Squaramide-Tethered Sulfonamides and Coumarins: Synthesis, Inhibition of Tumor-Associated CAs IX and XII and Docking Simulations

(1) Background: carbonic anhydrases (CAs) are attractive targets for the development of new anticancer therapies; in particular, CAs IX and XII isoforms are overexpressed in numerous tumors. (2) Methods: following the tail approach, we have appended a hydrophobic aromatic tail to a pharmacophore responsible for the CA inhibition (aryl sulfonamide, coumarin). As a linker, we have used squaramides, featured with strong hydrogen bond acceptor and donor capacities. (3) Results: Starting from easily accessible dimethyl squarate, the title compounds were successfully obtained as crystalline solids, avoiding the use of chromatographic purifications. Interesting and valuable SARs could be obtained upon modification of the length of the hydrocarbon chain, position of the sulfonamido moiety, distance of the aryl sulfonamide scaffold to the squaramide, stereoelectronic effects on the aromatic ring, as well as the number and type of substituents on C-3 and C-4 positions of the coumarin. (4) Conclusions: For sulfonamides, the best profile was achieved for the m-substituted derivative 11 (Ki = 29.4, 9.15 nM, CA IX and XII, respectively), with improved selectivity compared to acetazolamide, a standard drug. Coumarin derivatives afforded an outstanding selectivity (Ki > 10,000 nM for CA I, II); the lead compound (16c) was a strong CA IX and XII inhibitor (Ki = 19.2, 7.23 nM, respectively). Docking simulations revealed the key ligand-enzyme interactions.


Introduction
Squaric acid (3,4-dihydroxycyclobut-3-ene-1,2-dione), also called quadratic acid due to its shape-close to a perfect square-is a four-membered ring system that exhibits some astonishing properties, like strong acidity, and strong hydrogen bonding; the latter feature is responsible for its high melting point temperature and low water solubility [1]. Squaric acid can be easily transformed into squarate esters upon reaction with alcohols; squarates can be further transformed into mixed squaramate (reaction with one equivalent of an amine), and also into symmetrical and non-symmetrical squaramides by condensation with one (2.0 mol. equiv.) or two amines (1.0 mol. equiv. each) [1]. The most remarkable physical property of squaramides is their capacity of participating in bidirectional hydrogen bonding interactions (if prepared from primary amines), as acceptors (carbonyl groups), and as donors (NH groups) [2]. Moreover, it has been found that the intrinsic aromatic

Drug Design, Synthesis and Characterization
Encouraged by the remarkable properties of squaramides within the medicinal chemistry field, we decided to install it in a novel series of CA inhibitors and accomplish a comprehensive analysis of their bioactivities. Following the well-known tail approach [49], we envisioned the structures depicted in Figure 1. Aryl sulfonamides and coumarins were selected as the active pharmacophores for the inhibition of the metalloenzymes. Sulfonamides and isosteric sulfamates are reported to inhibit CAs by chelating the Zn 2+ ion from the catalytic active site [50], whereas coumarins behave as suicide inhibitors [51]; esterase activity of CAs provokes a hydrolysis of the lactam skeleton of the coumarin to give a 2-hydroxy cinnamic acid derivative which occludes the entry to the enzyme active site [52]. Encouraged by the remarkable properties of squaramides within the medicinal chemistry field, we decided to install it in a novel series of CA inhibitors and accomplish a comprehensive analysis of their bioactivities. Following the well-known tail approach [49], we envisioned the structures depicted in Figure 1. Aryl sulfonamides and coumarins were selected as the active pharmacophores for the inhibition of the metalloenzymes. Sulfonamides and isosteric sulfamates are reported to inhibit CAs by chelating the Zn 2+ ion from the catalytic active site [50], whereas coumarins behave as suicide inhibitors [51]; esterase activity of CAs provokes a hydrolysis of the lactam skeleton of the coumarin to give a 2-hydroxy cinnamic acid derivative which occludes the entry to the enzyme active site [52]. In order to favor the anchorage of the inhibitor to the CA, a hydrophobic tail was incorporated for binding the hydrophobic subsite of the enzyme. Appropriate elongation might allow interaction of the ligand, not only with the catalytic site, but also with the middle and outer rims of the enzyme; modification of the electronic effects of the substituents located on the aromatic residue might also modulate the inhibitory properties. Structural modifications on the pharmacophores were also considered. And last, but not least, the squaramide tether, behaving as a vinylogous amide, or urea linkage [15] can act both, as a strong hydrogen bonding donor and acceptor and might contribute to the interaction with the hydrophilic subsite of the enzyme. Moreover, the use of a conformationally-restricted linker, as a bioisostere of more conventional linkages, can minimize the entropy loss in the formation of the enzyme-inhibitor complex, and enhance its potency/selectivity; this is a well validated approach in the drug discovery strategies that can even hinder or retard the drug degradation upon metabolization [53].
