Isatin: a privileged scaffold for the design of carbonic anhydrase inhibitors

Abstract The isatin scaffold is the constitutive fragment of several natural and synthetic bioactive molecules. Albeit several benzene sulphonamide-based carbonic anhydrase inhibitors (CAIs) have been reported, only recently isatin benzene sulphonamides have been studied and proposed as CAIs. In this study we have designed, synthesised, and evaluated the biological activity of a series of differently substituted isatin-based benzene sulphonamides which have been designed for the inhibition of carbonic anhydrase isoforms. The activity of all the synthesised compounds was evaluated towards human carbonic anhydrase I, II, IX, and XII isozymes. Our results indicate that the nature and position of substituents on the isatin ring can modulate both activity and isozyme selectivity.


Introduction
The isatin nucleus could be considered as a privileged scaffold for the design of biologically active agents. The discovery and optimisation of isatin-based therapeutic agents have consistently attracted the interest of medicinal chemists and the chemistry, the biological properties and the therapeutic potential of isatin-based agents has been recently reviewed 1 . Several biological activities could be achieved by decoration of the isatin scaffold such as anti-cancer 2-7 , anti-oxidant 6 , HIV reverse transcriptase inhibition 8,9 , neuroprotective 10 , anti-fungal 11,12 , anti-bacterial 13,14 and antidiabetic 15 . Moreover, the isatin ring has been pointed out as an essential part of anticancer hybrid molecules 16,17 . Recently, the design of isatin-based carbonic anhydrase inhibitors (CAIs) has been reported 10,11,[18][19][20] . It is common knowledge that carbonic anhydrase isozyme family is involved in several physiological and/or pathological metabolic pathways [21][22][23][24][25][26] . They catalyse the simple reaction of the reversible hydration of carbon dioxide to bicarbonate and protons 27 , which is essential for the regulation of the different chemical species connected with CO 2 in the body and its transport across biological membranes such as the inter-, intra-and extra-cellular spaces 25,28 . Not surprisingly several CAIs have been reported and their therapeutic potential has been directed towards different pathologies [29][30][31] (Figure 1).

Materials and apparatus
Starting materials and reagents were obtained from commercial suppliers and were used without purification. All melting points were determined on a Stuart SMP11 melting points apparatus (Stone, UK) and are uncorrected. Electron ionisation mass spectra were obtained by a Fisons QMD 1000 mass spectrometer (Danvers, MA) (70 eV, 200 mA, ion source temperature 200 C). Samples were directly introduced into the ion source. Found mass values are in agreement with theoretical ones. Melting points, yield of reactions and the analytical data of derivatives are reported in Table 1. 1 H-NMR (Table 2) were registered on a Bruker 400 MHz spectrometer (Billerica, MA) or on a Varian 500 MHz (Palo Alto, CA) ( Table 2). All samples were measured in DMSO. Chemical shifts are reported referenced to the solvent in which they were measured. Coupling constants J are expressed in hertz (Hz). Elemental analyses were obtained on a Perkin-Elmer 240 B microanalyser (Waltham, MA). Analytical data of the synthesised compounds are in agreement within ±0.4% of the theoretical values. TLC chromatography was performed using silica gel plates (Merck F 254, Billerica, MA), spots were visualised by UV light.

Biological activity
Carbonic anhydrase inhibition assay The purification of cytosolic CA isoenzymes (CA I and CA II) were previously described with a simple one-step method by a Sepharose-4B-L tyrosine-sulphanilamide affinity chromatography 44 . The protein quantity in the column effluents was determined spectrophotometrically at 280 nm. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) was applied with a Bio-Rad Mini Gel system Mini-PROTEIN V R system (Hercules, CA), Bio-Rad Laboratories, Inc., China after purification of both CA isoenzymes. Briefly, it was performed in acrylamide for the running (10%) and the stacking gel (3%) contained SDS (0.1%), respectively. Activities of CA isoenzymes were determined according to a method by Verporte et al. 45 The increase in absorbance of reaction medium was spectrophotometrically recorded at 348 nm. Also, the quantity of protein was determined at 595 nm according to the Bradford method 46 . Bovine serum albumin was used as standard protein. The IC 50 values were obtained from activity (%) versus compounds plots 47 . For calculation of K I values, three different concentrations were used. The Lineweaver-Burk curves were drawn and calculations were realised 47 . The biological data are reported in Table 3.

