Investigation of novel alkyl/benzyl (4-sulphamoylphenyl)carbamimidothioates as carbonic anhydrase inhibitors

Abstract A library of novel alkyl/benzyl (4-sulphamoylphenyl)carbamimidothioates was synthesised by selective S-alkylation of the easily accessible 4-thioureidobenzenesulphonamide. The compounds were assayed as inhibitors of four human (h) carbonic anhydrase isoforms hCA I, II, VII, and XIII, as well as three bacterial enzymes belonging to the β-CA class, MscCA from Mammaliicoccus (Staphylococcus) sciuri and StCA1 and StCA2, from Salmonella enterica (serovar Typhimurium). Most compounds investigated here exhibited moderate to low nanomolar inhibition constants against hCA I, II, and VII. The cytosolic hCA XIII was also inhibited by these compounds, but not as effective as hCA I, II, and VII. Several compounds were very effective against MscCA and StCA1. StCA2 was less inhibited compared to MscCA and StCA1. Some compounds showed considerable selectivity for inhibiting some CA isoforms. They may thus be considered as interesting starting points for the discovery and development of novel therapeutic agents belonging to this class of enzyme inhibitors.


Introduction
Enzymes are drug targets for at least 50% of the clinically used drugs 1 . At leasta third of all known enzymes contain one or more metallic ions (such as Ca, K, Mg, Mn, Fe, Co, Ni, Cu, Zn, and Mo), that are essential to their biological functions 2,3 . Among the zinccontaining metalloenzymes, the carbonic anhydrases (or carbonate dehydratases) (CA, EC 4.2.1.1) are abundantly present in most life forms, e.g. mammalian tissues, plants, algae as well as all types of microorganisms, such as bacteria, protozoans, diatoms, etc 4 . Human CAs occur in at least 15 isoforms (CA I, II, III, IV, VA, VB, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV) depending upon the tissue, organ, or cell-type they are distributed in 5 . This enzyme assists the chemical interconversion of carbon dioxide and water to bicarbonate and H þ ions, which are thereafter involved in mammals in both physiological and pathological processes, including respiration, pH homeostasis, signal transduction, electrolyte secretion, bone resorption, lipogenesis, calcification, etc 6-8 . Due to broad roles of carbonic anhydrases in metabolism, selective inhibition of their activity is an excellent therapeutic target for several diseases 5 , including glaucoma (CA II, IV), edoema (CA II), obesity (CA VA), cancer (CA IX, XII), sterility (CA XIII), haemolytic anaemia (CA I), altitude sickness (CA II), and more recently in the management of neuropathic pain (CA VII) or as antiinfectives (when pathogenic organisms CAs are being targeted) 9 .
Sulphonamide-containing compounds are the main class of CA inhibitors 5 . More than 70 currently marketed drugs contain this privileged moiety 10 , and many researchers have been working to explore it, due to the potential of sulphonamides against diverse diseases. In Scheme 1, some of the effective CA inhibitors of the sulphonamide/sulphamate types (compounds 1-4) are shown.
In continuation of our work on the developments of carbonic anhydrase inhibitors 11 , in this study we report the synthesis of a panel of 15 novel alkyl/benzyl (4-sulphamoylphenyl)carbamimidothioates, for which we explored their inhibitory effects against a panel of human and bacterial CA isoforms involved in various pathologies.

Chemistry
The synthetic route to obtain the target compounds reported here is outlined in Scheme 2. The 4-thioureidobenzenesulphonamide intermediate 6, prepared according to the literature procedure 12,13 was upon treatment of sulphanilamide 5 with KSCN in a refluxing aqueous solution of HCl. The target alkyl/benzyl (4-sulphamoylphenyl)carbamimidothioates 8a-o were selectively synthesised by reaction of 4-thioureidobenzenesulphonamide 6 with the corresponding alkyl/benzyl halides 7 at elevated temperatures. Except for a few compounds which were purified by column chromatography, most of them were purified by simple extraction using ethyl acetate, which afforded the desired products with high purity and yields ranging from 62% to 89%. The analytical and spectroscopic data ( 1 H and 13 C NMR chemical shifts and mass spectra) of the purified compounds are in agreement with the purposed structures (see Experimental section for details).

