Novel sulphonamides incorporating triazene moieties show powerful carbonic anhydrase I and II inhibitory properties

Abstract A series of compounds incorporating 3-(3-(2/3/4-substituted phenyl)triaz-1-en-1-yl) benzenesulfonamide moieties were synthesised and their chemical structure was confirmed by physico-chemical methods. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the compounds were evaluated against human isoforms hCA I and II. KI values of these sulphonamides were in the range of 21 ± 4–72 ± 2 nM towards hCA I and in the range of 16 ± 6–40 ± 2 nM against hCA II. The 4-fluoro substituted derivative might be considered as an interesting lead due to its effective inhibitory action against both hCA I and hCA II (KIs of 21 nM), a profile rarely seen among other sulphonamide CA inhibitors, making it of interest in systems where the activity of the two cytosolic isoforms is dysregulated.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes that catalyse the interconversion reaction between carbon dioxide and bicarbonate 1,2 . CAs catalyse this reaction by using a metal hydroxide nucleophilic mechanism [3][4][5] . The metal ions present within the active site of these enzymes (Zn(II), Cd(II), Co(II), Fe(II) or Mn(II) for the i-CAs) are coordinated by three amino acid residues and a water molecule which is activated by the metal ion and the hydrophobic environment, becoming highly nucleophilic [3][4][5] . This metal hydroxide species nucleophilically attacks CO 2 and promotes its hydration to bicarbonate very efficiently at almost a neutral pH [3][4][5] . There are eight genetically distinct CA families, namely a-, b-, c-, d-, f-g-, h-, and i-CAs 3,4,6,7 . In humans, there are 12 catalytically active a-CA isoforms which have different catalytic activities 6 . These enzymes are distributed in many organs and tissues and they play important roles in many physiological and pathological processes such as acid-base regulation, biosynthetic reactions, electrolyte secretion, and calcification. Thus, human carbonic anhydrases (hCAs) are targets for the design of new drugs to use in the diagnosis and/or treatment of many diseases. For example, hCA II, IV, XII, and XIV inhibitors are used as diuretics, whereas hCA II, IV, and XII inhibitors are used as anti-glaucoma drugs 1,2,8,9 . hCA I plays a role in the regulation of retinal pathologic processes, and its inhibition is a strategy for the treatment of such conditions. hCA II inhibitors are used for the treatment of glaucoma, oedema, and epilepsy 5,8 . The cytosolic, highly abundant isoforms hCA I and II are in fact both drug targets for a multitude of diseases, as highlighted above, but due to their role in pH homeostasis and wide distribution in many tissues, are also frequently off-targets, when other isoforms (e.g. CA VA/B, VII, IX or XII) should be selectively inhibited 7,9 . Although CA inhibitors (CAIs) have been often used in clinics, the first and second-generation drugs have several undesired side effects because of their low selectivities for various isozymes with medicinal chemistry applications 5,8,10,11 . Therefore, there is a need for compounds with higher selectivity to diverse hCAs compared to the available drugs used clinically nowadays. Compounds containing sulphonamide group (R-SO 2 NH 2 ) and its isosteres sulfamide (R-NH-SO 2 NH 2 ) and sulfamate (R-OSO 2 NH 2 ) are among the most important classes of CAIs 12 . Some of these derivatives have been used as drugs for many years 13 . More recently, a compound bearing an ureido-substituted aryl-sulphonamide (SLC-0111) ( Figure 1) was reported to show remarkable CA inhibitory effects and is presently in Phase Ib/II clinical trials as an antitumor/antimetastatic agent 14,15 .
Triazenes are an interesting group of compounds which has many applications in the synthesis of various products, some of which possess pharmacological applications 16,17 . Dacarbazine and temozolomide (Figure 1), both incorporating a triazene moiety, are in the clinical use for cancer treatment, as they possess acceptable toxicity and good pharmacokinetic properties 17,18 . Additionally, the triazene group is an isostere of the ureido group and was recently reported to lead to interesting CA inhibitory derivatives by Akocak et al. 19 .
Some compounds bearing triazene substituted sulphanilamide or metanilamide (3-aminobenzene sulphonamide) were recently reported to possess CA inhibitory effects by one of our groups 13,20 , but there are few studies on this class of CA inhibitors. In this study, we synthesised new 1,3-diaryltriazene sulphonamide compounds 1-12 from the diazonium salt of metanilamide and different substituted aromatic amines and evaluated their inhibition profiles towards hCA I and II, in order to find out new drug candidates.

