4 − Arylbenzenesulfonamides as human carbonic anhydrase inhibitors (hCAIs): synthesis by Pd nanocatalyst − mediated Suzuki − Miyaura reaction, enzyme inhibition and X − ray crystallographic studies

Benzenesulfonamides bearing various substituted (hetero)aryl rings in the para − position were prepared by palladium nanoparticle − catalyzed Suzuki − Miyaura cross − coupling reactions and evaluated as human carbonic anhydrase (hCA) inhibitors against isoforms hCA I, hCA II, hCA IX and hCA XII. Almost all of the prepared 4 − arylbenzenesulfonamides showed low inhibition against hCA I isoform, whereas the other influence of the partially restricted aryl − aryl bond rotation on the activity/selectivity were rationalized by means of X − ray crystallography of the adducts of hCA II with several 4 − arylbenzenesulfonamides. 12 C 14 11 58.11; Found: C, 57.83;


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes present in most living organisms encoded by six genetically distinct families, the human α−, the β−, γ−, δ−, ζ− and the recently reported η−CAs. 1 They catalyze the reversible hydration of carbon dioxide to the bicarbonate ion and a proton, 2-3 a simple but essential reaction involved in respiration, electrolyte secretion, biosynthesis of several important molecules (urea, lipids, glucose, etc.), pH homeostasis and tumorigenicity, 4,5 and are thus targets for the design of activators and inhibitors. In humans, activators find their pharmacological application in pathologies connected with learning and memory impairment, 3,4,6,7 whilst inhibitors, originally used as diuretics, antiglaucoma agents or antiepileptics, are more recently further employed as antiobesity agents, antitumor drugs or diagnostic tools. 3,4,[6][7][8][9][10][11][12] In fact, the multiple pharmacological applications of CA inhibitors (CAIs) may be explained by the high number of isoforms and by their up− and down−regulation related to different pathologies. Humans express fifteen CA isoforms (hCAs), all containing a Zn(II) ion within the active site, but differing by their cellular localization (mitochondria, cytosol, cell membrane), tissues distribution and catalytic/inhibition features. [4][5][6][7][8][9][10]13 Even if the physiological role of relevant CAs in diverse pathologies is more and more precisely identified, one of the main difficulties concerning the development of selective inhibitors is how to manage off−target isoenzyme inhibition. Indeed, the active site of most CA isoenzymes is a rather large conical cavity where the Zn(II) ion is positioned at the bottom with two adjacent halves (one hydrophobic and one hydrophilic) and variability between isoforms is mainly observed on the edge/entrance of the active site. 13 Disruption of the catalytic process occurs following several possible mechanisms: the inhibitor can (i) bind directly to the zinc ion, 4,14-17 (ii) anchor to the zinc−coordinating water molecule/hydroxide ion, [18][19][20][21][22] or (iii) bind further away from the metal ion. 23,24 Zinc−binding drugs are widely investigated as inhibitors and the sulfonamide function, following the discovery of the exceptional 4 CA inhibitory activity of sulfanilamide, 25 represents one of the most potent zinc−binding group (ZBG). 26-28 Several primary sulfonamide derivatives, such as acetazolamide, methazolamide, ethoxzolamide, sulthiame, diclofenamide, dorzolamide, brinzolamide, sulpiride and zonisamide, have been developed and introduced in clinical use as diuretics, antiglaucoma, neuroleptic or antiepileptic agents (Supporting Information S1).
Structural characterization of benzenesulfonamides in complex with hCAs showed that the sulfonamide function, in its deprotonated form at physiological pH, binds to the active site with coordination of the negatively charged nitrogen atom to the Zn(II) ion. 4,27 Additionally, an extended network of hydrogen bonds involving residues Thr199 and Glu106 (conserved in all α−CAs) stabilizes the binding, whilst the phenyl ring participates in van der Waals interactions with other amino acid residues within the enzyme active site. 4,13,[29][30][31] Therefore, the binding mode of this pharmacophore appears quite similar, irrespective of the isoforms, making the rational design of isoenzyme−specific benzenesulfonamides CAIs a challenging program for medicinal chemists. 32 However, depending on the phenyl substituent (tail or linker moiety), more specific interactions can be established within the typical enzyme bipolar architecture, towards the middle part of the active site or towards its edge ( Figure 1). 4,13 physiological pH (7.4) with the active site of hCAs. The tail (aryl) interacts with hydrophobic or hydrophilic residues depending on its nature.
Herein, we report the synthesis of new 4−arylbenzenesulfonamide derivatives by using the so−called "tail approach", a drug design strategy based on appending scaffolds (tails) of different size, shape or nature to a ZBG containing pharmacophore, 4,13,31,[33][34][35][36] as opposed to the "ring approach" exploring several aromatic/heterocyclic fragments on which the ZBG is bound. 4,13,36,37 This modulation, based on the extension of benzenesulfonamide moiety by anchoring tails has been poorly investigated to date. The preparation of CAIs reported here was carried out by palladium mediated Suzuki−Miyaura cross−coupling reactions. Inhibition activity of the synthesized compounds was measured on tumor−associated isoenzymes hCA IX and hCA XII and on the physiologically dominant off−target isoforms hCA I and hCA II. To rationalize our drug design strategy, X−ray crystallography of several hCA II−sulfonamide adducts were also investigated.

