Design, Synthesis and Biological Assessment of Rhodanine-Linked Benzenesulfonamide Derivatives as Selective and Potent Human Carbonic Anhydrase Inhibitors

A novel series of twenty-five rhodamine-linked benzenesulfonamide derivatives (7a–u and 9a–d) were synthesized and screened for their inhibitory action against four physiologically relevant human (h) carbonic anhydrase (CA) isoforms, namely hCA I, hCA II, hCA IX, and hCA XII. All the synthesized molecules showed good to excellent inhibition against all the tested isoforms in the nanomolar range due to the presence of the sulfonamide as a zinc binding group. The target compounds were developed from indol-3-ylchalcone-linked benzenesulfonamide where the indol-3-ylchalcone moiety was replaced with rhodanine-linked aldehydes or isatins to improve the inhibition. Interestingly, the molecules were slightly more selective towards hCA IX and XII compared to hCA I and II. The most potent and efficient ones against hCA I were 7h (KI 22.4 nM) and 9d (KI 35.8 nM) compared to the standard drug AAZ (KI 250.0 nM), whereas in case of hCA II inhibition, the derivatives containing the isatin nucleus as a tail were preferred. Collectively, all compounds were endowed with better inhibition against hCA IX compared to AAZ (KI 25.8 nM) as well as strong potency against hCA XII. Finally, these newly synthesized molecules could be taken as potential leads for the development of isoform selective hCA IX and XII inhibitors.


Introduction
In recent years, studies of rhodanine (2-thioxothiazolidin-4-one) based molecules have been on the rise because of their wide spectrum of biological activities through different mechanisms of action, and they have become a very important group of heterocyclic compounds in drug discovery. Rhodanine consists of a five-membered ring with sulfide and amino groups at the first and third positions, respectively. The amino group could be responsible for interaction with the ligand binding site of target proteins through different types of interactions, such as hydrogen bonding as well as interactions with metal ions [1,2]. Rhodanine is mostly important in photochemistry, medicinal chemistry, biochemistry, and industry [3,4]. Several substituted rhodanine compounds have been investigated to evaluate their potential antidiabetic, anti-Alzheimer, anticancer, and antimicrobial activities [5,6]. Hence, the rhodanine structure, after different chemical modification and functionalization, could provide compounds with a broad range of biological activities [7][8][9][10].
Enzymes were usually targeted for drug discovery and development because of their vital role in many diseases. Therefore, researchers have focused on developing biochemistry, and industry [3,4]. Several substituted rhodanine compounds have been investigated to evaluate their potential antidiabetic, anti-Alzheimer, anticancer, and antimicrobial activities [5,6]. Hence, the rhodanine structure, after different chemical modification and functionalization, could provide compounds with a broad range of biological activities [7][8][9][10].
The synthesis of rhodanine derivatives and exploring their potential inhibitory actions against various diseases are popular areas of research. Unfortunately, few efforts have been made to investigate the potential of rhodanine derivatives as hCA inhibitors [7][8][9]. Hence, in the present study, we synthesized and investigated a novel series of benzenesulfonamide-linked rhodanine derivatives for their inhibitory effects on hCA isoenzymes. Moreover, to better explore the chemical space within this privileged scaffold and to take advantage of the good inhibitory potency displayed against hCA I, II, and XIII by indole-linked benzenesulfonamide derivatives [25][26][27][28][29], we designed our derivatives functionalizing the rhodanine nucleus with both aryl and isatin rings (tail approach) to orient the isoform selectivity against four different hCA isoforms. Hence, the target compound was developed from indol-3-ylchalcone-linked benzenesulfonamide (which was previously synthesized in our lab) where the indol-3-ylchalcone moiety was replaced with rhodanine-linked aldehydes or isatins (Figure 1) in order to keep the presence of a nitrogen in this portion of the scaffold constant or not constant.

Carbonic Anhydrase Inhibition: Structure-Activity Relationship (SAR) Studies
The inhibition data for the newly synthesized rhodanine-linked benzenesulfon derivatives were reported against two physiologically relevant cytosolic hCA I and II and two tumor-associated transmembrane hCA IX and hCA XII while using acet mide (AAZ) as the standard drug, and the results were summarized in Table 1. A isoenzymes belong to the human α-class of CAs. The following structure-activit tionships (SARs as reported in Figure 2) can be extrapolated as follows.
All the molecules tested against the cytosolic isoform hCA I showed good to ex inhibition in the nanomolar range. All the compounds showed inhibition with K nM, except for 7c with KI > 1000 nM. Out of twenty-five molecules, fifteen showed lent inhibition, with KI inferior to the reference drug AAZ (KI 250 nM). The most compounds were 7h (KI 22.4 nM), which was 11 times more potent than AAZ, wh (78.6 nM), 7n (84.0 nM), 7s (77.9 nM), 7t (77.5 nM), 7u (70.6 nM), 9a (41.6 nM), 9b

