Synthesis and biological evaluation of benzenesulphonamide-bearing 1,4,5-trisubstituted-1,2,3-triazoles possessing human carbonic anhydrase I, II, IV, and IX inhibitory activity

Abstract A library of benzenesulphonamides incorporating 1,2,3-triazole rings functionalised with ester, carboxylic acid, carboxamide, carboxyhydrazide, and hydroxymethyl moieties were synthesised. The carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV, and hCA IX. Among them, hCA II and IV are anti-glaucoma drug targets, being involved in aqueous humour secretion within the eye. hCA I was inhibited with Ki’s ranging between 8.3 nM and 0.8737 µM. hCA II, the physiologically dominant cytosolic isoform, was excellently inhibited by these compounds, with Ki’s in the range of 1.6–9.4 nM, whereas hCA IV was effectively inhibited by most of them, with Ki’s in the range of 1.4–55.3 nM. Thirteen of the twenty sulphonamides were found to be excellent inhibitors of tumour associated hCA IX with Ki’s ≤ 9.5 nM. Many of the new compounds reported here showed low nM inhibitory action against hCA II, IV, and IX, isoforms involved in glaucoma and some tumours, making them interesting candidates for further medicinal chemistry/pharmacologic studies.

Primary sulphonamide bearing heterocyclic compounds form a part of potent CAIs in which binding group binds to the Zn(II) ion as anion in a tetrahedral geometry. A large number of drugs belonging to this class, like acetazolamide (AZA), methazolamide (MZA), ethoxzolamide (EZA), dorzolamide (DZA), etc., are in clinical use from past many years targeting different therapeutic areas 21 .
General procedure: A mixture of appropriate b-diketoester 11a-11d (2.00 mmol) and piperidine (5 mol%) dissolved in 3 ml DMSO were heated at 70 C in silicon oil bath for 5 min followed by addition of 4-azidobenzenesulphonamide (2.02 mmol). After addition, reaction mixture was allowed to stir at 70 C for 4-6 h. Reaction was monitored through thin-layer chromatography. After completion, reaction mixture was poured into water after cooling to afford required product 4a-4d. The crude product thus obtained was recrystalised with ethanol.    Synthesis of 1-[4-(aminosulfonyl)phenyl]-5-alkyl/aryl-1H-1,2,3triazole-4-carboxylic acid (5a-5d) General procedure: An appropriate 1,2,3-triazolic ester 4a-4d was dissolved in 20% aq NaOH solution (5 ml) and refluxed for 4 h. Then cooled the solution to room temperature, added ice to it and neutralised with concd HCl which resulted into the precipitation of a white solid. The solid was filtered off, washed with water, dried and recrystallised from appropriate solvent which afforded the pure products 5a-5d.

4-[4-(Hydrazinocarbonyl)-5-(4-methoxyphenyl)-
General procedure: Appropriate 1,2,3-triazolic ester 4a-4d (3.22 mmol) was dissolved in dry tetrahydrofuran (20 ml) and a suspension of LiAlH 4 (6.44 mmol) in dry tetrahydrofuran (5 ml) was slowly added under anhydrous conditions. After 20 min of reaction at 20 C, reaction mixture was refluxed for 2 h. After completion of the reaction, an aqueous solution of HCl 1 N was added dropwise until a neutral pH. The reaction mixture was concentrated under reduced pressure; the residue was taken into ethyl acetate and washed with water and brine. The organic layer was dried over MgSO 4 and concentrated under reduced pressure. The residue was recrystalised in ethanol.