Results and discussion
Inhibitor Design. The benzenesulfonamide scaffold is known for its inhibitory potency against CAs and 2−substituted, 2,4−disubstituted and 3,4−disubstituted derivatives were shown in many cases to act as weaker inhibitors compared to 4−substituted derivatives. 4 Up until now, according to the "tail approach", esters, amides, imines, urea and thiourea functions were commonly used linkers to covalently attach the tail fragment to the benzene ring. 4,26,27 This approach gives higher flexibility to molecules but requires multi−step chemical pathways to obtain the desired compounds. The rationale for our drug design strategy was, on the contrary, the extension of the benzene−ZBG pharmacophore by anchoring an aryl moiety as a tail directly to the para position of the benzene ring, in the absence of any additional linker. This straightforward single−step approach, was 6 realized by using palladium catalyzed Suzuki−Miyaura cross−couplings of a unique aryl halide, 4−iodobenzenesulfonamide 2, with a series of aryl boronic acids or esters 3a−x (Scheme 1 and   Supporting Information, Table S1). In fact, this reaction represents a very efficient method for the formation of new carbon−carbon bonds, 38 applicable to a wide range of aryl halides and boronic acids and esters 39 and hereupon can be further exploited for the synthesis of molecules of biological interest. 40,41 In this study, we prepared a library of twenty four new 4−arylbenzenesulfonamides good performance up to five catalytic cycles. Moreover, due to the heterogeneity of the catalytic system, this nanomaterial was easily removed from the reaction medium by a simple filtration. As a test reaction, coupling of 2 with phenylboronic acid 3a was therefore carried out using both the homogeneous palladium catalyst PdCl2(dppf) . CH2Cl2 and the heterogeneous nanocatalyst MWNT/PdNP@SC12H25 (Supporting Information, Table S1). The desired cross−coupled product 4a was obtained in only 56% yield using the PdCl2(dppf) . CH2Cl2 catalyst while near quantitative yield (97%) was observed for the same reaction catalyzed by the more performant MWNT/PdNP@SC12H25 catalyst. Moreover, the high chemical yield achieved in the case of heterogeneous nanocatalyst is significant; sulfonamides obtained by traditional homogeneous catalysis often require additional purification by column chromatography, whereas the cross−coupling reaction using MWNT/PdNP@SC12H25 afforded compound 4a in pure form directly by crystallization from the reaction mixture after removal of the catalyst by filtration and subsequent solvent evaporation. In view of these improvements in synthetic approach, we decided to apply the MWNT/PdNP@SC12H25 nanocatalyst to the synthesis of all novel sulfonamides in this study. We prepared twenty four 4−arylbenzenesulfonamides coupling boronic acids or esters possessing electron withdrawing or electron donating groups and substituted at the ortho, meta or para positions (Supporting Information, Table S1). The products of the cross−coupling reactions were obtained in a range of yields from 56% to 98% ( Figure 2 and Supporting Information, Table   S1) illustrating the improved performance and versatility of this catalyst and its compatibility with heteroaromatic scaffolds. Indeed, only compounds 4o and 4p, obtained from the reaction with less reactive boronic acids 3o and 3p, were isolated in lower yields and required long reaction time as compared to the more reactive substrates (Supporting Information, Table S1). A long reaction time was also required for cross−couplings with sterically hindered ortho-substituted boronic acids 3b, 8 3c and 3t (Supporting Information, Table S1) but the corresponding products were isolated in good yields, confirming the efficiency of our heterogeneous catalyst in different experimental conditions.  Table 1 and compared to the inhibitory activity of the standard sulfonamide inhibitor acetazolamide (AAZ). The structure−activity relationships (SARs) can thus be summarized as follows. 10 (i) The cytosolic and ubiquitous isoform hCA I was poorly inhibited by the majority of the compounds reported in this current study which exhibited KIs in a micromolar range (1−9.5 µM).
Only derivatives 4c and 4s were between 2 and 3 times more potent than AAZ, with KIs of 146 and 83.6 nM respectively and compounds 4a, 4p and 4q were rather effective hCA I inhibitors, possessing KI values in a nanomolar range ( which, possessing KIs in the range of 324−764 nM, were low potency inhibitors. Therefore, they were generally found to be similar or slightly better hCA II inhibitors compared to AAZ (KI = 12 nM). Although these compounds possess a rather extensive molecular diversity, SAR is almost impossible to define as all substituted moieties lead to potent inhibition of this isoform. In the phenylsulfonylindolyl series, regioisomerism seems to play a significant role in enzyme inhibition. Indeed, 4t displayed weaker inhibition potency against hCA IX relative to its It is also important to note that this second transmembrane tumor−associated isoform, hCA XII, was inhibited by the new series of investigated benzenesulfonamides much more than the other transmembrane tumor−associated isoform, hCA IX. Indeed, the presence of a substituent in the aryl moiety (tail) at the ortho position, likely affecting the rotation ability of the benzenesulfonamide derivatives, appears to reduce the inhibitory activity against the hCA IX isoform and improve the inhibition potency against the hCA XII (compare, as an example, compounds 4a, 4b and 4c). The selectivity ratio for inhibiting the target hCA XII isoform over the cytosolic and off−target hCA II for most of this series of derivatives is rather low compared to that shown by the standard AAZ, except for derivatives 4l−n. In particular, derivative 4m represents the best selective inhibitor of the series against the tumor−associated isoform hCA XII over the cytosolic hCA II.
We can conclude that most of the sulfonamides belonging to this new series of derivatives obtained using an innovative synthetic pathway, have shown to possess high inhibition potency against the tumor−associated target isoforms, having on the other hand good selectivity ratios over the off−target cytosolic isoforms, in particular against hCA I. Crystallography. X−ray crystallography of hCA II adducts with sulfonamides 4c, 4g and 4h were investigated in this study ( Figure 3 and Supporting Information, Table S2). At physiological pH conditions, the three inhibitors were found within the hCA II active site in the deprotonated  (Table 1).
Benzenesulfonamide 4c bearing an ortho− isopropyl group on the tail phenyl ring orients differently as compared to derivatives 4g and 4h. In fact, as illustrated in Figure 3, the ortho−isopropylphenyl ring system better accommodates the hydrophobic pocket as compared to the other two derivatives, establishing van der Waals interactions with both residues Phe131 and Val121. This more efficient fitting may be the origin of a higher inhibition potency of 4c (KI = 0.98 nM) against hCA II.  16 We selective hCA inhibitors and on the basis of these preliminary results, the design of next generation inhibitors will be engaged.