Results and discussion
As a part of our ongoing research in the field of CAIs 37 and to achieve a better understanding of the structural requirements for the selective inhibition of the different CA isoforms, we have synthesised a series of 4-(3-methyl-4-oxo-5-(2-oxoindolin-3-ylidene)thiazolidin-2-ylideneamino)-benzenesulphonamides indicated as compounds EMAC 10020 a, c, d, l, m, n, and o. All the synthesised compounds bear a differently substituted isatin scaffold linked, by the interposition of a thiazolidinone spacer, to a benzene-sulphonamide moiety, as zinc binder group. The synthesis of compounds EMAC 10020 was performed as illustrated in Figure 2.
The procedure consists of two steps. The first step is the synthesis of the 4-sulphamoylphenyl-thiourea derivative (1 of Figure  2) by simple reaction of the 4-aminobenzensulphonamide with methylisothiocyanate. The second step of the synthetic route consists of the formation of the thiazolidinone spacer 2 which can be obtained by reacting 1 with ethyl-bromoacetate. Desired compounds were obtained by reacting compound 2 with the appropriate isatin. We attempted to perform step two and three in a one step one pot reaction, but our efforts only gave poor yields with respect to the three step procedure which was therefore preferred. Compounds EMAC 10020 were submitted to enzymatic assay to evaluate their activity and selectivity towards human CA (hCA) isozymes I, II, IX, XII. The results are illustrated in Table 3. As shown in Table 3 some of the tested compounds could be considered as hCA IX preferential inhibitors. However, the nature and position of the substituents on the isatin scaffold played a crucial role in  determining the activity and the isozyme selectivity. In the case of compound EMAC 10020 a, bearing an un-substituted isatin, almost no selectivity can be observed, but for a very poor activity towards XII isozyme. With respect to isozyme IX, the introduction of an electron withdrawing (EW) group (compounds EMAC 10020 c, EMAC 10020 d, and EMAC 10020 n) in the position 5 of the isatin lead to a decrease of activity, albeit some selectivity towards hCA IX could be observed for compound EMAC 10020 c. However, the isosteric replacement of the chlorine atom and of the trifluoro-methyl moiety by a methyl group lead to an increase of activity (K I 3.0 nM) and selectivity. According to these preliminary results, in the case hCA IX, the presence of an EW group in the position 5 of the isatin is not tolerated while the introduction of an electron donating substituent in the same position is beneficial for the activity and selectivity. Also in the case of compound EMAC 10020 m, bearing a fluorine atom in the position 7 of the isatin ring, a good activity (K I 4.1 nM) and selectivity towards hCA IX was observed. Conversely the introduction of a bulkier EW group in the position 7 of the isatin scaffold, as for compound EMAC 10020 o, leads to a decrease of activity towards all isozymes. All together these data corroborate the hypothesis that the hybridisation of isatin scaffold with the benzene-sulphonamide moiety could be advantageous for the design of new therapeutic agents targeting the tumour associated hCA IX isoform.

Conclusions
We have synthesised a small library of isatin/benzene-sulphonamides hybrids for the inhibition of hCA isoforms. The activity of the newly synthesised derivatives has been evaluated towards hCA I, II, IX, and XII. Compounds EMAC 10020 c, EMAC 10020 l, and EMAC 10020 m could be considered as preferential hCA IX inhibitors. Our data indicated that the nature and the position of the substituents play a crucial role in tuning activity and selectivity towards hCA isozyme. Overall these data support the hypothesis that isatin hybrid molecules could represent a valuable starting point for the design of active and selective hCAIs.