Continued Structural Exploration of Sulfocoumarin as Selective Inhibitor of Tumor-Associated Human Carbonic Anhydrases IX and XII

A series of new 3- and 7-substituted sulfocoumarins was obtained by several cyclization reactions and subsequent derivatization for screening as prodrug inhibitors of the human (h) cancer-associated carbonic anhydrases (CAs) IX and XII. All products were ineffective inhibitors against the off-target hCA I and II, whilst hCAs IX and XII were inhibited with inhibition constants (KIs) spanning from low nanomolar to the high micromolar range, according to the sulfocoumarin derivatization pattern. In particular, sulfocoumarin 15 turned out to be the most potent and selective inhibitor herein reported (hCA I and II: KI > 100 µM; hCA IX: KI = 22.9 nM; hCA XII: KI = 19.2 nM). Considering that hCA IX and XII validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here may be useful for identifying suitable drug candidates for clinical trials.


Introduction
Carbonic anhydrases are a ubiquitous metalloenzyme family present in all living organisms. Until now, eight genetically distinct CA subfamilies have been identified [1][2][3]. In mammals, 16 CA isoforms belonging to α-CAs are characterized, and in humans 15 CA (hCA) isoforms are expressed, showing differences in kinetic properties, subcellular localization and tissue distributions [4,5]. Twelve such isoforms are catalytically active (CAs I-IV, VA, VB, VI, VII, IX, XII and XIV), whereas the remaining three isoforms (VIII, X, XI), called CA-related proteins (CARPs), have no activity. hCAs can be further categorized into four different subsets depending on their subcellular localization as cytosolic (hCA I, II, III, VII, VIII, X, XI, XIII), mitochondrial (hCA VA and VB), secreted (hCA VI) and membrane-bound (hCA IV, IX, XII, and XIV) [5][6][7]. Four distinct CA inhibition mechanisms have been reported and detailed to date with both kinetic and X-crystallographic studies [8,9]. They include (a) the metal ion binders (anions, sulfonamides and their bioisosteres, dithiocarbamates, xanthates, etc.); (b) compounds that anchor to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines); (c) compounds occluding the active site entrance, such as coumarins and their isosteres (sulfocoumarins); and (d) compounds binding out of the active site, such as an aromatic carboxylic acid derivative [8][9][10]. Coumarins, such as 1 (natural product isolated from the Australian plant Leionema ellipticum) and 2 (the simple unsubstituted coumarin), were discovered as a new chemotype that can inhibit the metalloenzyme carbonic anhydrase a decade ago [11][12][13]. Many differently substituted coumarins were subsequently screened for their inhibitory activity against all the 13 catalytically active mammalian CA isoforms, CA I-VII, IX, XII-XV [14][15][16][17][18]. Many of these isoforms are established drug targets for designing agents with various applications, such as diuretics, antiglaucoma drugs, anticonvulsants, antiobesity agents or antitumor drugs/cancer diagnostic tools [17,18]. To explain the inhibitor mechanism subsequently screened for their inhibitory activity against all the 13 catalytically active mammalian CA isoforms, CA I-VII, IX, XII-XV [14][15][16][17][18]. Many of these isoforms are established drug targets for designing agents with various applications, such as diuretics, antiglaucoma drugs, anticonvulsants, antiobesity agents or antitumor drugs/cancer diagnostic tools [17,18]. To explain the inhibitor mechanism of coumarins 1 and 2, they were cocrystallized with human CA II, and the electron density data showed the presence of the hydrolyzed derivatives 4 and 5, respectively (Figure 1). The most notable aspect of this inhibition mechanism is the fact that the 4 and 5 occlude the enzyme active site binding at the entrance of the cavity. Over the last several years, a rather large number of new classes of CAIs were reported, starting from coumarins as the lead, among which are thiocoumarins, sulfocoumarins, 2-thioxo-coumarins, coumarin oximes, 5-/6-membered/thio)lactones, etc. [19][20][21][22][23][24][25][26][27][28]; coumarins and sulfocoumarins were also shown to be