Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors

The involvement of carbonic anhydrases (CAs) in a myriad of biological events makes the development of new inhibitors of these metalloenzymes a hot topic in current Medicinal Chemistry. In particular, CA IX and XII are membrane-bound enzymes, responsible for tumour survival and chemoresistance. Herein, a bicyclic carbohydrate-based hydrophilic tail (imidazolidine-2-thione) has been appended to a CA-targeting pharmacophore (arylsulfonamide, coumarin) with the aim of studying the influence of the conformational restriction of the tail on the CA inhibition. For this purpose, the coupling of sulfonamido- or coumarin-based isothiocyanates with reducing 2-aminosugars, followed by the sequential acid-promoted intramolecular cyclization of the corresponding thiourea and dehydration reactions, afforded the corresponding bicyclic imidazoline-2-thiones in good overall yield. The effects of the carbohydrate configuration, the position of the sulfonamido motif on the aryl fragment, and the tether length and substitution pattern on the coumarin were analysed in the in vitro inhibition of human CAs. Regarding sulfonamido-based inhibitors, the best template turned out to be a d-galacto-configured carbohydrate residue, meta-substitution on the aryl moiety (9b), with Ki against CA XII within the low nM range (5.1 nM), and remarkable selectivity indexes (1531 for CA I and 181.9 for CA II); this provided an enhanced profile in terms of potency and selectivity compared to more flexible linear thioureas 1–4 and the drug acetazolamide (AAZ), used herein as a reference compound. For coumarins, the strongest activities were found for substituents devoid of steric hindrance (Me, Cl), and short linkages; derivatives 24h and 24a were found to be the most potent inhibitors against CA IX and XII, respectively (Ki = 6.8, 10.1 nM), and also endowed with outstanding selectivity (Ki > 100 µM against CA I, II, as off-target enzymes). Docking simulations were conducted on 9b and 24h to gain more insight into the key inhibitor–enzyme interactions.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes (most of them Zn(II)-dependent) [1] that catalyse a simple but yet essential reaction, the reversible hydration of CO 2 , to furnish HCO 3 − and a proton [2]. The rate of the spontaneous, non-catalysed process was found to be pivotal in respiration [3] for the homeostasis of physiological pH [4], ureagenesis, or gluconeogenesis [5] among other biochemical events. However, it is not fast enough to meet the metabolic demand [6]. With a turnover number as high as 10 6 s −1 , CAs account for one of the fastest biocatalysts found in nature [7].
Carbonic anhydrases can be categorized into eight genetic families, and are named with Greek letters, that is, α, β, γ, δ, ζ, η, θ, and ι [8]; the latter family was discovered just very recently [9]. Among them, the α-CA family can be divided into 16 isoforms [10]. It is the only one found in mammals and, therefore, suitable to be used as a therapeutic target in numerous diseases, either with inhibitors or activators [11]. In this context, many α-CA inhibitors have been designed and tested against a variety of diseases, such as glaucoma [12], epilepsy [13], obesity [14], diabesity (the simultaneous presence of diabetes and obesity) [15], articular inflammatory diseases (e.g., arthritis [16]), or neuropathic pain [17]. However, undoubtedly the most pursued activity of CA inhibitors is as anticancer agents. A series of CA inhibitors are currently used as drugs, with various clinical uses as diuretics (e.g., acetazolamide), antiglaucoma agents (e.g., acetazolamide, dichlorphenamide, dorzolamide, brinzolamide), antiobesity agents (e.g., the combination of topiramate and a sympathomimetic agent), or against epilepsy (e.g., sulthiame) [18].
The human isoforms CA IX (almost absent in healthy cells) and XII are overexpressed in hypoxic tumours due to the action of the hypoxia-inducible factor-1 (HIF-1). They are responsible, together with anaerobic glycolysis, for the acidification of the tumour microenvironment, as well as for tumour survival and proliferation [19]. Moreover, CA XII inhibition has been associated [20] with the deactivation of the machinery associated to P-glycoprotein (P-gp).