The key synthetic intermediate for preparing the target compounds is 3,4-dimethoxycyclobut-3-ene-1,2-dione (dimethyl squarate, 2), which is accessible in a multigram scale by treatment of commercially available squaric acid 1 with methanolic trimethyl orthoformate [54] under refluxing conditions (Scheme 1). In order to favor the anchorage of the inhibitor to the CA, a hydrophobic tail was incorporated for binding the hydrophobic subsite of the enzyme. Appropriate elongation might allow interaction of the ligand, not only with the catalytic site, but also with the middle and outer rims of the enzyme; modification of the electronic effects of the substituents located on the aromatic residue might also modulate the inhibitory properties. Structural modifications on the pharmacophores were also considered. And last, but not least, the squaramide tether, behaving as a vinylogous amide, or urea linkage [15] can act both, as a strong hydrogen bonding donor and acceptor and might contribute to the interaction with the hydrophilic subsite of the enzyme. Moreover, the use of a conformationally-restricted linker, as a bioisostere of more conventional linkages, can minimize the entropy loss in the formation of the enzyme-inhibitor complex, and enhance its potency/selectivity; this is a well validated approach in the drug discovery strategies that can even hinder or retard the drug degradation upon metabolization [53].
Sulfonamide-containing ureido/peptide-mimetics 5, 6, 8 and 11 were obtained in a one-pot, two-step methodology (Schemes 1 and 2) by sequential nucleophilic displacement of the two methoxy groups with amino-derivatives 3 (anilines) and 4, 7, 9 (sulfonamides); final compounds were obtained as highly crystalline derivatives just by filtration, without the need of chromatographic purifications.
Reactions proceeded in higher rate and yields if the least nucleophilic amino compound was added in the first step. This is the reason for starting the synthesis with anilines 3 in Scheme 1, or with aminobenzenesulfonamides 7 and 9 in Scheme 2. 13 C-NMR supported the proposed structures; thus, resonances at roughly 180-184 ppm (assigned to C=O) and at 164-169 ppm (C-3 and C-4) are in agreement with reported squaramides bearing N,N -diaryl or N-alkyl-N -aryl substituents [55]. Squaramides 5 derived from sulfonamide 4 were obtained under refluxing conditions for both steps, in a short period of time and high yields (70-89%). Dimeric derivative 6 was obtained in a similar fashion by using 2.0 mol. equivalents of sulfonamide 4 (83% yield). Sulfonamide-containing ureido/peptide-mimetics 5, 6, 8 and 11 were obtained in a one-pot, two-step methodology (Schemes 1 and 2) by sequential nucleophilic displacement of the two methoxy groups with amino-derivatives 3 (anilines) and 4, 7, 9 (sulfonamides); final compounds were obtained as highly crystalline derivatives just by filtration, without the need of chromatographic purifications. Reactions proceeded in higher rate and yields if the least nucleophilic amino compound was added in the first step. This is the reason for starting the synthesis with anilines 3 in Scheme 1, or with aminobenzenesulfonamides 7 and 9 in Scheme 2. 13 C-NMR supported the proposed structures; thus, resonances at roughly 180-184 ppm (assigned to C=O) and at 164-169 ppm (C-3 and C-4) are in agreement with reported squaramides bearing N,N′-diaryl or N-alkyl-N′-aryl substituents [55].
