.73 (d, 2H, C–H aromatic,
J ¼ 8.7 Hz), 7.64–7.61 (d, 2H, C–H aromatic, J ¼ 8.4 Hz), 7.22 (s,
Procedure for synthesis of 4-ureidobenzenesulfonamide (1)
2H, C–H SO2NH2); 13C NMR (100 MHz, DMSO-d6): ꢀ ¼ 152.7,
In a 250-mL beaker, sulfanilamide (0.03 mol) was dissolved in 141.9, 139.1, 136.9, 128.7, 126.7, 123.9, 122.3, 120.8, 117.8.; LC-
a mixture of glacial HOAc (13 mL) and hot water (30 mL)48. MS (ESI; M)+: 397.13 (M + 2)+: 399.09; IR (KBr) cmꢂ1: 3371,
A solution of sodium cyanate (0.05 mol) in hot water (25 mL) was 3338 (N–H str of NH2), 3254 (N–H str), 3097 (aromatic C–H
added to the above mixture with continuous stirring. It was str),1695 (C ¼ O str) 1587 (C ¼ N str), 1531 (aromatic C ¼ C str),
allowed to stand for 30 min, then cooled in ice-bath and vacuum 1410, 1340, 1157 (S ¼ O str), 702 (C-Br str).
filtered, dried and recrystallized from EtOH. Yield: 70%; mp:
175 ꢁC(178–179 ꢁC)49; 1H NMR (400 MHz, DMSO-d6) ꢀ ppm: 2-(4-Nitrobenzylidene)-N-(4-sulfamoylphenyl)
8.20 (s, 2H, CO–NH2), 7.15 (s, 2H, SO2–NH2), 7.66–7.64 (d,1H, hydrazinecarboxamide (3c)
C–H aromatic), 7.62–7.59 (d, 1H, C–H aromatic), 7.55–7.51 (d,
Purity (HPLC): 97%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm: 11.0
1H, C–H aromatic), 7.52–7.40 (d, 1H, C–H aromatic), 6.01 (s, 1H,
(s, 1H, CO–NH), 9.27 (s, 1H, Ar–NH), 7.94 (s, 1H, N ¼ CH),
8.28–8.25 (d, 2H, C–H aromatic, J ¼ 9 Hz), 8.16–8.14 (d, 2H,
C–H aromatic, J ¼ 8.7 Hz), 7.88–7.85 (d, 2H, C–H aromatic,
J ¼ 8.7 Hz), 7.77–7.47 (d, 2H, C–H aromatic, J ¼ 8.7 Hz), 7.22 (s,
2H, SO2NH2); 13C NMR (100 MHz, DMSO-d6): ꢀ ¼ 152.7, 142.0,
141.5, 139.1, 137.2, 131.2, 128.9, 126.7, 126.4, 118.4. LC-MS
(ESI; M + H)+: 364.08; IR (KBr) cmꢂ1: 3350 (N–H str of NH2),
Ar–NH); IR (KBr) cmꢂ1: 3462 (N–H str of NH2), 3365 (N–H str),
3227 (aromatic C–H str), 1693 (C ¼ O str), 1533 (aromatic C ¼ C
str), 1409, 1321, 1155 (S ¼ O str).
Procedure for synthesis of 4-aminosulfonylphenyl
semicarbazide (2)
In a 250-mL RBF, equimolar quantities of 1 (0.05 mol) and 3246 (N–H str), 3086 (aromatic C–H str), 1687 (C ¼ O str), 1589
hydrazine hydrate (0.05 mol) in EtOH (2.5 ml) were refluxed for (C ¼ N str), 1537 (aromatic C ¼ C str), 1410, 1338, 1147 (S ¼ O
27 h48. The progress of the reaction was monitored by TLC using str), 1323 (C–NO2 str).
