Evaluation of sulphonamide derivatives acting as inhibitors of human carbonic anhydrase isoforms I, II and Mycobacterium tuberculosisβ-class enzyme Rv3273

Abstract A series of novel sulphonamide derivatives was obtained from sulphanilamide which was N4-alkylated with ethyl bromoacetate followed by reaction with hydrazine hydrate. The hydrazide obtained was further reacted with various aromatic aldehydes. The novel sulphonamides were characterised by infrared, mass spectrometry, 1H- and 13C-NMR and purity was determined by high-performance liquid chromatography (HPLC). Human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and II and Mycobacterium tuberculosis β-CA encoded by the gene Rv3273 (mtCA 3) inhibition activity was investigated with the synthesised compounds which showed promising inhibition. The KIs were in the range of 54.6 nM–1.8 µM against hCA I, in the range of 32.1 nM–5.5 µM against hCA II and of 127 nM–2.12 µM against mtCA 3.


Introduction
Sulphonamides are interesting biologically active compounds. There are numerous sulphonamide drugs available on the markets for the treatment of various diseases 1 . Sulphonamide derivatives such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide have been clinically used for decades as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). A diverse research trend in the past years has led to the obtaining of diuretic, anti-glaucoma, anti-cancer, anti-convulsant, anti-diabetic, and anti-obesity agents based on CA inhibitors (CAIs) of the sulphonamide type 2-6 . Sulphonamides act as strong CAIs by binding as anions to the zinc metal ion within the enzyme active site 7 .
CA has various roles in physiological events such as carbon dioxide and bicarbonate transport processes, respiration, pH balancing, CO 2 homeostasis, electrolyte secretion, biosynthetic reactions 1 . Distinct, evolutionarily non-related gene families of CAs are present in various organisms, out of which the a-class is present in humans, as 15 different isoforms (hCA I-XIV). hCA I is present in red blood cells and in many tissues but its physiological function is still unknown; however, it is known that hCA I is associated with retinal and cerebral edema, and the inhibition of CA I may be helpful in curing such conditions [1][2][3][4][5] . hCA II, the physiologically dominant isoform, is another enzyme which is associated with several disease conditions such as epilepsy, edema, glaucoma and altitude sickness 1-5 . Furthermore, it has also emerged in the past few years that these enzymes can be used as potential target for designing anti-infective drugs with a novel mechanism of action 6 .
In this article, we report a synthetic strategy for the generation and characterisation of some sulphonamide derivatives. The novel sulphonamides were purified and characterised using IR, Mass spectrometry, 1 H and 13 C-NMR for confirmation of their structure, and purity of the compounds was determined by using HPLC techniques. The newly synthesised compounds were analysed as inhibitors of human hCA I and II, and the bacterial, b-class enzyme from Mycobacterium tuberculosis (mtCA 3) encoded by the gene Rv3273 8 . carbonate (0.6 g) were added and the reaction mixture was heated under reflux for 12 h. The potassium salt was filtered off and excess of ethanol was removed under reduced pressure. The residue solidifies on cooling to give compound (2). Yield: 70%, R f : 0.72 (chloroform:methanol 9:1); M.P.: 141-144 C; IR (ATR) cm À1 : 3356 (N-H str of NH 2 ), 3271 (N-H str), 2993 (Ar C-H str), 2904 (aliphatic C-H str), 1728 (C ¼ O str), 1597 (C ¼ C str), and 1138 (S ¼ O str).
Using the above general procedure, the following compounds were prepared and characterised.

CA activity/inhibition studies
An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic activity of various CA isozymes for CO 2 hydration reaction 20 . Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) or 10 mM Tris (pH 8.5) as buffers, and 0.1 M Na 2 SO 4 (for maintaining constant ionic strength, which is not inhibitory against these enzymes), following the CA-catalysed CO 2 hydration reaction for a period of 10 s at 25 C. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and activation 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 determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionised diluted to 1 nM using the assay buffer. Inhibitor and enzyme solutions were pre-incubated together for 15 min (standard assay at room temperature) prior to assay, in order to allow for the formation of the enzyme-inhibitor complex. The inhibition constant (K I ), was obtained by considering the classical Michaelis-Menten equation and the Cheng-Prusoff algorithm by using non-linear least squares fitting as reported earlier 21-24 .