Squaramides 5 derived from sulfonamide 4 were obtained under refluxing conditions for both steps, in a short period of time and high yields (70-89%). Dimeric derivative 6 was obtained in a similar fashion by using 2.0 mol. equivalents of sulfonamide 4 Scheme 1. Preparation of sulfonamide-containing squaramides 5 and 6. Sulfonamide-containing ureido/peptide-mimetics 5, 6, 8 and 11 were obtained in a one-pot, two-step methodology (Schemes 1 and 2) by sequential nucleophilic displacement of the two methoxy groups with amino-derivatives 3 (anilines) and 4, 7, 9 (sulfonamides); final compounds were obtained as highly crystalline derivatives just by filtration, without the need of chromatographic purifications. Reactions proceeded in higher rate and yields if the least nucleophilic amino compound was added in the first step. This is the reason for starting the synthesis with anilines 3 in Scheme 1, or with aminobenzenesulfonamides 7 and 9 in Scheme 2. 13 C-NMR supported the proposed structures; thus, resonances at roughly 180-184 ppm (assigned to C=O) and at 164-169 ppm (C-3 and C-4) are in agreement with reported squaramides bearing N,N′-diaryl or N-alkyl-N′-aryl substituents [55].
Squaramides 5 derived from sulfonamide 4 were obtained under refluxing conditions for both steps, in a short period of time and high yields (70-89%). Dimeric derivative 6 was obtained in a similar fashion by using 2.0 mol. equivalents of sulfonamide 4 (83% yield). Attempts to reproduce the aforementioned conditions for pand m-substituted aminobenzenesulfonamides 7 and 9 proved to be unsuccessful, as desired compounds were obtained in a non-resolved mixture of compounds, including symmetrical dimeric species. In order to overcome such problems, and reduce the rate of side-products, both nucleophilic displacements on dimethyl squarate 2 were attempted at rt during significantly more prolonged reaction times (5 days for 8 and 17 days for 11).
In sulfonamides 5, 6, 8 and 11 several key structural motifs were modified in order to get valuable structure-activity relationships concerning their inhibitory properties against CAs: on the one hand, the stereoelectronic effects of the appended substituents on the aromatic scaffold (unsubstituted, electron-donating and electron-withdrawing, compounds 5a-e); unfortunately, the p-fluorophenyl derivative could not be obtained in a pure form; on the other hand, the distance between the arylsulfonamido moiety and the squaramide (5a vs. 8); and finally, the position of the sulfonamido motif (8 vs. 11).
In sulfonamides 5, 6, 8 and 11 several key structural motifs were modified in order to get valuable structure-activity relationships concerning their inhibitory properties against CAs: on the one hand, the stereoelectronic effects of the appended substituents on the aromatic scaffold (unsubstituted, electron-donating and electron-withdrawing, compounds 5a-e); unfortunately, the p-fluorophenyl derivative could not be obtained in a pure form; on the other hand, the distance between the arylsulfonamido moiety and the squaramide (5a vs. 8); and finally, the position of the sulfonamido motif (8 vs. 11).
We also envisioned the possibility of exploring a different pharmacophore for targeting CAs; we therefore replaced the arylsulfonamido motif, which very frequently leads to moderate selectivities, with coumarins (2H-chromen-2-ones), a natural and privileged structure within medicinal chemistry [56]. The coumarin skeleton, decorated with different substituents on C-3 and C-4 positions (compounds 14), is easily accessible using the acid-catalyzed Pechmann condensation [57] between resorcinol 12 and the appropriate β-ketoesters 13 (Scheme 3). Williamson synthesis on the free phenolic hydroxyl group of 14 at C-7 with an excess of α,ω-dibromoalkanes, followed by nucleophilic displacement of the terminal bromine atom with NaN3 and Pd-catalyzed hydrogenolysis afforded amino-coumarins 15, which were anchored on the cyclobutene-1,2-dione motif (Scheme 3) after reaction of dimethyl squarate 2 with different anilines 3 (R 1 = H, OMe, F, Cl, Br, I). This gave access to squaramido-containing coumarins 16 in moderate to excellent yields (26-93%). Regarding the squaramide moiety, similar spectroscopic data as those obtained for arylsulfonamides were observed.