isoform-selective CA inhibitors. Particularly, in 2012, the sulfocoumarins were identified as CAIs using kinetic and X-ray crystallographic studies, in which it has been also revealed that although structurally related to the coumarins, sulfocoumarins possess a different CA inhibition mechanism (Figure 2) [29]. As reported in Figure 1 for compound 3, sulfocoumarin were hydrolyzed by α-CAs to give trans-2-hydroxyphenyl-ω-ethenylsulfonic acid. Afterwards, the formed sulfonic acid binds to the CA II active site by anchoring the SO3H group to the zinc-coordinated water molecule/hydroxide ion ( Figure 2) [30]. After this discovery, many more derivatives were synthesized and analyzed for their interaction with different CA isoforms [31][32][33][34][35][36][37]. Over the last several years, a rather large number of new classes of CAIs were reported, starting from coumarins as the lead, among which are thiocoumarins, sulfocoumarins, 2thioxo-coumarins, coumarin oximes, 5-/6-membered/thio)lactones, etc. [19][20][21][22][23][24][25][26][27][28]; coumarins and sulfocoumarins were also shown to be isoform-selective CA inhibitors. Particularly, in 2012, the sulfocoumarins were identified as CAIs using kinetic and X-ray crystallographic studies, in which it has been also revealed that although structurally related to the coumarins, sulfocoumarins possess a different CA inhibition mechanism (Figure 2) [29]. As reported in Figure 1 for compound 3, sulfocoumarin were hydrolyzed by α-CAs to give trans-2-hydroxyphenyl-ω-ethenylsulfonic acid. Afterwards, the formed sulfonic acid binds to the CA II active site by anchoring the SO 3 H group to the zinc-coordinated water molecule/hydroxide ion ( Figure 2) [30]. After this discovery, many more derivatives were synthesized and analyzed for their interaction with different CA isoforms [31][32][33][34][35][36][37].
subsequently screened for their inhibitory activity against all the 13 catalytically active mammalian CA isoforms, CA I-VII, IX, XII-XV [14][15][16][17][18]. Many of these isoforms are established drug targets for designing agents with various applications, such as diuretics, antiglaucoma drugs, anticonvulsants, antiobesity agents or antitumor drugs/cancer diagnostic tools [17,18]. To explain the inhibitor mechanism of coumarins 1 and 2, they were cocrystallized with human CA II, and the electron density data showed the presence of the hydrolyzed derivatives 4 and 5, respectively ( Figure 1). The most notable aspect of this inhibition mechanism is the fact that the 4 and 5 occlude the enzyme active site binding at the entrance of the cavity. Over the last several years, a rather large number of new classes of CAIs were reported, starting from coumarins as the lead, among which are thiocoumarins, sulfocoumarins, 2-thioxo-coumarins, coumarin oximes, 5-/6-membered/thio)lactones, etc. [19][20][21][22][23][24][25][26][27][28]; coumarins and sulfocoumarins were also shown to be isoform-selective CA inhibitors. Particularly, in 2012, the sulfocoumarins were identified as CAIs using kinetic and X-ray crystallographic studies, in which it has been also revealed that although structurally related to the coumarins, sulfocoumarins possess a different CA inhibition mechanism (Figure 2) [29]. As reported in Figure 1 for compound 3, sulfocoumarin were hydrolyzed by α-CAs to give trans-2-hydroxyphenyl-ω-ethenylsulfonic acid. Afterwards, the formed sulfonic acid binds to the CA II active site by anchoring the SO3H group to the zinc-coordinated water molecule/hydroxide ion ( Figure 2) [30]. After this discovery, many more derivatives were synthesized and analyzed for their interaction with different CA isoforms [31][32][33][34][35][36][37].  The substitution pattern, and especially the position of the substituent on the heterocyclic ring system of the sulfocoumarin, are the main factors influencing CA inhibitory properties [30]. In this paper, we expanded the structure-activity relationships of the sulfocoumarin scaffold describing the synthesis and the evaluation of more than 30 sulfocoumarin belonging to two different classes: (a) 7-benzyloxysulfocoumarin and (b) 3-amidosulfocoumarin, obtained for the first time in this work.