The most widely studied family of CA inhibitors is comprised of Zn(II) chelators, mainly sulfonamides and their isosters sulfamates and sulfamides, which complex the metal through their deprotonated forms [21]. The cavity of the enzyme active site has an amphiphilic nature; accordingly, hydrophilic or hydrophobic interactions between the inhibitor and the active site could be established (tail approach) [22].
Coumarins (2H-chromen-2-ones) are secondary metabolites widely distributed in nature and considered as privileged structures in Medicinal Chemistry [23]. This is due to their numerous biological properties exhibited, including CA inhibition. The slow mode of inhibition observed for coumarins suggested that these compounds behaved as suicide inhibitors and were actually pro-drugs [24]. The intrinsic esterase activity of CAs provokes the hydrolysis of the lactone functionality of coumarins, furnishing a 2-hydroxycinnamic derivative; such a hydrolysed structure occludes the entry to the active site [24].
Moreover, the appendage of O-unprotected carbohydrates to pharmacophores responsible for the CA inhibition has been reported to be a valid approach for targeting selective CA IX and XII inhibition [25]. This is due to the fact that such isoforms are membraneanchored enzymes, and the highly hydrophilic carbohydrate tail precludes the entrance of the inhibitor into the cell. This fact avoids the inhibition of cytosolic CA enzymes and can therefore improve selectivity.
In this context, some of us reported [26,27] the preparation of glyco-sulfonamides connected through a flexible thiourea linker (1-4, Figure 1) on C-1 or C-2 positions of the carbohydrate residue.
Our main target herein has been the conformational restriction of the carbohydrate tail of 1-4 and to analyse the influence of this issue on the inhibitory properties of the new compounds. For this purpose, we have used not only arylsulfonamides as Zn-chelators, but also coumarin derivatives. In this context, we have included different substitution patterns and tether lengths for the connection with the carbohydrate residue.
connected through a flexible thiourea linker (1-4, Figure 1) on C-1 or C-2 positions of the carbohydrate residue.
Our main target herein has been the conformational restriction of the carbohydrate tail of 1-4 and to analyse the influence of this issue on the inhibitory properties of the new compounds. For this purpose, we have used not only arylsulfonamides as Zn-chelators, but also coumarin derivatives. In this context, we have included different substitution patterns and tether lengths for the connection with the carbohydrate residue.

Drug Design and Chemistry
Herein, we have accomplished the preparation of a series of conformationally restricted glycoconjugates for targeting CAs. For that purpose, we envisioned the general structure depicted in Figure 2. The CA-directed pharmacophore (arylsulfonamide, coumarin) is linked to a carbohydrate residue, which acts as the hydrophilic tail, through a bicyclic thiourea (imidazolidine-2-thione).
The restriction of the conformational flexibility of a drug is a well-validated approach that might provide several advantages. It minimizes the entropy penalty in ligand-protein interactions, furnishes improved selectivity towards certain isoforms, or reduces the drug metabolization [28]. Access to targeted bicyclic imidazolidine-2-thiones was carried out by using the methodology developed by some of us for preparing pseudo-nucleosides [29][30][31] and their selenium isosters [32]. Such a synthetic pathway involves the preparation of a transient thiourea on the C-2 of a reducing carbohydrate (Scheme 1), derived from 2-deoxy-2-amino-D-sugars. When R = arilsulfonamido, thioureas could be isolated upon coupling arylsulfonamide isothiocyanates with the corresponding O-unprotected 2-aminosugar [27], as no spontaneous cyclization was observed.
Nevertheless, for R = alkyl or aryl groups lacking the sulfonamido moiety, a spontaneous intramolecular cyclization was previously observed [29] to give a

Drug Design and Chemistry
Herein, we have accomplished the preparation of a series of conformationally restricted glycoconjugates for targeting CAs. For that purpose, we envisioned the general structure depicted in Figure 2. The CA-directed pharmacophore (arylsulfonamide, coumarin) is linked to a carbohydrate residue, which acts as the hydrophilic tail, through a bicyclic thiourea (imidazolidine-2-thione).