CA inhibition
All the newly synthesised 4-functionalised 1,2,3-triazole compounds (4a-4d, 5a-5d, 6a-6d, 7a-7d, and 8a-8d) were evaluated against cytosolic isoenzymes hCA I & hCA II and membrane bound isoenzymes hCA IV & hCA IX for their CA inhibition potential by using stopped-flow CO 2 hydrase assay method 49 and acetazolamide (AZA) was chosen as reference drug for the assay. In general, all the assayed compounds have shown significant inhibitory action against the reported isoforms, with low nanomolar inhibition constant (Ki). Inhibition data of the compounds as given in Table 1 let the following insights regarding CAs inhibitory properties.
i. The cytosolic isoform hCA I was in general significantly inhibited by all the newly synthesised compounds (4a-4d, 5a-5d, 6a-6d, 7a-7d, and 8a-8d) with Ki in the range 8.3 nM-0.8737 mM (Table 1). It is pertinent to mention that 5-CH3 substituted derivatives of newly synthesised compounds except 8a were most effective inhibitors of hCA I with Ki 15.1nM as compared to corresponding 5-aryl derivatives. At the same time some compounds namely 5b, 5c, 6c, 6d, 8a, and 8c showed weaker inhibition potential as compared to reference drug AZA (Ki ¼250 nM) against hCA I that is off-target while inhibiting hCA II and IV in glaucoma and hCA IX in tumours.
ii. Nearly all the newly synthesised compounds (4a-4d, 5a-5d, 6a-6d, 7a-7d, and 8a-8d) showed better inhibitory potential in low nanomolar range with Ki 9.4 nM except three compounds namely 5b, 7b, and 8a against the most abundant isoform hCA II as compared to standard drug AZA (Ki ¼12.1 nM). Some of the tested compounds mainly 5-CH3 derivatives (4a, 5a, and 6a) and two other compounds (7d and 8c) exhibited inhibitory potency (Ki 4 nM) several times better than AZA (Table 1). iii. All the tested compounds except some 5-CH3 derivatives namely 5a, 6a, 7a, and 8a showed excellent inhibition potential with Ki in the range of 1.4-55.3 nM against membrane bound isoform, hCA IV as compared to standard drug AZA. Most of the compounds (4a-4d, 5c-5d, 6b-6d, 7c-7d, and 8b-8d) have their inhibitory potency (Ki 8.4 nM) several folds superior than AZA (Ki 74 nM) against hCA IV which is one of the drug target for designing antiglaucoma drugs (Table 1). iv. In general, all the tested compounds except few (4c, 5b, 5c, 6b, 7b, 8a, and 8b) have shown better activity profile (Ki 9.5 nM) against tumour associated membrane bound isozyme hCA IX as compared to reference drug AZA (Ki ¼25.8 nM). It is significant to mention here that, in the broader sense, derivatives with 5-CH3and 5-(naphtha-2-yl) substitution have shown better activity as compared to other derivatives (Table 1). v. Interestingly compounds possessing rather bulky scaffolds were milder inhibitors of cytosolic isoform hCA I, over other isoforms (hCA II, IV, and IX) and is mainly due to the fact that the active site cavity of hCA I is smaller than other isozymes hCA II, IV and IX, because of the presence of two His residues (i.e. His 200 and His 67) 50 . Overall comparison of activity, in terms of SAR, reveals that all the compounds except derivatives with 5-CH3 group were better selective for hCA II and IV over hCA I in the broader sense. Therefore, these compounds can be important candidates for designing antiglaucoma drugs. However, their good activity against both hCA II and hCA IX suggests that structure of compounds needs further modification for getting better selectivity for tumour associated hCA IX over hCA II.

Conclusions
In this paper, we report a series of twenty novel compounds of 4functionalised 1-aryl-5-alkyl/aryl-1,2,3-triazole compounds (4a-4d, 5a-5d, 6a-6d, 7a-7d, and 8a-8d) bearing a primary sulphonamide group on the phenyl ring at N-1 position of 1,2,3-triazole scaffold with different functionalities at C-4, such as ester, carboxylic acid, carboxamide, hydrazinocarbonyl, and hydroxymethyl which were evaluated against four isozymes, hCA I, II, IV, and IX. Most of the compounds performed better against aforementioned isoforms showing low nanomolar potency as compared to reference drug acetazolamide. Out of twenty newly synthesised compounds, seventeen compounds (except 5 b, 7 b, and 8a) showed low nanomolar affinity (Ki 9.4 nM) for hCA II, sixteen compounds except the derivatives with 5-CH 3 substitution have displayed excellent activity (Ki 55.3 nM) for hCA IV and thirteen compounds (except 4c, 5 b, 5c, 6 b, 7 b, 8a, and 8 b) have shown better activity (Ki 9.5) for hCA IX while most of compounds with bulkier substitution at C-5 were medium to weaker inhibitors of hCA I with Ki values in the range of 56.2-873.7 nM. In short, reported compounds have shown remarkable activity against hCA I, II, IV, and IX isoforms from which it can be concluded that 1,2,3-triazoles scaffold deserve to be investigated further as a novel scaffold for CAIs.