Carbonic anhydrase inhibition
Sulphonamides 8a-o incorporating carbamimidothioate moieties have been tested as inhibitors of four human (h) CA isoforms, the cytosolic hCA I, II, VII and XIII, as well as the bacterial b-CAs from Mammaliicoccus (Staphylococcus) sciuri 14 and Salmonella enterica (serovar Typhimurium), StCA1 and StCA2 15 . It should be mentioned that the first bacterial enzyme, MscCA, was originally reported by us as Staphylococcus aureus b-CA, SauCA based on a genomic sequence annotated in the data bases in 2017 14 . A recent reanalysis of that sequence revealed that the original annotation was erroneous, and that the sequence encodes a b-class CA from another Staphylococcaceae family member, i.e. Staphylococcus sciuri, which is a Gram-positive, oxidase-positive, coagulase-negative member of these infectious bacteria known to provoke disease in humans and animals (it was originally isolated from the squirrel) 16 . In 2020, the species was renamed as belonging to a new genus, as Mammaliicoccus sciuri 16 . The hCAs included in the study were the ubiquitous hCA I and II, as well as hCA VII and XIII which are found in fewer tissues compared to hCA I and II, and are involved in several pathologies in the CNS (hCA VII) or reproductive tract (hCA XIII) 17,18 .
The following structure-activity relationship (SAR) can be evidenced from the data of Table 1.
(i) The slow cytosolic human isoform hCA I was strongly inhibited by all the synthesised carbamimidothioate derivatives with K I ranging between 38.5 and 92.5 nM. These results indicated that all compounds reported here are more potent than the standard drug acetazolamide (AAZ) on this isoform. Overall the relative inhibitory rates of reported carbamimidothioates followed the order: allyl carbamimidothioate ! benzyl carbamimidothioates %alkyl carbamimidothioates ! propargyl carbamimidothioates.
Although the SAR for the benzylic derivatives was not flat, the aliphatic derivatives showed a very flat SAR where the inhibition increased as the chains got longer.   (ii) The ubiquitous cytosolic human isoform hCA II was also effectively inhibited by all compounds in low to moderate nanomolar range with K I values ranging between 1.7 and 85.8 nM. Compound 8m having a 3,5-difluorobenzyl substitution was found to be the most effective amongst all reported derivatives, whereas the least effective inhibitor was the methyl-substituted compound 8a. The results showed that the presence of electron-withdrawing functionalities on the phenyl ring periphery of benzylic carbamimidothioates increased their inhibitory activities, whereas electrondonating groups reduced their potency. In the cases of aliphatic carbamimidothioates, it seems that the inhibitory activities increased with the increasing length of the carbon chain. For instance, the heptyl-substituted carbamimidothioate 8c was 34 times more potent as a CA II inhibitor compared to the methylsubstituted analogue 8a.
(iii) The brain-associated cytosolic isoform hCA VII showed a rather similar inhibition profile with hCA II, with most compounds investigated here. Thus, the 3,5-difluorobenzyl-substituted compound 8m was the strongest inhibitor with a K I of 1.2 nM whereas 3,5-dimethylbenzyl-, methyl-, and butyl-substituted carbamimidothioates 8o, 8a, and 8b, respectively, showed the weakest inhibitory capacities, with inhibition constants in the range of 13.8-25.6 nM (but they are still highly effective inhibitors). It is worth mentioning that more than half of the compounds investigated here (8c, 8e, 8g-8j, 8l, 8m), showed better inhibitory activities against hCA VII in comparison with the standard drug AAZ. Among them, half (8g-8j) showed more than 10 times a better selectivity against hCA VII vs hCA II.
(iv) The other cytosolic isoform hCA XIII was weakly inhibited by all investigated compounds (K I in the range of 69.3-925.9 nM) compared to AZA, which showed a K I of 16.0 nM. The best inhibitors were the 3,4-dichlorobenzyl-and 3,5-difluorobenzyl-substituted carbamimidothioates, 8n, 8m, which were however almost four times less potent inhibitor than acetazolamide.
(v) Against the bacterial b-CA isoform MscCA, except the benzyl-and 4-chlorobenzyl-substituted carbamimidothioates 8g, 8h, with K I values of 360.8 and 618.9 nM, respectively, the other compounds acted as weak inhibitors (K I in the range of 658.8-8761 nM) compared to the clinically used sulphonamide acetazolamide. It is noteworthy that while the allylic carbamimidothioate 8e showed the best result against hCA I, the worst result against MscCA were observed for this derivative. These results clearly indicated that the interaction of inhibitors with aand b-CAs is rather different.
(vi) The bacterial b-CA isoform StCA1 was also inhibited by the synthesised compounds 8a-8n in a moderate nanomolar range with K I values of 52.2 to 750.9 nM. Similar to MscCA, the most potent inhibitor against this isoform was compound 8g with a potency similar to that of AAZ. Interestingly, the less effective inhibitor was again the benzyl-substituted derivative 8i with 4-bromo substitution. These results revealed that the inhibitory activity of benzyl-substituted carbamimidothioates 8g-8o against StCA1 significantly depended on the electronic effects of substituent on the phenyl ring. Generally, unsubstituted and strongly electron-withdrawing groups (e.g. CN, NO 2 ) at the benzylcarbamimidothioate moiety induced higher inhibitory activities compared to electron-donating (e.g. Me) or less electron-withdrawing groups (e.g. Cl, Br).
(vii) StCA2 was the least inhibited bacterial CA among the three such enzymes investigated in this study. Indeed, the newly prepared compounds showed K I ranging between 270.2 and 9697 nM. Therefore, all of them were generally poor StCA2 inhibitors compared to AAZ. Interestingly, unlike StCA1, for which increasing the carbon chain-length of aliphatic carbamimidothioates led to an increased inhibition, for StCA2, the inhibition decreased by increasing the carbon chain-length in the synthesised derivatives.