The restriction of the conformational flexibility of a drug is a well-validated approach that might provide several advantages. It minimizes the entropy penalty in ligand-protein interactions, furnishes improved selectivity towards certain isoforms, or reduces the drug metabolization [28]. carbohydrate residue.
Our main target herein has been the conformational restriction of the carbohydrate tail of 1-4 and to analyse the influence of this issue on the inhibitory properties of the new compounds. For this purpose, we have used not only arylsulfonamides as Zn-chelators, but also coumarin derivatives. In this context, we have included different substitution patterns and tether lengths for the connection with the carbohydrate residue.

Drug Design and Chemistry
Herein, we have accomplished the preparation of a series of conformationally restricted glycoconjugates for targeting CAs. For that purpose, we envisioned the general structure depicted in Figure 2. The CA-directed pharmacophore (arylsulfonamide, coumarin) is linked to a carbohydrate residue, which acts as the hydrophilic tail, through a bicyclic thiourea (imidazolidine-2-thione).
The restriction of the conformational flexibility of a drug is a well-validated approach that might provide several advantages. It minimizes the entropy penalty in ligand-protein interactions, furnishes improved selectivity towards certain isoforms, or reduces the drug metabolization [28]. Access to targeted bicyclic imidazolidine-2-thiones was carried out by using the methodology developed by some of us for preparing pseudo-nucleosides [29][30][31] and their selenium isosters [32]. Such a synthetic pathway involves the preparation of a transient thiourea on the C-2 of a reducing carbohydrate (Scheme 1), derived from 2-deoxy-2-amino-D-sugars. When R = arilsulfonamido, thioureas could be isolated upon coupling arylsulfonamide isothiocyanates with the corresponding O-unprotected 2-aminosugar [27], as no spontaneous cyclization was observed.
Nevertheless, for R = alkyl or aryl groups lacking the sulfonamido moiety, a spontaneous intramolecular cyclization was previously observed [29] to give a Access to targeted bicyclic imidazolidine-2-thiones was carried out by using the methodology developed by some of us for preparing pseudo-nucleosides [29][30][31] and their selenium isosters [32]. Such a synthetic pathway involves the preparation of a transient thiourea on the C-2 of a reducing carbohydrate (Scheme 1), derived from 2-deoxy-2-amino-D-sugars. When R = arilsulfonamido, thioureas could be isolated upon coupling arylsulfonamide isothiocyanates with the corresponding O-unprotected 2-aminosugar [27], as no spontaneous cyclization was observed.
We envisioned the possibility of transforming the reducing thioureas 3 into the corresponding bicyclic counterparts 8 (Scheme 2). For that purpose, benzenesulfonamide isothiocyanates 6 were prepared using the reported experimental conditions (thiophosgene, HCl for sulfonamides 5a,b [34,35]; CS2, DCC for sulfonamide 5c [36]). The structural arrangement on 8 would allow us to analyse the influence of the position of the sulfonamido motif on the aromatic ring, and of the distance between the aromatic and the carbohydrate residues on the inhibitory properties.
Coupling 6a-c with D-glucosamine hydrochloride in the presence of NaHCO3 afforded thioureas 3a-c. The preparation of targeted bicyclic derivatives 8a-c was accomplished by in situ heating the thioureas at 90 °C in the presence of AcOH in aqueous EtOH. Transient 5-hydroxy-imidazolidine-2-thiones 7 were not isolated (Scheme 2).
We envisioned the possibility of transforming the reducing thioureas 3 into the corresponding bicyclic counterparts 8 (Scheme 2). For that purpose, benzenesulfonamide isothiocyanates 6 were prepared using the reported experimental conditions (thiophosgene, HCl for sulfonamides 5a,b [34,35]; CS 2 , DCC for sulfonamide 5c [36]). The structural arrangement on 8 would allow us to analyse the influence of the position of the sulfonamido motif on the aromatic ring, and of the distance between the aromatic and the carbohydrate residues on the inhibitory properties.