Chemistry
The key intermediate 1 (ethyl 2-((4-sulphamoylphenyl) amino) acetate) was obtained in good yields by the reaction of sulphanilamide with ethyl bromoacetate in the presence of potassium carbonate (no sulphonamide N-alkylation occurred), Scheme 1. During optimisation, the same reaction was performed with sodium carbonate instead of potassium carbonate. The results were not satisfactory, and whence the equimolar ratio of both the reactants in the presence of K 2 CO 3 and ethanol as a solvent was used to obtain compound 1. 4-((2-Hydrazinyl-2-oxoethyl)amino) benzene sulphonamide (2) was obtained by the reaction of hydrazine hydrate with 1 in equimolar ratios. The reaction was refluxed for 3 h at 70 C producing the compound with yields of 70-72%. Reaction of 2 with various substituted aromatic aldehydes afforded derivatives 6. When using unsaturated aldehydes, a cyclisation reaction occurred after the Schiff base formation, leading to compounds 5 (Scheme 1).
resonance (NMR). The structures of compounds were analysed by FTIR spectra which revealed that N-H stretches of amines in the region 3500-3000 cm À1 . Spectra revealed presence of C ¼ O stretching vibrations of amide in the region 1695-1630 cm À1 , aliphatic C-H stretching vibration was observed in 2920-2800 cm À1 and C ¼ N stretching vibration was observed at 1615-1564 cm À1 . Further evidence for formation of target compound was obtained from 1 H-NMR spectra which provided diagnostic tool for the positional elucidation of the protons. The Ar-NH proton was appeared at d ¼ 6.58 ppm as triplet. The formed pyrazoline was confirmed with doublet of doublet for CH 2 giving signal d ¼ 4.0-3.8 ppm (H a ), H b at d 7.1-6.6 ppm (compounds 5l-5u) and at d ¼ 3.26-3.13 ppm (compounds 5a-5k), CH giving signal at d ¼ 7.4-7.1 ppm (H x of series 5l-5u) and d ¼ 5.61-5.56 ppm (H x of series 5a-5k). The characteristic doublet signal of aromatic protons was observed between d ¼ 7.9 and 6.6 ppm. The NH protons of SO 2 -NH 2 as singlet between d ¼ 6.94 and 6.80 ppm was observed. Singlet in the range of d ¼ 6.59-6.2 ppm for Ar-NH protons was observed. The characteristic singlet signal of CH 3 protons in series 5l-5u was observed between d ¼ 2.1 and 2.04 ppm. Mass spectroscopy was done for newly synthesised compounds. The base peak m/z for the compounds were found as (M þ 1) þ with respective to their molecular weight except for 5i it is was found to be (M) þ .
High-performance liquid chromatography (HPLC) was done for newly synthesised compounds. Using area normalisation method, the percent purity for the compounds was found to be above 88%. The structures of novel Schiff bases 6a-6j were confirmed by MS, FTIR and 1 H and 13 C-NMR. The IR spectra displayed an intense absorption band in the range of 1615-1630 cm À1 , characteristic of the carbonyl groups. Additionally, intense bands, originating from the stretching vibration of the C ¼ N group of the azomethine were observed at 1685 and 3313 cm À1 for N-H str of NH 2 following the aliphatic C-H str displayed vibration at 3022 cm À1 . Further, we observed 1 H and 13 C-NMR interpretation for singlet peak at chemical shift d range in 7.95-8.02 ppm and 143.71-144.10 ppm, confirming the presence of azomethine group in the compound, respectively (Tables 1 and 2).