Biological Assessments
The vast panel of compounds reported herein, that is, sulfonamides 5, 6, 8 and 11, and coumarins 16, were tested in vitro against membrane-bound CAs IX and XII, with therapeutic interest against hypoxic tumors [58], and their activities were compared with off-target isoforms I and II (cytosolic) in order to calculate the selectivity index (S.I.). For that purpose, the stopped-flow CO 2 hydration assay was used ( Table 1). The drug acetazolamide (AAZ) was used as the reference compound. To the best of our knowledge, there is only one reported example of the use of squaramides in the inhibition of CAs; in this case, the squaramide motif was decorated with a bis-benzoxaborole fragment and turned out to be a moderate inhibitor of CAs [59]. therapeutic interest against hypoxic tumors [58], and their activities were compared with off-target isoforms I and II (cytosolic) in order to calculate the selectivity index (S.I.). For that purpose, the stopped-flow CO2 hydration assay was used ( Table 1). The drug acetazolamide (AAZ) was used as the reference compound. To the best of our knowledge, there is only one reported example of the use of squaramides in the inhibition of CAs; in this case, the squaramide motif was decorated with a bis-benzoxaborole fragment and turned out to be a moderate inhibitor of CAs [59]. therapeutic interest against hypoxic tumors [58], and their activities were compared with off-target isoforms I and II (cytosolic) in order to calculate the selectivity index (S.I.). For that purpose, the stopped-flow CO2 hydration assay was used ( Table 1). The drug acetazolamide (AAZ) was used as the reference compound. To the best of our knowledge, there is only one reported example of the use of squaramides in the inhibition of CAs; in this case, the squaramide motif was decorated with a bis-benzoxaborole fragment and turned out to be a moderate inhibitor of CAs [59]. and coumarins 16, were tested in vitro against membrane-bound CAs IX and XII, with therapeutic interest against hypoxic tumors [58], and their activities were compared with off-target isoforms I and II (cytosolic) in order to calculate the selectivity index (S.I.). For that purpose, the stopped-flow CO2 hydration assay was used ( Table 1). The drug acetazolamide (AAZ) was used as the reference compound. To the best of our knowledge, there is only one reported example of the use of squaramides in the inhibition of CAs; in this case, the squaramide motif was decorated with a bis-benzoxaborole fragment and turned out to be a moderate inhibitor of CAs [59]. Regarding sulfonamides, incorporation of substituents on the p-position of the phenyl ring (Table 1, derivatives 5b-e vs. 5a) yielded an impairment of the inhibition of off-target enzymes (Ki = 373-910 nM); nevertheless, this observation was translated into an improvement of selectivity just for p-bromophenyl derivative 5d, which turned out to be the strongest inhibitors of this first series against CA IX (Ki = 67.6 nM) and XII (Ki = 85.5 nM). Insertion of electron-donating groups (OMe, 5b) was particularly unfavorable for the inhibition of CA XII.
Dimeric sulfonamide 6, despite keeping a good inhibitory level against tu- Regarding sulfonamides, incorporation of substituents on the p-position of the phenyl ring (Table 1, derivatives 5b-e vs. 5a) yielded an impairment of the inhibition of offtarget enzymes (K i = 373-910 nM); nevertheless, this observation was translated into an improvement of selectivity just for p-bromophenyl derivative 5d, which turned out to be the strongest inhibitors of this first series against CA IX (K i = 67.6 nM) and XII (K i = 85.5 nM). Insertion of electron-donating groups (OMe, 5b) was particularly unfavorable for the inhibition of CA XII.