Coupling 6a-c with D-glucosamine hydrochloride in the presence of NaHCO 3 afforded thioureas 3a-c. The preparation of targeted bicyclic derivatives 8a-c was accomplished by in situ heating the thioureas at 90 • C in the presence of AcOH in aqueous EtOH. Transient 5-hydroxy-imidazolidine-2-thiones 7 were not isolated (Scheme 2).
The same methodology was used for the preparation of D-galacto-configured imidazolidine-2-thiones 9a and 9b, using D-galactosamine hydrochloride as the reducing 2-aminosugar (Scheme 3). Attempts to extend this series to the D-manno configuration failed, as complex and non-resolved mixtures were obtained.
New hybrid carbohydrate-coumarins were also accessed using the above synthetic methodology. In order to establish structure-activity relationships, some key structural motifs were modulated. Thus, the carbohydrate configuration, distance between the sugar and the coumarin residues, and C-3/C-4 substitution pattern on the coumarin scaffold were accordingly modified. Moreover, some thioureas on the C-2 position were also prepared with non-reducing carbohydrates in order to analyse the influence of the bicyclic structure on the biological properties. The same methodology was used for the preparation of D-galacto-configured imidazolidine-2-thiones 9a and 9b, using D-galactosamine hydrochloride as the reducing 2-aminosugar (Scheme 3). Attempts to extend this series to the D-manno configuration failed, as complex and non-resolved mixtures were obtained.  New hybrid carbohydrate-coumarins were also accessed using the above synthetic methodology. In order to establish structure-activity relationships, some key structural motifs were modulated. Thus, the carbohydrate configuration, distance between the sugar and the coumarin residues, and C-3/C-4 substitution pattern on the coumarin scaffold were accordingly modified. Moreover, some thioureas on the C-2 position were also prepared with non-reducing carbohydrates in order to analyse the influence of the The same methodology was used for the preparation of D-galacto-configured imidazolidine-2-thiones 9a and 9b, using D-galactosamine hydrochloride as the reducing 2-aminosugar (Scheme 3). Attempts to extend this series to the D-manno configuration failed, as complex and non-resolved mixtures were obtained.  New hybrid carbohydrate-coumarins were also accessed using the above synthetic methodology. In order to establish structure-activity relationships, some key structural motifs were modulated. Thus, the carbohydrate configuration, distance between the sugar and the coumarin residues, and C-3/C-4 substitution pattern on the coumarin scaffold were accordingly modified. Moreover, some thioureas on the C-2 position were also prepared with non-reducing carbohydrates in order to analyse the influence of the Firstly, imidazolidine-2-thione 16 and its linear counterpart 13 were obtained in good to excellent yields (95% and 71%, respectively), using the synthetic pathway depicted in Scheme 4. These compounds lack a linker, so the coumarin residue was directly attached to the glucofurano-imidazolidine. In both cases, the key intermediate was coumarin-derived isothiocyanate 11 [37], obtained in almost quantitative yield by the treatment of commercially available 7-amino-4-methylcoumarin 10 with thiophosgene. Coupling 11 with methyl 2-amino-2-deoxy-α-D-glucopyranoside 12 furnished 13 (Scheme 4). Aminoglycoside 12 was obtained in a three-step procedure starting from D-glucosamine hydrochloride: N-benzoylation [38], Amberlite IR-120(H + )-catalysed Fischer glycosylation [39], and Ndeprotection (NaOH). Alternatively, the coupling of 11 and D-glucosamine hydrochloride in the presence of NaHCO 3, followed by refluxing in aq. EtOH containing AcOH afforded imidazolidine-2-thione 16. Its formation took place through transient thiourea 14 and 5-hydroxy-imidazolidine-2-thione 15.
For achieving such structural diversity, Pechmann condensation [40] provided three different coumarin sets; in turn, they were subjected to a Williamson synthesis with α,ω-dibromoalkanes under basic conditions (K2CO3) to furnish ω-bromoalkyl derivatives 17a−i. Subsequent nucleophilic displacement with NaN3, Pd/C-catalysed hydrogenolysis, and isothiocyanation reaction with thiophosgene afforded isothiocyanate derivatives 20a−i in good overall yields (Scheme 5). With the aim of increasing the structural diversity of the carbohydrate-coumarin template, a flexible hydrocarbon linker with different lengths was introduced on C-7. The substituents on C-3 and C-4 positions were also modified, including alkyl, aryl, and halogen fragments (H, CH 3 , Ph, Cl).