Chemistry
All chemicals and solvents were purchased from Sigma-Aldrich and Merck. The nuclear magnetic resonance (NMR) spectra ( 1 H NMR, 13

Synthesis of 1,3-diaryltriazene sulphonamide derivatives
To the solution of 3-aminobenzene sulphonamide (5 mmol) in water (3 ml), concanrate HCl (1.5 ml) was added then the mixture was cooled to 0-5 C and stirred for 5 min. To this mixture, sodium nitrite (7 mmol) in water (3 ml) was added dropwise during about 10-15 min at 0-5 C. This mixture was stirred about 15-20 min at 0-5 C. Then, this mixture (diazonium solution) was added to a suitable aniline (5 mmol) solution (in 5 ml methanol) by adjusting the pH between 6 and 7 with the simultaneous addition of saturated sodium acetate in water. The reaction mixture was stirred at 0-5 C for 3 h and then overnight at room temperature in dark 13 . The precipitated solid product was collected by filtration and washed several times with cold water. The crude compounds were air-dried then purified by crystallization from methanol. The chemical structures of the compounds 1-12 were characterised by 1 H NMR, 13 C NMR, and HRMS.

Carbonic anhydrase inhibitory effects
CA inhibition with compounds 1-12 on hCA I and hCA II are shown in Table 1. Acetazolamide (AZA) was used as a reference drug. According to Table 1, K I values (inhibitory potency) of the compounds 1-12 were in the range of 21 ± 4-72 ± 2 nM towards hCA I while they were in the range of 16 ± 6-40 ± 2 nM towards hCA II. K I values of AZA were 19 ± 2 nM and 17 ± 4 nM towards hCA I and hCA II, respectively (by an esterase method, using 4nitrophenyl acetate as substrate).
It can be seen from the results in Table 1 that all compounds in the series had a higher K I compared to AZA towards hCA I. According to the Table 1 lowest K I values (21 ± 4 nM and 28 ± 6 nM, respectively) in the series towards hCA I. The 4-fluoro substituted derivative compound 2 had the most effective inhibitory activity with a similar K I value (21 ± 4 nM) with AZA against hCA I.
According to the K I values of the compounds against hCA II, the most active compounds in the series were the 4-ethoxy substituted derivative compound 4 and the 4-ethyl substituted derivative compound 6 with their lower K I values than AZA (16 ± 6 nM and 16 ± 2 nM, respectively). On the other hand, the non-substituted derivative 1 and the 4-bromo substituted derivative 3 had similar K I values, comparable to those of the reference drug, AZA (18 ± 5 nM and 19 ± 4 nM, respectively). When the K I values towards hCA II in Table 1 are evaluated, it was observed that substitution at the para position of the phenyl ring leads to more effective inhibitors compared to the ortho or meta substitutions. Probably, this is due to the steric impairments in which the latter two substitution patterns participate within the costrained active site of the enzyme, as compared to the less sterically impaired para substitution, as observed for many other types of CAIs 32-41 .

Conclusions
We report a series of 1,3-diaryltriazene substituted metanilamide derivatives 1-12, acting as CA inhibitors against the widely spread cytosolic isoforms hCA I and II. According to the inhibition results, the 4-fluoro substituted derivative compound 2 can be considered as a lead molecule due to its interesting inhibition profile against both hCA I and hCA II, making it of interest in systems where the activity of the two cytosolic isoforms is dysregulated.