CHCl3:MeOH (8:2) as the mobile phase. The 2/3rd volume of
EtOH was removed under reduced pressure and then the reaction
2-(2,4-Dichlorobenzylidene)-N-(4-sulfamoylphenyl)
mixture was poured onto crushed ice. The resultant precipitate
hydrazinecarboxamide (3d)
was filtered, washed with H2O and dried. The crude product was
Purity (HPLC): 97%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
11.17 (s, 1H, CO–NH), 9.32 (s, 1H, Ar–NH), 8.32 (s, 1H,
N ¼ CH), 8.86–8.83 (d, 2H, C–H Aromatic), 8.44–8.41 (d, 1H, C–
H Aromatic), 7.76–7.33 (d, 2H, C–H Aromatic), 7.52–7.50 (dd,
1H, C–H Aromatic), 7.53 (s, 1H, C–H Aromatic), 7.21 (s, 2H,
SO2NH2); 13C NMR (100 MHz, DMSO-d6): ꢀ ¼ 152.6, 148.2,
recrystallized from EtOH. Yield: 56%; mp: 207 ꢁC; 1H NMR
(300 MHz, DMSO-d6) ꢀ ppm: 8.98 (s, 1H, CO–NH), 7.15 (s, 2H,
SO2–NH2), 4.40 (s, 2H, NH2), 7.60–7.68 (m, 5H, Ar–NH and
C–H aromatic); IR (KBr) cmꢂ1: 3365 (N–H str of NH2), 3281
(N–H str), 3074 (aromatic C–H str), 1676 (C ¼ O str), 1521
(aromatic C ¼ C str), 1408, 1301, 1153 (S ¼ O str).

DOI: 10.3109/14756366.2014.986118
Inhibition of CA isoforms with Schiff’s bases
3
142.0, 139.0, 137.5, 136.1, 132.8, 129.6, 126.3, 123.4, 121.3, C–H aromatic), 7.65–7.64 (d, 1H, C–H thiophene), 7.46–7.45 (d,
118.9; LC-MS (ESI; M)+: 387.10, (M + 2)+: 389.05; IR 1H, C–H thiophene), 7.20 (s, 2H, SO2NH2), 7.13–7.10 (t, 1H, C–
(KBr) cmꢂ1: 3390, 3346 (N–H str of NH2), 3265 (N–H str), H thiophene); 13C NMR (100 MHz, DMSO-d6): ꢀ ¼ 158ꢃ9, 152.8,
2966 (aromatic C–H str), 1697 (C ¼ O str), 1587 (C ¼ N str), 142.2, 141.7, 137.1, 129.8, 128.5, 126.4, 125.1, 118.4; LC-MS
1537 (aromatic C ¼ C str), 1410, 1327, 1157 (S ¼ O str), 678 (ESI) (M + H)+: 325.10; IR (KBr) cmꢂ1: 3367 (N–H of NH2 str),
(C–Cl str).
3261 (N–H str), 3084 (aromatic C–H str), 1687 (C ¼ O str), 1589
(C ¼ N str), 1531 (aromatic C ¼ C str), 1411, 1327, 1149 (S ¼ O
str), 761 (C–S str).
2-(4-Hydroxybenzylidene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3e)
2-(Anthracen-9-ylmethylene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3j)
Purity (HPLC): 99%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
10.71 (s, 1H, CO–NH), 9.84 (s, 1H, Ar–NH), 9.13 (s, 1H, OH),
7.88 (s, 1H, N ¼ CH), 7.87–7.85 (d, 2H, C–H Aromatic), 7.74– Purity (HPLC): 98%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
7.71 (d, 2H, C–H Aromatic), 7.69–7.66 (d, 2H, C–H Aromatic), 11.12 (s, 1H, CO–NH), 9.21 (s, 1H, Ar–NH), 9.14 (s, 1H,
6.86–6.82 (d, 2H, C–H Aromatic), 7.21 (s, 2H, SO2NH2); 13C N ¼ CH), 8.7 (s, 1H, C–H aromatic), 8.52–8.50 (d, 2H, C–H
NMR (100 MHz, DMSO-d6): ꢀ ¼ 152.8, 142.1, 140.1, 137.3, aromatic), 8.17–8.15 (d, 2H, C–H aromatic), 7.80–7.58 (m, 8H,
130.6, 130.42, 129.0, 126.3, 118.6, 115.8; LC-MS (ESI; M + H)+: C–H aromatic), 7.19 (s, 2H, SO2NH2); 13C NMR (100 MHz,
335.16; IR (KBr) cmꢂ1: 3446 (O–H str), 3369, 3315 (NH str of DMSO-d6): ꢀ ¼ 152.7, 142.1, 140.0, 139.1, 137.4, 136.9, 133.3,
NH2), 3227 (N–H str), 3095 (aromatic C–H str), 1693 (C ¼ O str), 131.3, 129.1, 128.7, 126.5, 126.3, 122.7, 118.7; LC-MS (ESI;
1587 (C ¼ N str), 1529 (aromatic C ¼ C str), 1415, 1327, 1159 M + H)+: 419.15; IR (KBr) cmꢂ1: 3414 (NH str of NH2), 3282
(S ¼ O str).