Chemistry
Due to the difficulties in designing and synthesizing selective sulfonamide inhibitors against each isoform, such as SLC-0111 ( Figure 3) [38], a potent and selective zinc binder CAI against hCA IX and XII, scientists opted for the development of novel chemotypes among which are sulfocoumarins, the preferred CAI scaffold adopted in this project. The general strategy of Zalubovskis group [16,29], for the preparation of 6substituted sulfocoumarins and validated by Nocentini et al. [36], in 2015, for the synthesis of 7-substituted such derivatives, was applied in this manuscript to extend the structureactivity relationships, whereas the general strategy of Liu's group [39], for the designed of 3-substituted sulfocoumarins, was validated in this project in order to synthesize 3amido derivatives for the first time. To start, 2-hydroxy-4-methoxybenzaldehyde 7 or 2′hydroxy-4′-methoxyacetophenon 8 were synthesized with the sulfocoumarin scaffold 9 and 10. After that, their phenol moiety was released in the presence of BBr3 in dry DCM to obtain 11 and 12 in high yield and high purity. Finally, a nucleophilic substitution was performed in the presence of different benzyl bromides with K2CO3 as a base in dry DMF at RT to give compounds 13-22 (Scheme 1).  The general strategy of Zalubovskis group [16,29], for the preparation of 6-substituted sulfocoumarins and validated by Nocentini et al. [36], in 2015, for the synthesis of 7-substituted such derivatives, was applied in this manuscript to extend the structure-activity relationships, whereas the general strategy of Liu's group [39], for the designed of 3-substituted sulfocoumarins, was validated in this project in order to synthesize 3-amido derivatives for the first time. To start, 2-hydroxy-4-methoxybenzaldehyde 7 or 2 -hydroxy-4 -methoxyacetophenon 8 were synthesized with the sulfocoumarin scaffold 9 and 10. After that, their phenol moiety was released in the presence of BBr 3 in dry DCM to obtain 11 and 12 in high yield and high purity. Finally, a nucleophilic substitution was performed in the presence of different benzyl bromides with K 2 CO 3 as a base in dry DMF at RT to give compounds 13-22 (Scheme 1). 4BCW), derived from the CA-mediated hydrolysis of sulfocoumarin 3. The Zn(II) is shown as a gre sphere that is bound to the protein His ligands (labels not shown). Water molecules are represente as red spheres. H-bonds are represented as black dashed lines.
The substitution pattern, and especially the position of the substituent on th heterocyclic ring system of the sulfocoumarin, are the main factors influencing CA inhibitory properties [30]. In this paper, we expanded the structure-activity relationship of the sulfocoumarin scaffold describing the synthesis and the evaluation of more than 3 sulfocoumarin belonging to two different classes: (a) 7-benzyloxysulfocoumarin and (b 3-amidosulfocoumarin, obtained for the first time in this work.