Dimeric sulfonamide 6, despite keeping a good inhibitory level against tumor-associated CAs, provoked a considerable improvement in the inhibition of the off-target enzymes, and thus, a sharp decrease in the selectivity ( Table 1).
The shortening of the distance between the aryl sulfonamide and the squaramide moieties (8 vs. 5a) led to an increased inhibitory activity against CAs I and II, but also for CA IX and XII, affording a clear selectivity improvement, particularly for CA XII, reaching the low nanomolar range (K i = 6.57 nM). Thus, S.I. (I/XII) were found to be 2.2 and 15 for compounds 5a and 8, respectively, and S.I. (II/XII), 7.4 and 10 for the same two compounds ( Table 1).
Further SAR analysis revealed that m-regioisomers 10 (intermediate compound), and 11 kept within the low nM inhibition range for CA XII. Interestingly, the latter compound can be considered the lead compound of the sulfonamide series, as a clear impairment of activity against CA I and II was observed, improving the selectivity indexes of the reference drug AAZ (Table 1). Table 2 depicts the same kind of data for squaramide-containing coumarins 16a-p; the first relevant conclusion that can be reached is that outstanding selectivities are observed for all compounds of this second series, as no relevant activities were detected for concentrations as high as 10 µM against CA I and II. This observation constitutes a significant difference with related sulfonamido counterparts.
Compounds 16a-d, with an unsubstituted phenyl residue, provided information about the influence of the length of the hydrocarbon chain connecting the coumarin and squaramide moieties. Clearly, elongation led to an improvement of activity against tumor associated CAs, reaching the highest activities for n = 12 (16d) and 9 (16c) for CA IX and XII, respectively. Low nanomolar activities were achieved (K i = 19.2, 18.1 nM for CA IX; 7.23, 7.91 nM for CA XII).
The second sub-series of compounds is comprised of derivatives 16e-i, where the hydrocarbon chain was kept unchanged (n = 5), and the stereoelectronic effects (electron withdrawing and donating effects) on the p-position of the phenyl ring were modified, and the effects, compared with the unsubstituted counterpart 16b. Data shown in Table 2 indicate that such substituents do not exert much influence on the bioactivities; just a moderate impairment of activity was observed for 16e (R = OMe), 16g (R = Cl), 16i (R = I).
The third set of compounds includes coumarins 16j-o, which were designed for analyzing the influence of a disubstituted pattern on C-3 and C-4 positions of the coumarin on the inhibition profile. The same hydrocarbon chain length (n = 5) and substitution pattern on the phenyl ring as previously indicated was settled. Comparison with unsubstituted 16b revealed ( Table 2) an impairment of activity for CA IX and XII in all the members of the series, CA IX being more affected. Therefore, an increase in the steric hindrance was found to be detrimental on the activity.
The same conclusion can be reached by replacing the Me group on 16b with a Ph residue (16p); although being monosubstituted just on the C-4 position, such group is endowed with a high steric demand, that furnished a roughly 6.1-fold decreased activity for both enzymes when compared with 16b.
Some representative compounds (5a-e, 16b, 16e-i) were also tested as potential antiproliferative agents against a panel of six human tumor cell lines: A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast), WiDr (colon). Unfortunately, no relevant activity was found for the tested compounds. Derivatives 16g-i were not soluble under the assay conditions; compounds 5a-e, 6, 16e and 16f had GI 50 > 100 µM for all the cell lines. Compound 16b had GI 50 = 94 ± 10 µM for line A549, and >100 µM for the rest. impairment of activity against CA I and II was observed, improving the selectivity indexes of the reference drug AAZ (Table 1). Table 2 depicts the same kind of data for squaramide-containing coumarins 16a-p; the first relevant conclusion that can be reached is that outstanding selectivities are observed for all compounds of this second series, as no relevant activities were detected for concentrations as high as 10 µM against CA I and II. This observation constitutes a significant difference with related sulfonamido counterparts. Previous data were generated under normoxic conditions, when small concentrations of CAs are present; however, under hypoxic conditions, CA IX/XII are overexpressed and constitute a survival mechanism of the tumor cells. There are extensive data in literature indicating that under hypoxic conditions, the inhibition of such enzymes constitutes a potent antitumor mechanism [60][61][62][63][64].