CA inhibition
The compounds 5 and 6 reported here were investigated as inhibitors of three CAs involved in crucial physiologic processes and known to act as drug targets, i.e. the human (h) isoforms hCA I and II (belonging to the a-CA class) and the bacterial enzyme mtCA3 from Mycobacterium tuberculosis (a b-class CA) (Table 3). Acetazolamide (AAZ), a clinically used sulphonamide has been employed as standard inhibitor in the assay.
As seen from data of Table 3, all investigated compounds inhibited the three enzymes, but generally with a medium potency. Thus, the inhibition constants (K I s) were in the range of 54.6 nM-1.8 mM against hCA I, in the range of 32.1 nM-5.5 mM against hCA II and of 127 nM-2.12 mM against mtCA 3, showing a quite flat structure-activity relationship, except for some particular cases which will be discussed in detail.
Thus, for hCA I, the best inhibitors were 5d, 5j and 6c, with K I s ranging between 54.6 and 93.4 nM, being thus much better Table 1. Physicochemical properties of 4-((2-(3-alky/aryl-5-ary/heteroaryl-4,5-dihydro-1H-pyrazol-1-yl inhibitors compared to the standard acetazolamide (K I of 250 nM, Table 3). These compounds incorporate p-chlorophenyl and p-tolyl moieties (5d), p-chlorophenyl and 9-anthranyl (5j) moieties, and the 2-hydroxyphenyl fragment in the case of 6c, which are in fact not very different from those found in compounds showing a much worse inhibitory pattern (e.g. 5c, 5k, 6e, etc.). Thus, the explanation that we propose is that the quite long and flexible linker between the benzenesulphonamide fragment and the imine or heterocylic parts of the molecule, affords for a multitude of diverse orientation of the tail present in these compounds, which is probably detrimental to a tight binding, except for the few cases mentioned above, i.e. 5d, 5j and 6c, for which probably some of these conformations assure good interactions with the enzyme active site. However, for the majority of these derivatives, these various conformations/orientations may be not favourable, which explain why most of them have inhibition constants in the high nanomolar-micromolar range (Table 3). More or less the same situation was observed for the inhibition of hCA II, but for this isoform the most effective inhibitors were 5e, 5r, 5s and 6c, with K I s ranging between 32.1 and 83.1 nM (Table 3). Compound 5e is the only derivative incorporating a 4pyridyl moiety, which seems to be effective in inducing strong hCA II inhibitory effects, whereas the remaining ones incorporate 2-or 4-hydroxyphenyl groups. However, the change of these groups to halogens or to methoxy leads to a strong loss of inhibitory effects. The explanation we propose is the same as above for the discussion of hCA I inhibition data.mtCA3 was also effectively inhibited by several of the new compounds, such as 5e, 5g, 5j and 5k, which showed K I s ranging between 127 and 157 nM (acetazolamide has an inhibition constant of 104 nM, being only slightly more effective compared to these sulphonamides). However, the largest majority of these derivatives showed K I s in the range of 250 nM-2.12 mM, being thus much less effective inhibitors.

Conclusion
We report here a new series of sulphonamide derivatives, which was obtained by reaction of a hydrazide derivative with aromatic/ heterocyclic aldehydes, followed by an eventual cyclisation to a Table 2. Physicochemical properties of 4-((2-(arylmethylidene)hydrazinyl)-2-oxoethyl)amino) benzene sulphonamide derivatives (6a-6j Table 3. hCA I, II and mtCA 3 inhibition data of compounds 5 and 6 reported in the article, by a stopped-flow CO 2 hydrase assay 20 .
five-membered heterocylic system. 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 hCA I and hCA II and the bacterial enzyme mtCA3 from Mycobacterium tuberculosis. The compounds acted as effective-medium potency inhibitors, with K I s in the range of 54.6 nM-1.8 mM against hCA I, in the range of 32.1 nM-5.5 mM against hCA II and of 127 nM-2.12 mM against mtCA 3.