Chemistry
Due to the difficulties in designing and synthesizing selective sulfonamide inhibitor against each isoform, such as SLC-0111 ( Figure 3) [38], a potent and selective zinc binde CAI against hCA IX and XII, scientists opted for the development of novel chemotype among which are sulfocoumarins, the preferred CAI scaffold adopted in this project. The general strategy of Zalubovskis group [16,29], for the preparation of 6 substituted sulfocoumarins and validated by Nocentini et al. [36], in 2015, for the synthes of 7-substituted such derivatives, was applied in this manuscript to extend the structure activity relationships, whereas the general strategy of Liu's group [39], for the designe of 3-substituted sulfocoumarins, was validated in this project in order to synthesize 3 amido derivatives for the first time. To start, 2-hydroxy-4-methoxybenzaldehyde 7 or 2 hydroxy-4′-methoxyacetophenon 8 were synthesized with the sulfocoumarin scaffold and 10. After that, their phenol moiety was released in the presence of BBr3 in dry DCM to obtain 11 and 12 in high yield and high purity. Finally, a nucleophilic substitution wa performed in the presence of different benzyl bromides with K2CO3 as a base in dry DM at RT to give compounds 13-22 (Scheme 1). Compound 23 (2-bromobenzenesulfonyl chloride) was reacted with 3-methoxyphenol in dry DCM in the presence of Et 3 N as a base to obtain intermediate 24; after that, a cyclization was performed in dry DMA with Pd(OAc) 2 as a catalyst at 150 • C to give the sulfocoumarin 25. To prepare the next reaction, the methoxy group was released in the presence of BBr3 in dry DCM between −10 • C and RT to synthesize intermediate 26, and then a nucleophilic substitution was performed in dry DMF at RT with K 2 CO 3 as a base to give us compounds 27-31 (Scheme 2).
Compound 23 (2-bromobenzenesulfonyl chloride) was reacted with 3methoxyphenol in dry DCM in the presence of Et3N as a base to obtain intermediate 24; after that, a cyclization was performed in dry DMA with Pd(OAc)2 as a catalyst at 150 °C to give the sulfocoumarin 25. To prepare the next reaction, the methoxy group was released in the presence of BBr3 in dry DCM between −10°C and RT to synthesize intermediate 26, and then a nucleophilic substitution was performed in dry DMF at RT with K2CO3 as a base to give us compounds 27-31 (Scheme 2). A cyclization reaction was performed between starting materials 32, or salicylaldehyde, or 33, 4-methoxysalicylaldehyde, and ethyl 2-chlorosulfonylacetate in DCE at 90 °C in the presence of dry pyridine as a base; after that, the obtained ethyl esters (34,35) were hydrolyzed in EtOH and NaOH 5M at reflux to release the carboxylic acid moiety of compounds 36 and 37. Finally, a coupling reaction was performed in dry DMF between appropriate anilines and intermediates 36 and 37 to give us products 38-47 with an amide as linker (Scheme 3).

Carbonic Anhydrase Inhibition
Sulfocoumarins 13-22, 27-31, 38-47 were screened in vitro for the inhibition of four physiologically relevant hCA isoforms, the cytosolic hCAI and II and the trans-membrane tumor-associated hCA IX and XII [4][5][6]8,[40][41][42][43]; acetazolamide (AAZ) was used as standard CAI. CA I is the main off-target isoform for most therapeutic applications of CAIs, whilst CA II is considered off-target in many pathologies to reduce side effects resulting from systematic CA inhibition as much as possible. Table 1 shows the inhibition data obtained after a period of incubation of 6 h of the enzyme and inhibitors. A cyclization reaction was performed between starting materials 32, or salicylaldehyde, or 33, 4-methoxysalicylaldehyde, and ethyl 2-chlorosulfonylacetate in DCE at 90 • C in the presence of dry pyridine as a base; after that, the obtained ethyl esters (34,35) were hydrolyzed in EtOH and NaOH 5M at reflux to release the carboxylic acid moiety of compounds 36 and 37. Finally, a coupling reaction was performed in dry DMF between appropriate anilines and intermediates 36 and 37 to give us products 38-47 with an amide as linker (Scheme 3).
Compound 23 (2-bromobenzenesulfonyl chloride) was reacted with 3methoxyphenol in dry DCM in the presence of Et3N as a base to obtain intermediate 24; after that, a cyclization was performed in dry DMA with Pd(OAc)2 as a catalyst at 150 °C to give the sulfocoumarin 25. To prepare the next reaction, the methoxy group was released in the presence of BBr3 in dry DCM between −10°C and RT to synthesize intermediate 26, and then a nucleophilic substitution was performed in dry DMF at RT with K2CO3 as a base to give us compounds 27-31 (Scheme 2). A cyclization reaction was performed between starting materials 32, or salicylaldehyde, or 33, 4-methoxysalicylaldehyde, and ethyl 2-chlorosulfonylacetate in DCE at 90 °C in the presence of dry pyridine as a base; after that, the obtained ethyl esters (34,35) were hydrolyzed in EtOH and NaOH 5M at reflux to release the carboxylic acid moiety of compounds 36 and 37. Finally, a coupling reaction was performed in dry DMF between appropriate anilines and intermediates 36 and 37 to give us products 38-47 with an amide as linker (Scheme 3).