Moreover, it is important to mention that a series of inhibitors of CA XII have also been found to inhibit the drug efflux transporter P-glycoprotein (P-gp) [65], one of the most common mechanisms for elimination of xenobiotics, and thus, responsible for the development of chemoresistance [66], one of the major challenges to be overcome in the development of new anticancer therapies.

Docking Simulations
The lead compounds in each series (m-benzenesulfonamide 11 and coumarin 16c) were subjected to docking simulations in order to predict the key interactions involved in their inhibition of CA XII, what can be of interest for future drug design. Squaramate 10, although not exhibiting an optimal selectivity profile, was also included in the calculations, as it was endowed with the strongest inhibition of CA XII within both sub-series. Sulfonamides are reported [37] to interact with CAs through its deprotonated form, and thus, as anionic species; accordingly, such observations were taken into consideration for the in silico study. Figure 2 shows the predicted interactions of one of the most favorable poses for compound 10 binding the active site of CA XII; in this pose, the deprotonated sulfonamido motif exhibits interactions with the Zn 2+ cation through the NH residue. Hydrogen bonding interactions between one of the oxygen atoms of the sulfonamido moiety and Thr 198, Thr 199 residues are also observed. This situation is similar to the interactions shown in crystal structures of arylsulfonamides-CA (II, IX) complexes [67]. Some other favourable poses also predict interaction, together with NH, of the SO 2 residue and the Zn(II), due to the existence of a partial negative charge on the oxygen atoms. and 16f had GI50 > 100 µM for all the cell lines. Compound 16b had GI50 = 94 ± 10 µM for line A549, and >100 µM for the rest.
Previous data were generated under normoxic conditions, when small concentrations of CAs are present; however, under hypoxic conditions, CA IX/XII are overexpressed and constitute a survival mechanism of the tumor cells. There are extensive data in literature indicating that under hypoxic conditions, the inhibition of such enzymes constitutes a potent antitumor mechanism [60][61][62][63][64].
Moreover, it is important to mention that a series of inhibitors of CA XII have also been found to inhibit the drug efflux transporter P-glycoprotein (P-gp) [65], one of the most common mechanisms for elimination of xenobiotics, and thus, responsible for the development of chemoresistance [66], one of the major challenges to be overcome in the development of new anticancer therapies.

Docking Simulations
The lead compounds in each series (m-benzenesulfonamide 11 and coumarin 16c) were subjected to docking simulations in order to predict the key interactions involved in their inhibition of CA XII, what can be of interest for future drug design. Squaramate 10, although not exhibiting an optimal selectivity profile, was also included in the calculations, as it was endowed with the strongest inhibition of CA XII within both sub-series. Sulfonamides are reported [37] to interact with CAs through its deprotonated form, and thus, as anionic species; accordingly, such observations were taken into consideration for the in silico study. Figure 2 shows the predicted interactions of one of the most favorable poses for compound 10 binding the active site of CA XII; in this pose, the deprotonated sulfonamido motif exhibits interactions with the Zn 2+ cation through the NH residue. Hydrogen bonding interactions between one of the oxygen atoms of the sulfonamido moiety and Thr 198, Thr 199 residues are also observed. This situation is similar to the interactions shown in crystal structures of arylsulfonamides-CA (II, IX) complexes [67]. Some other favourable poses also predict interaction, together with NH, of the SO2 residue and the Zn(II), due to the existence of a partial negative charge on the oxygen atoms. When shifting to structurally related m-squaramide 11, the larger size of the appended phenyl residue compared to the methoxy group of 10, provokes its orientation towards the exit of the cavity. Similarly, to 10, interactions of the deprotonated sulfonamido group with Zn 2+ (NH) and Thr 198, Thr 199 Leu 197 (oxygen atoms of the sulfonamido moiety) were observed in the most favorable poses (Figure 3). When shifting to structurally related m-squaramide 11, the larger size of the appended phenyl residue compared to the methoxy group of 10, provokes its orientation towards the exit of the cavity. Similarly, to 10, interactions of the deprotonated sulfonamido group with Zn 2+ (NH) and Thr 198, Thr 199 Leu 197 (oxygen atoms of the sulfonamido moiety) were observed in the most favorable poses (Figure 3). When shifting to structurally related m-squaramide 11, the larger size of the appended phenyl residue compared to the methoxy group of 10, provokes its orientation towards the exit of the cavity. Similarly, to 10, interactions of the deprotonated sulfonamido group with Zn 2+ (NH) and Thr 198, Thr 199 Leu 197 (oxygen atoms of the sulfonamido moiety) were observed in the most favorable poses (Figure 3).  As previously indicated, coumarins behave as prodrugs, as the lactone functionality is hydrolyzed by CAs to furnish the corresponding 2-hydroxycinnamic acid derivative [52], presumably with E-configuration (Scheme 4). This inhibitory mechanism was previously demonstrated both by X-ray crystallography and mass spectrometry [68].