(NH str), 2916 (aromatic C–H str), 1647 (C ¼ O str) 1595
(imine C ¼ N str), 1545 (aromatic C ¼ C str), 1471, 1311, 1155
(S ¼ O str).
2-(4-Methylbenzylidene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3f)
2-(3-Nitrobenzylidene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3k)
Purity (HPLC): 99%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
10.85 (s, 1H, CO–NH), 9.19 (s, 1H, Ar–NH), 7.93 (s, 1H,
N ¼ CH), 7.88–7.85 (d, 2H, C–H aromatic), 7.78–7.74 (d, 2H, C– Purity (HPLC): 98%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
H aromatic), 7.76–7.71 (d, 2H, C–H aromatic), 7.26–7.23 (d, 2H, 11.13 (s, 1H, CO–NH), 9.40 (s, 1H, Ar–NH), 8.66 (s, 1H,
C–H aromatic), 7.22 (s, 2H, SO2NH2), 2.4 (s, 3H, CH3); 13C N ¼ CH), 7.22 (s, 2H, SO2NH2), 8.33 (d, 1H, C–H aromatic),
NMR (100 MHz, DMSO-d6): ꢀ ¼ 152.6, 148.2, 147.3, 140.5, 8.24–8.23 (d, 1H, C–H aromatic), 8.10 (s, 1H, C–H aromatic),
138.9, 137.5, 136.1, 132.7, 126.3, 123.4, 118.9, 39.6; LC-MS 7.77–7.74 (d, 2H, C–H aromatic), 7.75–7.73 (d, 2H, C–H
(ESI; M + H)+: 333.18; IR (KBr) cmꢂ1: 3363 (N–H str), 3257 aromatic), 7.70 (s, 1H, C–H aromatic); 13C NMR (100 MHz,
(N–H str), 3105 (aromatic C–H str), 1689 (C ¼ O str), DMSO-d6): ꢀ ¼ 159.3, 152.6, 142.1, 137.4, 133.9, 131.1, 126.9,
1589 (C ¼ N str), 1533 (aromatic C ¼ C str), 1411, 1325, 1161 126.3, 124.3, 121.8, 121.7, 118.8; LC-MS (ESI; M + H)+:
(S ¼ O str).
364.0718; IR (KBr) cmꢂ1: 3362 (N–H str of NH2), 3211 (N–H
str), 3082 (aromatic C–H str), 1703 (C ¼ O str), 1589 (C ¼ N str),
1535 (aromatic C ¼ C str), 1410, 1350, 1155 (S ¼ O str), 1327
(C–NO2 str).
2-((1H-Indol-3-yl)methylene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3g)
Purity (HPLC): 99%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
11.56 (s, 1H, CO–NH), 10.54 (s, 1H, Ar–NH), 8.91 (s,1H, NH of
indole), 8.23–7.17 (m, 1H of CH ¼ N, 2H, SO2NH2 and 9H of C–
2-(2-Fluorobenzylidene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3l)
H aromatic); 13C NMR (100 MHz, DMSO-d6): ꢀ ¼ 158ꢃ2, 152.6, Purity (HPLC): 99%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
142.1, 137.5, 134.8, 132.8, 131.4, 130.5, 127.4, 127.3, 127.0, 11.08 (s, 1H, CO–NH), 9.27 (s, 1H, Ar–NH), 8.20 (s, 1H,
126.7, 126.3, 118.8; LC-MS (ESI; M + H)+: 358.14; IR (KBr) N ¼ CH), 7.23 (s, 2H, SO2NH2), 8.36–8.30 (t, 1H, C–H aromatic),
cmꢂ1: 3344 (N–H str), 3277 (N–H str), 3115 (aromatic C–H str), 7.87–7.85 (d, 2H, C–H aromatic), 7.76–7.73 (d, 2H, C–H
1695 (C ¼ O str), 1585 (C ¼ N str), 1531 (aromatic C ¼ C str), aromatic), 7.50–7.29 (m, 3H, C–H aromatic); 13C NMR
1410, 1317, 1155 (S ¼ O str).
