2-((1H-Benzo[d]imidazol-2-yl)amino)benzo[d]thiazole-6-sulphonamides: a class of carbonic anhydrase II and VII-selective inhibitors

Abstract A small library of substituted cyclic guanidine incorporated benzothiazole-6-sulphonamides was synthesized. All obtained compounds were investigated for their inhibitory activity against the key brain-associated human carbonic anhydrase isoform hCA VII (a promising target for the treatment of neuropathic pain) and three isoforms expressed in brain and other tissues, hCA I, II, and IV. Sulphaguanidine derivatives 9a–d were inactive on the all investigated isoforms while the primary sulphonamide containing guanidines 6a–c and 7a–c were inactive towards hCA IV but displayed inhibiting properties on hCA I, II, and VII with KIs values in the low nanomolar to micromolar ranges. The results indicated that isoforms hCA II and VII were potently and selectively inhibited by these compounds, whereas the cytosolic hCA I was less sensitive to inhibition. The derivatives reported in this study might be useful for design of more potent and selective inhibitors of hCA II and VII.


Introduction
Human carbonic anhydrases (hCA,EC 4.2.1.1), are a large family of zinc-metalloenzymes with twelve catalytically active isoforms (hCA I, hCA II, hCA III, hCA IV, hCA VA, hCA VB, hCA VI, hCA VII, hCA IX, hCA XII, CA XIII, hCA XIV), which are vital for respiration, pH regulation, ion transport, metabolism, biosynthetic reactions and other physiological processes 1 . However, the abnormal activity of each isoform stimulates the initiation of various pathological processes in which a disturbed cellular pH buffering or metabolism plays a significant role 2 . Therefore, hCAs are regarded as promising therapeutic targets for many diseases, and the design of isoform-selective inhibitors has attracted the attention of researchers over the last 40 years 3 . In this context, brain-associated isoform CA VII, has been recently validated as an important target in neuropathic pain 4 , a disease which results from damage or dysfunction of the nervous system and whose patients suffer from the lack of effective treatment options 5,6 . Sulphonamides are the largest group of sulphur-containing drugs and the chief class of zinc-binding carbonic anhydrases inhibitors (CAIs) 7,8 . Acetazolamide, methazolamide, ethoxzolamide, brinzolamide, and dorzolamide were the earliest drugs approved as carbonic anhydrase inhibitors and still currently used in the treatment of various diseases such as glaucoma, duodenal ulcers, epilepsy and as diuretics 8 . As shown in Figure 1, the structural similarity of all five drugs is that they share the sulphonamide moiety bound to a heterocycle. High inhibitory potency against hCA VII is another similarity of these clinically used drugs 9 . However, they are not isoform selective inhibitors and are active against the majority of isoforms, with inhibition constants in low nanomolar range. Due to their poor selectivity against a single isoform, another thing they have in common is the multitude of side effects. Ethoxzolamide is the most potent hCA VII inhibitor in this series (K I of 0.8 nM). However, it also inhibits hCA I, II, IV-VII, IX, and XII-XIV in moderate to low nanomolar ranges. Therefore, the search for structural improvement of this drug to produce less-toxic, more efficient, and isoform selective agents is an ongoing struggle 10 . Very recently, we disclosed that incorporation of the guanidine moieties as tails to classical benzenesulphonamide scaffolds improved selectivity against hCA VII versus ubiquitous hCA I and CA II (Figure 2(a)) 11 . Considering all these facts and in connection with our works in the development of selective CAIs 12 , with the objectives of development of hCA VII selective inhibitors, in this study, we present the synthesis of three different sets of novel cyclic guanidine incorporated benzothiazole-6-sulphonamides ( Figure 2(b)) and evaluate their capability to inhibit three cytosolic isozymes (hCA I, II, and VII) and one membranebound isozyme (hCA IV).

Chemistry
The rationale for obtaining the novel compounds reported here consisted in replacing phenyl ring in the 4-guanidinobenzenesulphonamide scaffold, a structure recently reported by our group as selective inhibitor of hCA VII 11 , by a heteroaryl ring, leading thus to guanidine-heteroarene sulphonamide which presumably show enhanced hCAs inhibitory activity.