Ca inhibitory assay
An applied photophysics stopped-flow instrument has been used for assaying the CA-catalysed CO 2 hydration activity 19 . 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 for a-CAs or 20 mM TRIS (pH 8.4) as buffer for b-CAs, 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 distilleddeionised water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6 h 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 12,13,20,21 , and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier 22,23 and their concentrations in the assay system ranged between 7.6-12.5 nM.

Conclusions
A new series of novel alkyl/benzyl (4-sulphamoylphenyl)carbamimidothioates (8a-o) was designed, synthesised and evaluated for their ability to inhibit four pharmacologically significant cytosolic hCA isozymes, hCA I, hCA II, hCA VII and hCA XIII as well as three b-CAs from the bacterial pathogens Mammaliicoccus (Staphylococcus) sciuri, MscCA, and Salmonella enterica (serovar Typhimurium), StCA1 and StCA2. Listed below are some important information of the bioassays: (i) all investigated compounds 8a-o showed strong inhibitory activities against hCA I (K Is , 38.5-92.5 nM) compared to that of AAZ (K I , 250.0 nM); (ii) up to half of the compounds investigated here (8c-f, 8k-n) showed better inhibitory activities against hCA II (K Is , 1.7-9.4 nM) in comparison with AAZ (K I , 12.5 nM); (iii) Among the 15 compounds examined, 8 of them (8c, 8e, 8g-8j, 8l, and 8m) showed better inhibitory activities against hCA VII (K Is , 1.2-2.4 nM) in comparison with AAZ (K I , 2.5 nM). Importantly, among these 8 compounds, half (8g-8j) showed more than 10 times much better selectivity against hCA VII vs hCA II; (iv) All investigated compounds (8a-o) exhibited weaker inhibitory activity (K Is , 69.3-925.9 nM) than AAZ (K I , 16.0 nM); (v) Against the bacterial b-CA isoform MscCA, except compounds 8g and 8h with K I values of 360.8 and 618.9 nM, respectively, other compounds acted as weak inhibitors (K Is , 658.8-8761 nM) compared to AAZ (K I , 625 nM); (vi) Against the bacterial b-CA isoform StCA1, only benzyl-and 4-cyanobenzyl-substituted carbamimidothioates 8g and 8j with K I values of 52.2 and 55.7 nM, respectively, showed better inhibitory activities compared to AAZ (K I , 59 nM); and (vii) These compounds were generally poor inhibitors of StCA2 with K I values ranging between 270.2 and 9697 nM.

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
The work was supported by the European Union's Horizon 2020 research and innovation programme under grant agreement No 951883 within SPRINGBOARD project.