(100 MHz, DMSO-d6): ꢀ ¼ 142.1, 138.9, 137.1, 130.9, 129.5,
128.8, 128.3, 126.8, 125.5, 125.1, 124.5, 118.5; LC-MS (ESI;
M + H)+: 337.0773; IR (KBr) cmꢂ1: 3373 (N–H str), 3269 (N–H
str), 3099 (aromatic C–H str), 1687 (C ¼ O str), 1591 (C ¼ N str),
1537 (aromatic C ¼ C str), 1413, 1301, 1159 (S ¼ O str), 1103
(C–F str).
2-(2-Chlorobenzylidene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3h)
Purity (HPLC): 97%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
11.14 (s, 1H, CO–NH), 9.29 (s, 1H, Ar–NH), 8.38 (s, 1H,
N ¼ CH), 7.22 (s, 2H, SO2NH2), 7.87–7.84 (d, 2H, C–H
aromatic), 7.76–7.73 (d, 2H, C–H aromatic), 7.63–7.40 (m, 4H,
CA activity measurements and inhibition studies
An SLX Applied Photophysics stopped-flow instrument has been
used for measuring the catalytic activity and inhibition with a CO2
hydration assay method51. Phenol red (at a concentration of
0.2 mM) has been used as indicator, working at the absorbance
maximum of 557 nm, with 20 mM Hepes (pH 7.4) or 20 mM Tris
(pH 8.3) as buffers and 20 mM Na2SO4 or NaClO4 (for
maintaining constant ionic strength). The initial rates of the
CA-catalyzed CO2 hydration reaction were followed for a period
of 10–100 s. The concentrations of substrate (CO2) ranged from
1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants, with at least six traces of the initial 5–10% of
C–H aromatic); LC-MS (ESI; M + H)+: 353.04; IR (KBr) cmꢂ1
:
3375 (N–H str), 3230 (N–H str), 3095 (aromatic C–H str), 1695
(C ¼ O str), 1591 (C ¼ N str), 1537 (aromatic C ¼ C str), 1411,
1334, 1174 (S ¼ O str).
2-(2-Thienylidene)-N-(4-sulfamoylphenyl)
hydrazinecarboxamide (3i)
Purity (HPLC): 98%; 1H NMR (300 MHz, DMSO-d6) ꢀ ppm:
10.82 (s, 1H, CO–NH), 8.99 (s, 1H, Ar–NH), 8.18 (s, 1H,
N ¼ CH), 7.80–7.78 (d, 2H, C–H aromatic), 7.74–7.72 (d, 2H,

4
N. Singasane et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
the reaction being used for determining the initial velocity, for reaction with the appropriate heterocyclic/aromatic aldehydes in
each inhibitor. The uncatalyzed rates determined were subtracted the presence of glacial acetic acid and ethanol, which yielded the
from the total observed rates. Stock solutions of inhibitors Schiff’s bases 3a–l. The products obtained in this way were
(10 mM) were prepared in distilled-deionized water and dilutions purified by recrystallization from ethanol. Table 1 lists their
up to 0.01 nM were done with the assay buffer. Enzyme and relevant physicochemical data.
inhibitor solutions were pre-incubated prior to assay for 15 min (at
Structures of the synthesized compounds were confirmed by
room temperature), in order to allow for the formation of the E–I IR, 1H NMR, 13C NMR and LC-MS. In the IR spectra, formation
complex. The inhibition constants were obtained by non-linear of the Schiff’s bases was confirmed by disappearance of the –NH2
least-squares methods using PRISM 3 and the Cheng–Prusoff group and appearance of the imine N ¼ CH band, characterized by
equation as reported earlier by our groups52–55. The CAs were an absorption at 1589–1591 cmꢂ1. The semicarbazone derivatives
recombinant proteins obtained in-house as reported earlier50–52
.
presented N–H stretching vibration at 3344–3390 cmꢂ1, CO–NH
The concentrations of enzyme used in the assay were: 16.0 nM for stretching vibrations at 1687–1703 and 3211–3277 cmꢂ1, respect-
hCAI, 9.1 nM for hCA II; 9.5 nM for hCA IX and 12.6 nM for ively, in their IR spectra. Aromatic C ¼ C stretching vibrations
hCA XII, respectively.