Experimental section
General All solvents were of reagent grade and, when necessary, purified and dried by standard methods.     Table S2.

Structure determination
The crystal structure of hCA II (PDB accession code: 4FIK) without solvent molecules and other heteroatoms was used to obtain initial phases of the structures using Refmac5. 49 5% of the unique reflections were selected randomly and excluded from the refinement data set for the purpose of Rfree calculations. The initial |Fo − Fc| difference electron density maps unambiguously showed the inhibitor molecules. An electron density, which could be interpreted as a second molecule of inhibitor 4g, was present near the N−terminal region of the protein. Thus, a second 4g molecule was introduced in the model and refined with unitary occupancy. Moreover, after the introduction of molecule 4c, residual electron densities were present in the Fo − Fc map and they were interpreted 30 as water molecules at partial occupancy. Also, a partial occupancy was assigned to the atoms of compound 4c. Atomic models for inhibitors were calculated and the energy minimized using the program JLigand 1.0.39. Refinements proceeded using normal protocols of positional, isotropic atomic displacement parameters alternating with manual building of the models using COOT. 50 Solvent molecules were introduced automatically using the program ARP. 51 The quality of the final models were assessed with COOT and Rampage. 52 Crystal parameters and refinement data are summarized in the Supporting Information, Table S2. Atomic coordinates were deposited in the Protein Data Bank (PDB accession code: 5E28, 5E2K, 5E2S). Graphical representations were generated with Chimera. 53