Synthesis and biological evaluation of novel 3-(quinolin-4-ylamino)benzenesulfonamides as carbonic anhydrase isoforms I and II inhibitors

Abstract Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial metalloenzymes that are involved in diverse bioprocesses. We report the synthesis and biological evaluation of novel series of benzenesulfonamides incorporating un/substituted ethyl quinoline-3-carboxylate moieties. The newly synthesised compounds were in vitro evaluated as inhibitors of the cytosolic human (h) isoforms hCA I and II. Both isoforms hCA I and II were inhibited by the quinolines reported here in variable degrees: hCA I was inhibited with KIs in the range of 0.966–9.091 μM, whereas hCA II in the range of 0.083–3.594 μM. The primary 7-chloro-6-flouro substituted sulphfonamide derivative 6e (KI = 0.083 μM) proved to be the most active quinoline in inhibiting hCA II, whereas, its secondary sulfonamide analog failed to inhibit the hCA II up to 10 μM, confirming the crucial role of the primary sulphfonamide group, as a zinc-binding group for CA inhibitory activity.


Introduction
Carbonic anhydrases (CA) (CAs, EC 4.2.1.1) are zinc-containing metalloenzymes that are present in most organisms all over the tree of life 1,2 . These metalloenzymes efficiently catalyse the rapid interconversion of carbon dioxide and water to bicarbonate and protons. In humans, this fundamental reaction encompasses three simple chemical entities, CO 2 , HCO 3 À , and H þ , essential in a host of physiological and pathological processes, such as calcification, bone resorption, electrolyte secretion, pH and CO 2 homeostasis, tumorigenicity, and several biosynthetic reactions 3-5 . Eight distinct genetic enzymatic families were identified; the a-, b-, c-, d-, f-. g-, hand i-CAs 3-5 . To date, 15 human (h) isoforms of CA have been identified, which have all belong to the a-class and have different patterns of tissue distribution and cellular localisation as the following; cytosolic (I, II, III, VII, and XIII), membrane-bound (IV, IX, XII, and XIV), secreted (VI) and mitochondrial (VA and VB) forms 3-5 . CA I and II are present at high concentrations comparing to other CA isoforms in the erythrocytes cytosol and several other tissues.
Several important pathological consequences result from the dysfunction of hCA II activity, thus this isoform is an established drug target for a multitude of diseases, such as oedema 6 , epilepsy 7 , acute mountain sickness 8 , and glaucoma, where excessive aqueous humour is secreted within the eye, with the subsequent increase in the intraocular pressure (IOP) [9][10][11] . CA inhibitors (CAIs) are able to diminish IOP by decreasing the rate of bicarbonate formation and thus secretion of the aqueous humour. For more than 60 years, carbonic anhydrase inhibitors are in clinical use for the treatment of glaucoma, such as the topically acting dorzolamide and brinzolamide drugs, and the systemic acetazolamide and methazolamide drugs 9 (Figure 1).
Pertaining to its prevalence in diverse natural products, such as alkaloids, and in different pharmacologically active substances, quinoline stands out as a promising privileged scaffold that is endowed with a wide spectrum of biological activities. Just to name a few, antimalarial 12 , antileishmanial 13 , anti-tubercular 14 , antidepressant 15 , anticancer 16,17 and antiglaucoma 18 actions were reported for quinoline derivatives. Accordingly, medicinal chemists embarked on exploring various quinoline-based molecules comprehending their potential to develop promising and efficient bioactive compounds 19,20 . These efforts led to FDA approval for several quinoline-based drugs such as the anticancer agent lenvatinib, the anti-asthmathic drug montelukast, the antiviral Clioquinol, and the anaesthetic Dibucain.
In the present study, we report a new series of primary benzenesulfonamides incorporating un/substituted ethyl quinoline-3carboxylate (6a-6f, Figure 1) as well as the secondary benzenesulfonamide analogue (11, Figure 1), with the prime goal of developing effective quinoline-based antiglaucoma candidates targeting the cystolic isoform hCA II. These quinoline-based benzenesulfonamides were evaluated in vitro for their inhibitory activity towards the physiologically relevant hCA isoforms I and II, using stopped-flow CO 2 hydrase assay.

Chemistry
All reaction and manipulations were performed in nitrogen atmosphere using standard Schlenk techniques. All reaction solvents and reagents were purchased from commercial suppliers and used without further purification. Microwave-assisted synthesis was carried out in a Biotage Initiator þ apparatus operating in single mode, the microwave cavity producing controlled irradiation at 2.45 GHz (Biotage AB, Uppsala, Sweden). The reactions were run in sealed vessels. These experiments were performed by employing magnetic stirring and a fixed hold time using variable power to reach (during 1 À 2 min) and then maintain the desired temperature in the vessel for the programed time period. The temperature was monitored by an IR sensor focused on a point on the reactor vial glass. The IR sensor was calibrated to internal solution reaction temperature by the manufacturer. The NMR spectra were obtained on Bruker Avance 400 (400 MHz 1 H and 101 MHz 13 C NMR). 1 H NMR spectra were referenced to tetramethylsilane (d ¼ 0.00 ppm) as an internal standard and were reported as follows: chemical shift, multiplicity Column chromatography was performed on Merck Silica Gel 60 (230-400 mesh) and eluting solvents for all of these chromatographic methods were noted as appropriated-mixed solvent with given volume-to-volume ratios. TLC was carried out using glass sheets pre-coated with silica gel 60 F 254 purchased by Merk. Highresolution spectra were performed on Waters ACQUITY UPLC BEH C18 1.7 l-Q-TOF SYNAPT G2-Si High Definition Mass Spectrometry. Compounds 3a-f, 4a-f [21][22] and 10 23 were previously prepared.