Carbonic Anhydrase Inhibition: Structure-Activity Relationship (SAR) Studies
The inhibition data for the newly synthesized rhodanine-linked benzenesulfonamide derivatives were reported against two physiologically relevant cytosolic hCA I and hCA II and two tumor-associated transmembrane hCA IX and hCA XII while using acetazolamide (AAZ) as the standard drug, and the results were summarized in Table 1. All the isoenzymes belong to the human α-class of CAs. The following structure-activity relationships (SARs as reported in Figure 2) can be extrapolated as follows.
All the molecules tested against the cytosolic isoform hCA I showed good to excellent inhibition in the nanomolar range. All the compounds showed inhibition with K I < 900 nM, except for 7c with K I > 1000 nM. Out of twenty-five molecules, fifteen showed excellent inhibition, with K I inferior to the reference drug AAZ (K I 250 nM). The most active compounds were 7h (K I 22.4 nM), which was 11 times more potent than AAZ, while 7k (78.6 nM), 7n (84.0 nM), 7s (77.9 nM), 7t (77.5 nM), 7u (70.6 nM), 9a (41.6 nM), 9b (92.2 nM), and 9d (35.8 nM) were also more potent compared to AAZ. It was also observed that both electronwithdrawing and electron-donating substituents on the aromatic portion (R) contributed to good inhibition. Isatin-based series was slightly better than aryl-substituted rhodanines towards this isoform. nM), and 9d (35.8 nM) were also more potent compared to AAZ. It was also observed that both electron-withdrawing and electron-donating substituents on the aromatic portion (R) contributed to good inhibition. Isatin-based series was slightly better than aryl-substituted rhodanines towards this isoform. The other physiologically dominant isoform hCA II, which is also known as the antiglaucoma drug target, was inhibited by all the tested compounds in a good to excellent nanomolar range (4.3-954.2 nM) comparable to AAZ (12.1 nM). The most active compounds were 9a (4.3 nM, nearly three times better than AAZ), 9b (5.5 nM), 9d (5.2 nM), 7h (7.3 nM), 7k (12.7 nM), 7p (8.0 nM), 7t (9.6 nM), and 7u (8.9 nM). In this case, both the electron-withdrawing and electron-donating substituents on the aromatic ring (R) provided good inhibition. Compound 7c was also the least active within the series.
Excellent inhibitory activity was observed with all the synthesized compounds investigated in the low nanomolar range for the inhibition of the main tumor-associated transmembrane isozyme hCA IX. Four derivatives, namely 7b, 7l, 7m, and 7o, showed high hCA IX inhibitory activity, with KI ranging from 28.2 to 51.5 nM, which was comparable to AAZ, whereas the remaining 21 molecules showed better inhibition (4.2-23.6 nM) than AAZ (25.8 nM). The best hCA IX inhibitors, 7g (4.2 nM) and 9a (4.7 nM), were nearly six times better than the standard AAZ. Collectively, the trend was flat, indicating that The other physiologically dominant isoform hCA II, which is also known as the antiglaucoma drug target, was inhibited by all the tested compounds in a good to excellent nanomolar range (4.3-954.2 nM) comparable to AAZ (12.1 nM). The most active compounds were 9a (4.3 nM, nearly three times better than AAZ), 9b (5.5 nM), 9d (5.2 nM), 7h (7.3 nM), 7k (12.7 nM), 7p (8.0 nM), 7t (9.6 nM), and 7u (8.9 nM). In this case, both the electronwithdrawing and electron-donating substituents on the aromatic ring (R) provided good inhibition. Compound 7c was also the least active within the series.
Excellent inhibitory activity was observed with all the synthesized compounds investigated in the low nanomolar range for the inhibition of the main tumor-associated transmembrane isozyme hCA IX. Four derivatives, namely 7b, 7l, 7m, and 7o, showed high hCA IX inhibitory activity, with K I ranging from 28.2 to 51.5 nM, which was comparable to AAZ, whereas the remaining 21 molecules showed better inhibition (4.2-23.6 nM) than AAZ (25.8 nM). The best hCA IX inhibitors, 7g (4.2 nM) and 9a (4.7 nM), were nearly six times better than the standard AAZ. Collectively, the trend was flat, indicating that these rhodanine-based derivatives represent a suitable template to design potent and selective hCA IX inhibitors.
The molecules displayed selectivity towards hCA IX and XII compared to hCA I and II. The structure-activity relationships (Figure 2) showed that regardless of whether the molecules bared electron-donating or electron-withdrawing groups, they exhibited good inhibition. However, it was also observed that the molecules with aryl rings (7a-u) or isatins (9a-d) showed good inhibition for specific isoforms.