For achieving such structural diversity, Pechmann condensation [40] provided three different coumarin sets; in turn, they were subjected to a Williamson synthesis with α,ωdibromoalkanes under basic conditions (K 2 CO 3 ) to furnish ω-bromoalkyl derivatives 17a-i. Subsequent nucleophilic displacement with NaN 3 , Pd/C-catalysed hydrogenolysis, and isothiocyanation reaction with thiophosgene afforded isothiocyanate derivatives 20a-i in good overall yields (Scheme 5).
Finally, isothiocyanates 20a-i were transformed with excellent yields into both linear thioureas 21e,f, and into bicyclic counterparts 24a-i (Scheme 6). For that purpose, the same synthetic procedures as aforementioned for analogous 13 and 16 (Scheme 4) were followed. Compounds 24g,i could not be isolated pure and were not included in the study.
We also attempted to extend this reaction to other carbohydrate configurations in order to increase the structural diversity of the potential CA inhibitors. Thus, using coumarinderived isothiocyanate 20c and D-galactosamine hydrochloride 25 (Scheme 7), bicyclic derivative 26 was obtained in excellent yield (82%). A strong deshielding was observed for C-4 in 26 in comparison with 24c (87.4 vs. 79.3 ppm, respectively). This observation was reported for glycopyranosides of such configurations [41]. Unfortunately, attempts to obtain the corresponding D-manno isomer were again unsuccessful, and a non-resolved complex mixture was obtained. Finally, isothiocyanates 20a−i were transformed with excellent yields into both linear thioureas 21e,f, and into bicyclic counterparts 24a−i (Scheme 6). For that purpose, the same synthetic procedures as aforementioned for analogous 13 and 16 (Scheme 4) were followed. Compounds 24g,i could not be isolated pure and were not included in the study.
The CA selected for the assays can be categorized into two families: cytosolic (CA I, off-target; CA II, relevant against glaucoma [42]) and membrane-bound (CA IV, involved in rheumatoid arthritis [43]; CA IX and XII, overexpressed in hypoxic tumours [44]). The choice of such isoforms will provide information about selectivity against tumourassociated CA IX and XII compared to other relevant CAs. Promiscuous inhibitors can provoke severe side-effects. The obtained data are depicted in Table 1 (sulfonamides) and Table 2 (coumarins). We also attempted to extend this reaction to other carbohydrate configurations in order to increase the structural diversity of the potential CA inhibitors. Thus, using coumarin-derived isothiocyanate 20c and D-galactosamine hydrochloride 25 (Scheme 7), bicyclic derivative 26 was obtained in excellent yield (82%). A strong deshielding was observed for C-4 in 26 in comparison with 24c (87.4 vs. 79.3 ppm, respectively). This observation was reported for glycopyranosides of such configurations [41]. Unfortunately, attempts to obtain the corresponding D-manno isomer were again unsuccessful, and a non-resolved complex mixture was obtained. We also attempted to extend this reaction to other carbohydrate configurations in order to increase the structural diversity of the potential CA inhibitors. Thus, using coumarin-derived isothiocyanate 20c and D-galactosamine hydrochloride 25 (Scheme 7), bicyclic derivative 26 was obtained in excellent yield (82%). A strong deshielding was observed for C-4 in 26 in comparison with 24c (87.4 vs. 79.3 ppm, respectively). This observation was reported for glycopyranosides of such configurations [41]. Unfortunately, attempts to obtain the corresponding D-manno isomer were again unsuccessful, and a non-resolved complex mixture was obtained. Important differences in inhibitory properties were found when comparing the carbohydrate configuration (gluco vs. galacto, compounds 8 vs. 9), the regioisomeric position of the sulfonamido motif on the aromatic ring, and the presence or absence of the small tether connecting the bicyclic heterocycle and the arylsulfonamide scaffolds. A preference for the tumour-associated CA XII