General procedure for preparation of compounds 4a-f
A solution of compounds 3a-f (1.0 mmol) in POCl 3 (6 ml) was refluxed for 1 h. The mixture was evaporated in vacuo and the residue was extracted with methylene chloride, crushed ice and aqueous NH 3 . The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography (SiO 2 , ethyl acetate (EA): n-Hex 10: 1) to get key intermediates 4a-f 21,22 .
General procedures for preparation of the target quinolines 6a-f and 11 To a MW vial, were successively added the appropriate ethyl 4chloroquinoline-3-carboxylate derivative 4a-f (0.21 mmol), 3-aminobenzenesulfonamide 5 (0.036 gm, 0.21 mmol) or 3-amino-Nmethylbenzenesulfonamide 10 (0.040 gm, 0.21 mmol), and ethanol (12 ml) at room temperature. The MW vial was sealed and heated under MW conditions for 30 min at 150 C. The mixture was evaporated in vacuo and the residue was extracted with EA and NaHCO 3 (aq). The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography (SiO 2 , EA: n-Hex), in a gradient elution with 1:5 (EA: n-hex) ratio, to furnish quinolines 6a-f and 11, respectively.

CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO 2 hydration activity 24 . Phenol red (at a concentration of 0.2 mM) has been 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-catalyzed 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 pre-incubated 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 non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier 25-29 and represent the mean from at least three different determinations.
In Scheme 2, 3-amino-N-methylbenzenesulfonamide 10 was prepared as reported earlier 13 through a nucleophilic substitution for 3-nitrobenzenesulphonyl chloride 7 with methylamine, followed by a catalytic hydrogenation to the nitro function. The later reacted with the key intermediate 4e in refluxing ethanol to afford the target secondary benzenesulfonamide 11 (Scheme 2).
The structures of the newly prepared quinolines 6a-f and 11 were confirmed and elucidated by NMR spectroscopy and high resolution mass spectroscopy, which were in full agreement with the postulated structures (Supplementary material).

Carbonic anhydrase inhibition
The newly prepared 3-(quinolin-4-ylamino)benzenesulfonamides 6a-f and 11 were evaluated for their ability to inhibit the physiologically relevant hCA cytosolic isoforms, hCA I and II, by a stopped-flow CO 2 hydrase assay 24 . The inhibition data of the prepared quinolines and the sulfonamide acetazolamide AAZ (as a standard inhibitor) against the two examined isoforms are summarised in Table 1. The following structure-activity relationship (SAR) could be noted regarding the inhibition data reported in Table 1: i. The secondary sulfonamide reported here (11) failed to inhibit the tested hCA isoforms (hCA I and hCA II) up to 10 lM, which confirmed the crucial role of the primary sulfonamide as a zinc-anchoring group, with the additional two hydrogen bonds with Thr199 and Thr200 residues within the enzyme active site. ii. The data presented in Table 1 ascribed to the prepared primary sulfonamides (6a-6e) weak potency in inhibiting the ubiquitous cytosolic isoform hCA I with inhibition constants (K I s) in the micromolar range, in detail, between 4.233 and 9.091 lM, except for the 6-methoxy substituted analog 6c which arose as a submicromolar hCA I inhibitor with a K I equals 0.966 lM, which represents 3.8-fold decreased efficacy to the reference drug AAZ (K I equals 0.250 lM towards hCA I). On contrary, the 5,7-dichloro substituted primary sulfonamide 6f failed to inhibit the hCA I up to 10 lM. Noteworthy, the SAR outcomes highlighted that grafting the strong electron-donating 6-metoxy group (compound 6c; K I ¼ 0.966 lM) resulted in 4.4-fold efficacy enhancement in comparison to the unsubstituted analogue 6a (K I ¼ 4.233 lM). Regarding the impact of substitution of the quinoline moiety within the primary sulfonamides series 6a-6f; the inhibitory activities were decreased in the order of 6-OCH 3 >6-CH 3 >7-Cl-6-F > 6-Br >5,7-(Cl) 2 . iii. The second ubiquitous cytosolic isoform examined here was hCA II. It was apparent from the displayed results (Table 1) that the tested primary sulfonamides (6a-6e) effectively interfere with hCA II catalytic activities in submicromolar/micromolar concentration range (K I values of 0.083 -3.594 lM), whereas, no significant inhibition towards hCA II was revealed for quinoline 6f (K I >10 lM). Nevertheless, among the tested quinolines, 7-chloro-6-flouro substituted compound 6e (K I ¼ 0.083 lM) proved to be the most active quinoline in inhibiting hCA II. Moreover, grafting a 6-methoxy group within the quinoline scaffold (compound 6c; K I ¼ 0.083 lM) was advantageous for the inhibitory activity toward hCA II, similarly to the SAR for hCA I inhibition.

Conclusion
In summary, we successfully synthesised new benzenesulfonamides, bearing un/substituted ethyl quinoline-3-carboxylate scaffold (6a-f and 11), which were evaluated for their inhibition of hCA I and hCA II. Both the examined isoforms were inhibited by the quinolines reported here in variable degrees; hCA I was inhibited with K I s in the range of 0.966-9.091 lM, whereas hCA II in the range of 0.083-3.594 lM. Among the tested compounds, the primary 7-chloro-6-flouro substituted sulfonamide derivative 6e (K I ¼ 0.083 lM) proved to be the most active quinoline in inhibiting hCA II, whereas, its secondary sulfonamide analogue 11 failed to inhibit the hCA II up to 10 lM, confirming the crucial role of the primary sulphonamido group, as a ZBG, for CA inhibitory activity.

Disclosure statement
No potential conflict of interest was reported by the authors.