Carbonic Anhydrase Inhibition
Sulfocoumarins 13-22, 27-31, 38-47 were screened in vitro for the inhibition of four physiologically relevant hCA isoforms, the cytosolic hCAI and II and the trans-membrane tumor-associated hCA IX and XII [4][5][6]8,[40][41][42][43]; acetazolamide (AAZ) was used as standard CAI. CA I is the main off-target isoform for most therapeutic applications of CAIs, whilst CA II is considered off-target in many pathologies to reduce side effects resulting from systematic CA inhibition as much as possible. Table 1 shows the inhibition data obtained after a period of incubation of 6 h of the enzyme and inhibitors.

Carbonic Anhydrase Inhibition
Sulfocoumarins 13-22, 27-31, 38-47 were screened in vitro for the inhibition of four physiologically relevant hCA isoforms, the cytosolic hCAI and II and the trans-membrane tumor-associated hCA IX and XII [4][5][6]8,[40][41][42][43]; acetazolamide (AAZ) was used as standard CAI. CA I is the main off-target isoform for most therapeutic applications of CAIs, whilst CA II is considered off-target in many pathologies to reduce side effects resulting from systematic CA inhibition as much as possible. Table 1 shows the inhibition data obtained after a period of incubation of 6 h of the enzyme and inhibitors. Noteworthily, the assay inhibition performed within the usual 15 min incubation period (as for the sulfonamides) led to the very weak inhibition constants (data not shown) [43]. For this reason, we herein report a 6 h incubation time instead. The following structure-activity relationship (SAR) can be gathered from the inhibition data reported in Table 1. Noteworthily, the assay inhibition performed within the usual 15 min incubation period (as for the sulfonamides) led to the very weak inhibition constants (data not shown) [43]. For this reason, we herein report a 6 h incubation time instead. The following structureactivity relationship (SAR) can be gathered from the inhibition data reported in Table 1. The following structure-activity relationships (SAR) should be noted:  Compounds 11 and 12 showed a low micromolar inhibition, respectively K I = 2896 nM and K I = 3857 nM, whilst analogs 9 and 10 have the K I in the medium micromolar range, which can likely be associated to the presence of a free phenol moiety on products 11 and 12, which is protected in 9 and 10.

Chemistry
Anhydrous solvents and all reagents were purchased from Merck, Fluorochem and TCI. All reactions involving air-or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance (1H-NMR, 13C-NMR,) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d 6 . Chemical shifts are reported in parts per million (ppm), and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; bs, broad singlet; dd, doublet of doublets. The assignment of exchangeable protons was confirmed by the addition of D 2 O. Analytical thin-layer chromatography (TLC) was carried out on Sigma Aldrich silica gel F-254 plates. Flash chromatography purifications were performed on Sigma Aldrich Silica gel 60 (230-400 mesh ASTM) as the stationary phase and ethyl acetate/n-hexane or MeOH/DCM were used as eluents. Melting points (mp) were measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 appa-ratus and are uncorrected. The solvents used in mass spectrometry analysis were acetone, acetonitrile (Chromasolv grade), purchased from Sigma-Aldrich (Milan, Italy), and mQ water 18 MΩ cm, obtained from Millipore's Simplicity system (Milan, Italy). The HPLC-MS and MS/MS analysis was carried out using a Varian 500-MS ion trap system (Palo Alto, CA, USA) equipped by two Prostar 210 pumps, a Prostar 410 autosampler and an Electrospray source (ESI) operating in negative ions. Stock solutions of analytes were prepared in acetone at 1.0 mg mL-1 and stored at 4 • C. Working solutions of each analyte were freshly prepared by diluting stock solutions in a mixture of mQ water:acetonitrile 1:1 (v/v) up to a concentration of 1.0 µg mL −1 . The mass spectra of each analyte were acquired by introducing, via syringe pump at 10 µL min −1 , the working solution. Raw data were collected and processed by Varian Workstation Vers. 6.8 software.