were observed in the 1529–1537 cmꢂ1 region, whereas aromatic
C–H stretching vibration in the 2966–3115 cmꢂ1 region. In
addition, the strong SO2 stretching vibrations were observed in
the region 1410–1415, 1301–1350 and 1147–1159 cmꢂ1. The 1H-
NMR spectra of compounds 3a–l displayed singlets at ꢀ ¼ 7.94–
9.14 ppm (for N ¼ CH), proton which confirms the formation of
Schiff’s bases. The singlet in the range of ꢀ ¼ 10.5–11.56 ppm was
observed for CO–NH protons; the singlet at ꢀ ¼ 9.19–10.54 ppm
corresponding to Ar–NH protons; whereas the broad singlet at
ꢀ ¼ 7.19–8.20 ppm corresponding to the SO2NH2 protons. In all
compounds, a multiplet was observed within the range of
ꢀ ¼ 8.44–6.82 ppm for aromatic protons. In compound 3e, the
singlet at ꢀ ¼ 9.13 ppm was assigned to the OH proton and the
singlet at ꢀ ¼ 2.4 ppm for the CH3 protons of 3f. A singlet at
Results and discussion
Chemistry
The synthesis of target compounds was carried out as outlined in
Scheme 1. Sulfanilamide was reacted with sodium cyanate in the
presence of glacial acetic acid, leading to 4-ureidobenzenesulfo-
namide (1). 4-Ureidobenzenesulfonamide 1 was further used to
synthesize 4-aminosulfonylphenyl semicarbazide (2) through
condensation with hydrazine hydrate in ethanol. Compound 2
was then used to synthesize N4-(4-aminosulfonylphenyl)-N1-(aryl/
substituted–aryl/heteroaryl) semicarbazone derivatives 3a–l by
O
O
NH
HN
NH
HN
NHNH
2
2
2
a
b
c
H
H
N
N
N
Ar
O
SO NH
SO NH
SO NH
2 2
H NO S
2
2
2
2
2
2
Sulfanilamide
1
2
3a-3l
Scheme 1. Synthesis of compounds 3a–l. Reagents and conditions were: (a) NaCNO, glacial AcOH; (b) NH2NH2ꢃH2O, EtOH, reflux, 27 h;
(c). ArCHO, glacial AcOH, EtOH, reflux, 10–25 h.
Table 1. Structures and physicochemical properties of compounds 1–3 reported here.
H
H
N
N
N
Ar
O
H NO S
2
2
.
Compound code
Ar
Molecular formula
Mol. Wt.
Melting Point (ꢁC)
Rf*
Yield (%)
1
2
–
–
C7H9N3O3S
C7H10N4O3S
215.0
230.0
175
207
243
256
275
245
230
231
224
228
242
249
223
241
0.48
0.33
0.71
0.76
0.64
0.79
0.82
0.75
0.70
0.79
0.77
0.85
0.67
0.80
75
56
69
65
72
77
62
79
68
71
65
82
74
66
3a
3b
3c
3d
3e
3f
3g
3h
3i
4-F–C6H4
4-Br–C6H4
4-NO2–C6H4
2,4-Cl2–C6H3
4-OH–C6H4
4-CH3–C6H4
3-Indolyl
2-Cl–C6H4
2-Thienyl
9-Anthranyl
3-NO2–C6H4
2-F–C6H4
C14H13FN4O3S
C14H13BrN4O3S
C14H13N5O5S
C14H12Cl2N4O3S
C14H14N4O4S
C15H16N4O3S
336.11
397.25
363.09
387.25
334.36
332.39
357.4
C
16H15N5O3S
C14H13ClN4O3S
12H12N4O3S2
22H18N4O3S
352.8
C
324.39
418.48
363.09
336.1
3j
3k
3l
C
C14H13N5O5S
C14H13FN4O3S
*TLC mobile phase – CHCl3:MeOH (8:2).

DOI: 10.3109/14756366.2014.986118
Inhibition of CA isoforms with Schiff’s bases
5
ꢀ ¼ 8.91 ppm corresponding to NH of indole was observed for effective hCA XII inhibitors (KIs in the range of 21.5–23.8 nM),
compound 3g.
whereas the remaining derivatives were medium potency hCA XII
inhibitors (KIs in the range of 42.3–95.8 nM). The structure
activity relationship is thus rather flat, with a reduced variation in
the inhibition constants over the entire series of derivatives. This
may be due to the quite long linker between the sulfamoylphenyl
and arylimine fragments of the molecules investigated here,
which probably induces a too flexible conformation of the
inhibitor molecule when bound to the enzyme active site. This
leads to a destabilization of the enzyme–inhibitor adduct and as a
consequence the weaker inhibitory power of these Schiff’s bases
compared to similar derivatives17–25 possessing a shorter linker
between the two fragments mentioned above.