Carbonic anhydrase inhibition
The inhibition data against four human CA isoforms expressed in brain, hCA I, II, IV (off-target) and VII (target for neuropathic pain drug discovery) with the newly synthesised sulphonamides 6a-c, 7a-c, and 9a-d along with the reference drug acetazolamide (AAZ), are shown in Table 1.
The following structure activity relationship (SAR) can be figured out from the inhibition data of Table 1: i. Among the entire series, only compounds bearing primary sulphonamide moiety 6a-c and 7a-c were moderately inhibited the cytosolic isoform hCA I, whereas sulphaguanidine  derivatives 9a-d were entirely inactive against this isoform (K I > 100 mM). The SAR showed that compounds containing imidazoline core 6a-c displayed better inhibitory capability against this isoform, with K Is ranging between 442.4 and 7590 nM, as compared to the benzimidazoline-substituted ones 7a-c (K Is of 4927-9533 nM). Notably, the inhibitory potency was reduced significantly when any functionality was presented on the periphery of(benz)imidazoline rings. As shown in Table 1, 2-((4,5-dihydro-imidazol-2-yl)amino) benzothiazole-6-sulphonamide 6a exhibited the best activity of all the evaluated compounds, albeit approximately two-fold less effective than standard drug, AAZ. ii. The physiologically most relevant isoform hCA II was inhibited by compounds 6a-c and 7a-c more than hCA I (K Is in the range 37.6-577.6 nM). The results showed that sulphaguanidines 9a-d were also inactive against this isoform. Again imidazoline-incorporated sulphonamides 6a-c demonstrated superior inhibitory activities (K Is in the range 37.6-65.6 nM) compared to benzimidazoline-substituted derivatives 7a-c (K Is of 84.0-577.6 nM). Once more, the optimal substitution seems to be an unsubstituted imidazoline ring, in 6a, which is the most effective hCA II inhibitor in the series investigated here. iii. As seen from data of Table 1, none of the tested compounds showed inhibitory action against the transmembrane isoform hCA IV (K I > 100 mM), which is considered as being an off-target isoform in our study. Notably, AAZ shows low nanomolar inhibitory activity against this isoform with a K I of 74nM. iv. Similar to the inhibitory pattern of investigated compounds against ubiquitous CA I and CA II, sulphaguanidine derivatives 9a-d failed to inhibit the neuropathic pain associated hCA VII. However, the rest of the derivatives 6a-c and 7a-c effectively inhibited this isoform, with K Is values ranging from 37.4to 694.4 nM. Although again analogues 6a-c exhibited better inhibitory activity when compared to compounds 7ac, in this case, the SAR was not very flat. Indeed, compound 6a has the highest inhibitory effect for the off-target isoforms hCA I and hCA II while methyl-substituted compound 6b showed superior inhibitory potency towards hCA VII. Although this compound displayed 15-fold higher selectivity for hCA VII over hCA I, it did not show significant selectivity towards hCA VII versus hCA II (hCA VII/hCA II % 1.3).

Conclusion
We have synthesised three small sets of novel cyclic guanidine incorporated benzothiazole-6-sulphonamides (6a-c, 7a-c, and Table 1. Inhibition data of human CA isoforms CAI, II, IV and VII with benzothiazole-6-sulphonamide substituted five-membered (bi)cyclic guanidines 6, 7, 9 using acetazolamide (AAZ) as standard drug. 9a-d) and screened them against four human CA isoforms expressed in brain, hCA I, II, IV and VII. Among them, compounds bearing primary sulphonamide moiety 6a-c and 7a-c showed potent inhibitory activity against three cytosolic isoforms hCA I, II and VII whereas they did not display any inhibitory activity towards membrane-bound isoform hCA IV. The SAR indicated that generally compounds of series 6a-c displayed better hCA inhibitory activity compared to the derivatives 7a-c. It was also found that hCA II and VII were the considerably most sensitive to these inhibitors than hCA I. However, the newly developed compounds did not display remarkable selectivity towards hCA VII versus hCA II. Therefore, further optimisation and exploration of this kind of novel scaffolds required to development of new isoform-selective CAIs with better inhibition potency and fewer side effects.