General synthetic procedure for 7-methoxybenzo[e][1,2]oxathiine 2,2-dioxide (9) and 7-methoxy-4-methylbenzo[e][1,2]oxathiine 2,2-dioxide (10)
Et 3 N (1.5 equiv.) and Mesyl chloride (1.5 equiv.) were added slowly to a solution of 7 or 8 (3 g, 1 equiv.) in dry DCM (20 mL) at 0 • C under nitrogen atmosphere. The solution was stirred for 2 h at RT. Slush was added to the mixture and the reaction mixture was extracted in DCM (3 × 25 mL), dried with Na 2 SO 4 , filtered and evaporated to give us a pale yellow oil. DBU (1.2 equiv.) was added dropwise to a solution of the oil in dry DCM (20 mL) at 0 • C under nitrogen atmosphere. The solution was stirred o.n. at RT. Slush was added slowly and the reaction mixture was extracted in DCM (3 × 25 mL). The collected organic phases were dried with Na 2 SO 4 , filtered and evaporated under vacuum to give a brown oil. POCl 3 (1.5 equiv.) was added dropwise to a solution of the oil in dry Py (3 mL) at 0 • C under nitrogen atmosphere. The solution was stirred on at RT. Ice was added slowly to the reaction mixture and the resulting precipitate was filtered to give intermediates 9 and 10 as a light brown powder in high yield. 9 and 10 were purified by silica gel chromathography column (EtOAc/Hexane: from 20% to 60% v/v) to give white-grey powder.

Carbonic Anhydrase Inhibition
An Applied Photophysics stopped-flow instrument was used for assaying the CA catalyzed CO 2 hydration activity [44]. 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 were used for determining the initial velocity. The uncatalyzed 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-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6h 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 and represent the mean from at least three different determinations. The enzyme concentrations were in the range 5-16 nM. All CA isoforms were recombinant ones obtained in-house [47,48].

Conclusions
We report here a series of 7-substituted and 3-substituted sulfocoumarins, obtained by cyclization of 2-hydroxy-4-methoxybenzaldehyde (7), 2 -hydroxy-4 -methoxyacetophenon (8), 2-bromobenzenesulfonyl chloride (23), salicylaldehyde (32) or 4-methoxysalicylaldehyde (33) and possessing different substituents in position 3 or 7 of the heterocyclic ring. 7-Substituted sulfocoumarins resulted in being good inhibitors of hCA IX and XII, whilst 3-substituted sulfocoumarins showed a worsening in terms of inhibition potency on this isoform. A common feature of all the synthesized products was the absence of inhibition on hCA I and II. The most potent products belong to Type 1 group, probably due to the absence of the bulky group on the heterocyclic ring; particularly, sulfocoumarin 15 showed a nanomolar inhibition on hCA IX and XII and turned out to be the most potent inhibitor achieved (hCA IX: K I = 22.9 nM; hCA XII: K I = 19.2 nM). The structure-activity relationship for this class of CAIs has been expanded considering the synthesis of 3-substituted sulfocoumarines for the first time. The observed isoform-selective inhibition displayed here may be considered to be of interest for various biomedical applications.