In fact, the binding energy of the non-hydrolyzed form shows a remarkable impairment compared to the cinnamic acid derivative binding the enzyme in the active site (Table 3).   As previously indicated, coumarins behave as prodrugs, as the lactone functionality is hydrolyzed by CAs to furnish the corresponding 2-hydroxycinnamic acid derivative [52], presumably with E-configuration (Scheme 4). This inhibitory mechanism was previously demonstrated both by X-ray crystallography and mass spectrometry [68]. As previously indicated, coumarins behave as prodrugs, as the lactone functionality is hydrolyzed by CAs to furnish the corresponding 2-hydroxycinnamic acid derivative [52], presumably with E-configuration (Scheme 4). This inhibitory mechanism was previously demonstrated both by X-ray crystallography and mass spectrometry [68].
In fact, the binding energy of the non-hydrolyzed form shows a remarkable impairment compared to the cinnamic acid derivative binding the enzyme in the active site (Table 3).  In fact, the binding energy of the non-hydrolyzed form shows a remarkable impairment compared to the cinnamic acid derivative binding the enzyme in the active site (Table 3). In this context, we have accomplished two different docking simulations for derivative 16c, one on the cavity of the enzyme active site (Figure 4), and another one in the entry to the cavity ( Figure 5), in order to predict the most favorable binding region with the corresponding E-configured 2-hydroxycinnamic acid derivative.   Regarding the simulation within the active site, it is the concomitant carboxylate moiety that interacts with the Zn 2+ cation, whereas the bulky nonyl and squaramido moieties occlude the entry to the cavity of the catalytic site ( Figure 4).
Nevertheless, simulations conducted at the entry of the cavity (Figure 5), where the carboxylate moiety cannot interact with the Zn(II) cation, led to a significant impairment in the binding energy (Table 3, roughly −10 kcal/mol vs. −6.9 kcal/mol). This would predict that the hydrolyzed coumarin system has the appropriate shape and size to be accommodated within the enzyme active site and interact with the Zn(II) through ionic interactions. A similar situation was recently reported for psoralen derivatives [69]. Regarding the simulation within the active site, it is the concomitant carboxylate moiety that interacts with the Zn 2+ cation, whereas the bulky nonyl and squaramido moieties occlude the entry to the cavity of the catalytic site ( Figure 4).
Nevertheless, simulations conducted at the entry of the cavity (Figure 5), where the carboxylate moiety cannot interact with the Zn(II) cation, led to a significant impairment in the binding energy (Table 3, roughly −10 kcal/mol vs. −6.9 kcal/mol). This would predict that the hydrolyzed coumarin system has the appropriate shape and size to be accommodated within the enzyme active site and interact with the Zn(II) through ionic interactions. A similar situation was recently reported for psoralen derivatives [69].