Chemistry methods
Starting materials, reagents and solvents were purchased from commercial sources and used as received without any type of further purification. Thin-layer chromatography (TLC) was performed on silica gel, spots were visualised with UV light (254 and 365 nm). NMR spectra were recorded on Bruker 300 and 500 spectrometers in DMSO or DMF with chemical shifts values (d) in ppm relative to tetramethylsilane (TMS). High-resolution mass spectra (HRMS) were recorded on a mass spectrometer with a Q-TOF micro mass analyser using the ESI technique. (2) 4-Aminobenzensulphonamide (1) (30 g, 174.3 mmol) was dissolved in 3.5 M HCl (180 ml) under gentle warming. The solution was cooled down to r.t. and then KSCN (16.94 g,174.3 mmol) was added to the reaction mixture and the mixture was refluxed for 3 h. After cooling to room temperature, the reaction mixture was diluted with ice-cold water (approximately 200 ml). The formed solids were collected by filtration, washed with water and air dried to afford 2 (12.1 g, 31%) as white powder. 1 8, 127.3, 139.8, 143.9, 182.8

4-Thioureidobenzenesulphonamide
To a suspension of 4-thioureidobenzenesulphonamide (2) (10 g, 43.3 mmol) in CHCl 3 (120 ml) was added dropwise a solution of Br 2 (3.27 ml, 64.9 mmol) in CHCl 3 (15 ml) and stirred at 70 C for 4.5 h. After cooling to rt volatiles were removed under reduced pressure to give a lumpy solid that was dissolved in H 2 O (120 ml). The aqueous solution was treated with ammonium hydroxide until the pH adjusted to greater than 9-10 and then stirred at 90 C for 1 h. The formed precipitate was collected by filtration, washed with H 2 O and cold ethanol and dried under vacuum to afford compound 3 (7.9 g, 80%) as off-white solid. 1 (4) To an ice-cooled stirred solution of 2-aminobenzo[d]thiazole-6-sulphonamide (3) (20 g, 87.3 mmol) in DMF (160 ml), DMF-DMA (11.6 ml, 87.3 mmol) was added dropwise and stirring continued at 0 C for 2 h. After completion of the reaction, the mixture was extracted with DCM (3 Â 100 ml). The combined organic layers were washed with water (1 Â 100 ml) and concentrated under reduced pressure to 30 ml and then treated with CHCl 3 (250 ml). The precipitated solid was collected by filtration, washed with CHCl 3 (3 Â 25 ml) and dried to afford the target product 4 (9.3 g, 37%) as yellowish powder.
Note, due to the precipitation of considerable amounts of the desired product by adding Na 2 SO 4 to the organic extracts, extracts collected were not dried over Na 2 SO 4 and directly concentrated under vacuum. 1 (1.24 ml, 10.3 mmol) in DMSO (5.0 ml) was stirred at 120 C for 15 h. After completion of the reaction, the mixture was cooled to room temperature and treated with water (25 ml) and then extracted with EtOAc (3 Â 25 ml). The combined organic extracts were washed with water (3 Â 25 ml) and concentrated to obtain an orange solid that was dispersed in t-BuOMe (30 ml) and then filtered-off. The collected solution was treated with hexanes (40 ml) and the precipitated solids were collected by filtration and washed with hexanes and dried to afford compound 6c (0.177 g, 49%) as white powder.

2-((1H-benzo[d]imidazol-2-yl)amino)benzo[d]thiazole-6-sulphonamide (7a)
A mixture of dimethyl (6-(N-((dimethylamino) 4 g, 1.03 mmol) and o-phenylenediamine (0. 89 g, 8.24 mmol) in DMSO (5.0 ml) was stirred at 120 C for 15 h. After completion of the reaction, the mixture was cooled to room temperature, hydrazine hydrate (2 ml) was dropwise added and the reaction mixture was stirred for 1 h. Then water (30 ml) and ethyl acetate (30 ml) were added to the mixture and the solution was stirred for 10 min and then remained still for a few minutes. Subsequently, the solids formed in the organic phase were collected by filtration and washed with EtOAc to afford compound 7a (0.179 g, 50%) as white powder. 1

CA inhibition assays
An applied photophysics stopped-flow instrument has been used for assaying the CA-catalysed CO 2 hydration activity 13 . Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic 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 velocity. The uncatalysed rates were determined 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 nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier 14 and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier 15 .

Disclosure statement
No potential conflict of interest was reported by all author(s) except CTS. CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Funding
This work was supported by the European Regional Development Fund (ERDF, project