was observed in most of the bicyclic derivatives shown in Table 1, this effect being more strongly pronounced for galacto-derivatives 9a,b. In both families of compounds, an impairment of inhibitory properties against CA I, II, and IX was observed when shifting from para to meta substitution. This was observed in a more significant fashion for the galacto counterparts (9a, 9b), reaching submicromolar-micromolar activities for such enzymes (Table 1). Interestingly, for the latter compound, the inhibition of CA XII was kept in the low nM range (K i = 5.1 nM), thus affording remarkable selectivities for this enzyme (I/XII = 1531; II/XII = 181.9). The selectivities found for 9b far exceeded those found for the reference drug AAZ (I/XII = 43.9; II/XII = 2.1). Such an observation, that is, improved selectivities for the meta regioisomer, was also fulfilled, although to a lower extent, for gluco derivatives. The strong inhibition activity against CA XII exerted by 9a,b was not overpassed by any of the glyco-thioureas depicted as reference compounds (1)(2)(3)(4). Regarding activity against CA XII of the reference compounds, an opposed situation was observed for some of them. As a result, gluco-configured derivative was more potent than epimeric galacto counterparts (e.g., 1a vs. 2a; 3a vs. 4a).
The elongation of the structure by introducing a small ethylene-type tether between the carbohydrate and the aryl sulfonamide moieties (8a vs. 8c) led to an increase in activities for all the tested enzymes, except for CA IV (Table 1). Consequently, similar selectivities were found when comparing 8a and 8c. The latter one was proved to be a strong inhibitor of CA II (8.9 nM), an enzyme involved in glaucoma development [42].
With all the data in hand, compound 9b can, therefore, be considered as the lead compound within the first set of imidazolidine-2-thiones derived from arylsulfonamides. Table 1. Inhibition constants and selectivity indexes of sulfonamido-containing imidazolidine-2thiones 8, 9 against hCAs I, II, IV, IX, and XII compared with thioureas 1-4 and AAZ a .  An important difference found for coumarin derivatives ( Table 2) was their negligible activity towards CAs I and II, and their strong inhibition of tumour-associated membrane-anchored CA IX and XII, lying on the low-to mid-nanomolar range (6.8-177.3 nM) for CA IX; 10.1-260.3 nM for CA XII). As a result, an outstanding isoform selectivity compared to the reference drug AAZ was achieved, a fact found in some previous coumarin derivatives [45][46][47][48]. The following conclusions can be reached from the analysis of the remaining data depicted in Table 2: i.
For compounds lacking linkers, almost no difference in activities can be found for non-cyclic (13) and bicyclic (16)  An important difference found for coumarin derivatives ( Table 2) was their negligible activity towards CAs I and II, and their strong inhibition of tumour-associated membrane-anchored CA IX and XII, lying on the low-to mid-nanomolar range (6.8-177.3 nM) for CA IX; 10.1-260.3 nM for CA XII). As a result, an outstanding isoform selectivity compared to the reference drug AAZ was achieved, a fact found in some previous coumarin derivatives [45][46][47][48]. The following conclusions can be reached from the analysis of the remaining data depicted in Table 2: i.
For compounds lacking linkers, almost no difference in activities can be found for non-cyclic (13) and bicyclic (16)  An important difference found for coumarin derivatives ( Table 2) was their negligible activity towards CAs I and II, and their strong inhibition of tumour-associated membrane-anchored CA IX and XII, lying on the low-to mid-nanomolar range (6.8-177.3 nM) for CA IX; 10.1-260.3 nM for CA XII). As a result, an outstanding isoform selectivity compared to the reference drug AAZ was achieved, a fact found in some previous coumarin derivatives [45][46][47][48]. The following conclusions can be reached from the analysis of the remaining data depicted in Table 2: i.