CA inhibition
The CA inhibitory properties of sulfonamides 3a–l reported here
were investigated against four physiologically relevant isoforms,
hCA I, II (cytosolic) as well as hCA IX and XII (transmembrane,
tumor-associated enzymes). The following structure activity
relationship can be observed from the data of Table 2:
(i) The slow cytosolic isoform hCA I was inhibited with
medium efficacy by four sulfonamides investigated here, i.e. 3b,
g, h and i, which showed inhibition constants in the range of 71.2–
95.2 nM, being thus more effective than the standard drug
acetazolamide AAZ (Table 1). These derivatives incorporate 4-
and 2-Cl-phenyl- as well as heterocyclic (indolyl and thienyl)
moieties. It may be observed that changing the substitution pattern
at the Ar moiety has a detrimental effect on the hCA I inhibitory
activity, as compounds incorporating other moieties (4-nitro, 3-
nitro, 4-Me, 3,4-dichloro-, etc) are much weaker inhibitors of this
isoform, with KIs in the range of 250–6530 nM (Table 2).
(ii) The physiologically dominant hCA II (drug target for
antiglaucoma agents) was efficiently inhibited by many of the
sulfonamides investigated here, such as 3a–c, 3e–j and 3l, which
showed KIs in the range of 4.0–19.5 nM, being more potent or
equipotent the standard drug AAZ. Thus, unlike hCA I discussed
above, many substitution patterns of the Ar moiety in the
sulfonamides 3 lead to effective inhibitors of this isoform.
Only two compounds, 3d (2,4-dichlorophenyl derivative) and 3k
(3-nitrophenyl derivative) were medium potency hCA II inhibi-
tors, with KIs in the range of 51.6–75.0 nM. Thus, small
differences in the nature or substitution pattern of the Ar moiety
(e.g. the regiomers 3c and 3k, which only differ by the position of
the NO2 moiety) of these derivatives leads to drastic changes in
the inhibition pattern of the corresponding compounds (Table 2).
(iii) The hCA IX inhibition profile with the compounds
investigated here was less interesting, however. Only compound
3 a (4-fluorophenyl derivative) showed an activity similar to
AAZ, with a KI of 27.3 nM, whereas all other compounds were
much less effective as inhibitors of this transmembrane isoform,
with inhibition constants in the range of 90.4–424 nM (Table 2).
(iv) A rather similar behavior to that mentioned above for hCA
IX; was also observed for the inhibition of the second transmem-
brane isoform, hCA XII. Two compounds, 3i and 3j (incorporat-
ing 2-thienyl and 9-anthranyl moieties, respectively) were
Conclusion
We report here a new series of new Schiff’s bases, which was
obtained from the sulfanilamide semicarbazone (4-aminosulfo-
nylphenyl semicarbazide), by reaction with aromatic/heterocyclic
aldehydes. The compounds were designed to incorporate moieties
known to induce effective inhibitory for CA isoforms involved in
crucial physiologic or pathologic processes such as the cytosolic
CA I and II or the transmembrane, tumor-associated CA IX and
XII. The investigated compounds were medium potency – weak
CA I inhibitors, highly effective, low nanomolar CA II inhibitors,
but few of them inhibited effectively CA IX and XII. This may
probably due to the long spacer between the sulfamoylphenyl and
imine fragments of the molecules, which probably induces a
highly flexible conformation of the inhibitor bound to the active
site of the enzyme, with destabilizing effects for the adduct. The
detailed structure activity relationship for this class of inhibitors is
discussed.
Acknowledgements
The authors are thankful to Dr V. J. Kadam, Principal, Bharati
Vidyapeeth’s College of Pharmacy, Navi Mumbai for providing necessary
research facilities in the department. This work was financed in part by
two FP7 EU projects, Metoxia and Dynano. PSK thanks Ms. Sona
Warrier for her help during preparation of this manuscript.
Declaration of interest
Most authors declare no conflict of interest. CTS declares conflict
of interest, being author on many patents claiming CA inhibitors
with various pharmacologic applications.
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Supplementary material available online
Supplementary Figures S1–S36.