Prediction of Physicochemical Properties
A series of relevant molecular physicochemical properties, like logP, logS (Ali) [70], H-bond acceptors/donors, TPSA (SwissADME freeware suite) [71,72], or pKa (MolGpka) [73,74] were calculated for squaramides in order to predict their drug likeness properties (Table 4). As it can be observed from Table 4, all compounds have the required lipophilicity, polar surface, number of H-bond acceptors/donors and molecular size so as to fulfill the Lipinsky rule of five, and therefore, to be considered to have drug likeness properties. Only derivatives 16d,h,I,n,o slightly exceeded the limitation of 500 g/mol for their molecular weights. Sulfonamido-containing 5, 6, 8, 10, 11 are expected to be either moderately soluble, or soluble in water; coumarin derivatives 16a,b are predicted to be moderately soluble in water, while the rest compounds of the series, with longer tethers, a disubstituted pattern on C-3/C-4 positions of the coumarin, or bearing a Ph ring at C-4 position, are expected to have poor water solubility.
With the exceptions of dimeric sulfonamide 6 and coumarin derivative 16d, the rest of the compounds of the series are predicted to have high gastrointestinal absorption, in contrast with the reference drug AAZ. This feature might ensure an appropriate bioavailability of the title compounds.

General Methods
TLCs (Merck 60 F 254 , gel thickness 0.25 mm) were performed using aluminum-coated sheets, using the appropriate eluant. Spots were visualized by UV light (λ = 254 nm), and by charring with 10% ethanolic vanillin containing 1% H 2 SO 4 , or with 3% ninhydrin in EtOH. Column chromatography purifications were performed using silica gel stationary phase (Merck 60, particle size 40-63 µm), eluting by gravity, or with a mild pressure, using the eluant indicated in the experimental section.
NMR spectra were registered in the Centro de Investigación, Tecnología e Innovación de la Universidad de Sevilla (CITIUS), using Bruker Avance III 300 and 500 spectrometers (300 and 500 MHz for 1 H, 75.5 and 125.7 MHz for 13 C), and DMSO-d 6 as solvent (see Supplementary Material). Chemical shifts (δ) are expressed in ppm, and coupling constants (J), in Hz. Residual signals from the solvent are used as internal references [75]. Mass spectra were registered using a Q Exactive spectrometer, using Electrospray Ionization (ESI).

General Procedure for the Preparation of Squaramides 5a-e
A mixture of dimethyl squarate 2 (1.0 equiv.) and the corresponding aniline derivative 3 (1.0 equiv.) in MeOH (5 mL) was refluxed for 2 h. Then, 4-(2 -aminoethyl)benzenesulfonamide 4 (1.0 equiv.) was added, and refluxed for further 2 h. After that, the crude reaction mixture was filtered and the solid was washed with cold MeOH.

General Procedures for the Preparation of Coumarin Derivatives 16a-p
Method A. A solution of dimethyl squarate 2 (1.0 equiv.) and the corresponding aniline 3 (1.0 equiv.) in MeOH (5 mL) was heated in a Fisher-Porter tube at 100 • C for 2 h. Then, the corresponding amino-coumarin 15 (1.0 equiv.) was added and the mixture was heated in the Fisher-Porter tube at 100 • C for further 24 h. A precipitate was formed, which was filtered and washed with cold MeOH.
Method B. A solution of dimethyl squarate 2 (1.0 equiv.) and the corresponding aniline 3 (1.0 equiv.) in MeOH (7 mL) was refluxed for 4 h. Then, the corresponding amino-coumarin 15 (1.0 equiv.) was added and the reaction was refluxed for further 12 h. A precipitate was formed, which was filtered and washed with cold MeOH.
Method C. A solution of dimethyl squarate 2 (1.0 equiv.) and the corresponding aniline 3 (1.0 equiv.) in MeOH (7 mL) was kept at rt for 24 h. Then, the corresponding aminocoumarin 15 (1.0 equiv.) was added and the reaction was kept at the same temperature for further 24 h. A precipitate was formed, which was filtered and washed with cold MeOH.