Chemistry
The target compounds were synthesized from commercially available reagents without further purification. The solvents were distilled and dried following standard methods as necessary prior to their use. All the air and moisture sensitive reactions were kept under inert conditions using clean and dried glassware and a syringe technique to transfer the solutions. The reactions were monitored via thin layer chromatography (TLC) using Merck silica gel 60F-254 plates. The synthesized molecules were purified by washing them with a mixture of 5-10% ethylacetate in hexane. The melting points were acquired using Stuart digital melting point apparatus (SMP 30) in open capillary tubes, and they were uncorrected. Nuclear Magnetic Resonance ( 1 H NMR and 13 C NMR) spectra were recorded using an Avance Bruker 500 MHz and 125 MHz spectrometer in DMSO-d 6 as the solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts were represented as δ values in parts per million (ppm) and coupling constants (J) in Hz. Multiplicities were described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and dd (doublet of doublets). HRMS were determined using the Agilent QTOF mass spectrometer 6540 series instrument, and they were performed via ESI techniques at 70 eV.

General Procedure for the Synthesis of 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic Acid (3)
Compound 3 was obtained from one-pot, three component condensation reaction, where glycine (2 g, 26.6 mmol) and sodium hydroxide (2.3 g, 58 mmol) were dissolved in 15 mL of ethanol followed by the dropwise addition of carbon disulfide (1.5 mL) with vigorous stirring at room temperature for 6 h. Chloroacetic acid (3 g, 31 mmol) was then added and stirring continued at room temperature for 5 h. The resultant reaction mixture was acidified with dilute HCl until pH 1.0 was achieved and stirring continued for 1 h. The progress of the reaction was checked using TLC. Twenty to thirty mL of water was then added, and the cyclized product was extracted in ethyl acetate, dried over anhydrous sodium sulfate, and evaporated under vacuum. The residue was purified by washing it with a mixture of 5-10% ethylacetate in hexane to obtain compound 3 (4.2 g, 82%).

General Procedure for the Synthesis of Benzylidene/Oxoindolin-Containing
Thioxothiazolidin-Linked Benzenesulfonamides (7a-u, 9a-d) The appropriate benzaldehyde (6)/isatin (8) (0.23 mmol), sodium acetate (37 mg, 0.45 mmol), and glacial acetic acid (0.45 mmol) were added to a stirring solution of compound 5 (80 mg, 0.23 mmol) in ethanol and then refluxed for 4-6 h. The progress of the reaction was checked using TLC, and on completion, the reaction mixture was cooled to room temperature, followed by the addition of ice-cold water, and the precipitate was filtered off, washed with water, and then dried to afford the target compounds (7a-u/9a-d) in good to excellent yield (NMR data for intermediates and final compounds are reported as Supplementary Materials).  13

Carbonic Anhydrase Inhibition Assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument was used to assay the inhibition of various CA isozymes [33]. Phenol Red (at a concentration of 0.2 mM) was used as an indicator, working at an absorbance maximum of 557 nm with 10 mM Hepes (pH 7.4) as a buffer and 10 mM NaClO 4 to maintain constant ionic strength (this anion was not inhibitory in the used concentration), following the CA-catalyzed CO 2 hydration reaction for a period of 5-10 s. Saturated CO 2 solutions in water at 25 • C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-water 1:1, v/v) with dilutions up to 0.01 nM done with the assay buffer mentioned above. At least seven different inhibitor concentrations were used to measure the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay to allow for the formation of the E-I complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper were the means of these results. The inhibition constants were obtained via nonlinear, least-squares methods using the Cheng-Prusoff equation, as reported earlier [34], and they represented the means of at least three different determinations. All CA isozymes used in this study were recombinant proteins obtained by our group, as reported earlier, and their concentrations in the assay system were 5-12 nM [35][36][37].

Conclusions
The present research work explained the design and synthesis of novel benzenesulfonamidelinked rhodanine derivatives (7a-u, 9a-d) as crucial and selective human carbonic anhydrase inhibitors. All the tested hCA isoforms (I, II, IX, XII) were affected by the newly synthesized molecules in variable degrees of inhibition. All the molecules showed excellent inhibition in the nanomolar range. However, the selectivity was more pronounced towards hCA IX and XII. The most potent and efficient compounds against hCA I were 7h (22.4 nM) and 9d (35.8 nM) compared to standard AAZ (250.0 nM), whereas in the case of hCA II, the isatin derivatives 9a (4.3 nM), 9b (5.5 nM), and 9d (5.2 nM) exhibited excellent potency compared to standard AAZ (12.1 nM). The compounds which showed the best inhibition against isoform hCA IX were 7g (4.2 nM) and 9a (4.7 nM) compared to standard AAZ (25.8 nM). Conversely, for hCA XII, all the compounds exhibited moderate potencies, and the best among them were 7i (9.0 nM) and 7s (9.0 nM) compared to standard AAZ (5.7 nM). It was also noticed that both aryls and isatins with electron-donating and electron-withdrawing groups showed good inhibition potencies. From the results obtained in the present study, it is concluded that these newly synthesized benzenesulfonamide-linked rhodanine derivatives are very interesting lead molecules for the design and synthesis of isoform selective inhibitors of tumor-associated isoforms.