For compounds lacking linkers, almost no difference in activities can be found for non-cyclic (13) and bicyclic (16)  An important difference found for coumarin derivatives ( Table 2) was their negligible activity towards CAs I and II, and their strong inhibition of tumour-associated membraneanchored CA IX and XII, lying on the low-to mid-nanomolar range (6.8-177.3 nM) for CA IX; 10.1-260.3 nM for CA XII). As a result, an outstanding isoform selectivity compared to the reference drug AAZ was achieved, a fact found in some previous coumarin derivatives [45][46][47][48]. The following conclusions can be reached from the analysis of the remaining data depicted in Table 2: i.
For compounds lacking linkers, almost no difference in activities can be found for non-cyclic (13) and bicyclic (16) thioureas. ii.
The insertion of a linker between the carbohydrate and the coumarin residues of byclicic structures proved to be benefitial for the inhibition of both membranebound enzymes (16 vs . 24a-c). iii.
The presence of a Ph residue on C-3 both in linear thioureas (21e,f) and imidazolidine-2-thiones (24d,e) provoked an impariment of the inhibitory profile against CA IX and XII, reaching the submicromolar range. This is probably due to steric clash within the active site. iv.
Considering the effect of the substituents (n = 4), the observed order of activity is: v. The best template for the inhibition of CA XII was proved to be a short linkage (n = 3), and the monosubtitution of coumarin on C-3 with small substituents (Me, 24a), with K i = 10.1 nM vi.
Little differences in activity were found by changing the carbohydrate configuration (24c vs. 26).

Antiproliferative Activities
The compounds prepared herein were subjected to in vitro testing as potential antiproliferative agents against a panel of six human solid tumour cell lines, following minor modifications of the protocol from the US National Cancer Institute (NCI) [49]. They can be categorized into two groups: drug-sensitive cell lines (A549, HBL-100, HeLa, SW1573) and multidrug resistant cell lines (T-47D, WiDr). Compounds that exhibited more noticeable antiproliferative activity (from moderate to good) are depicted in Table 3 (GI 50 expressed in µM). Ph-derived thiourea 21e and imidazolidine-2-thione 24f were not included in the study. The remaining derivatives proved to have negligible activity at the maximum concentration tested (GI 50 > 100 µM).
The incorporation of a phenyl moiety, in both some of the thioureas and the bicyclic counterparts (21f and 24e), provided an increase in the antiproliferative potency, probably by improving the hydrophilic/hydrophobic balance for cell penetration. Interestingly, such a property was strongly dependent on the linker size, as the longer imidazolidine-2-thione derivative 24f (n = 6) exhibited GI 50 values > 100 µM for all the tested cell lines. Two of the tested compounds (21f, 24e) exhibited strong activity on the SW1573 cell line (GI 50 = 9.7, 5.7 µM, respectively). Thiourea 21f also showed the best profile for the other five lines, with good GI 50 values ranging from 23 to 36 µM.

Docking Simulations
Docking studies shed light on the molecular interactions that could take place between compounds and the different hCA isoforms. Arylsulfonamide 9b and coumarin derivative 24h were selected for such studies. D-Galacto-configured sulfonamide 9b was predicted to act as a zinc-chelating agent through its sulfonamido moiety (Figure 3). The deprotonated form of the sulfonamide interacts, through the NH moiety, with the Zn 2+ cation of CA XII. A hydrogen bond interaction is also established between Thr 198, Thr 199, and the sulfonamido scaffold. In the active form of the CA, Thr 198 is hydrogen bonded with the H 2 O/OHcoordinated with the zinc ion [50]. Although docking techniques do not allow the simulation of the displacement of water molecules, the interaction of 9b directly with Zn 2+ and the Thr 198 residue could explain the inhibitory effect towards the catalytic activity of the enzyme. The 2D-and 3D-predicted interactions of 9b and the active site of CA XII are depicted in Figures 3A and 3B, respectively.        Table 3. Antiproliferative activity (GI 50 , µM) of selected compounds (mean ± SD).  (21f and 24e), provided an increase in the antiproliferative potency, probably by improving the hydrophilic/hydrophobic balance for cell penetration. Interestingly, such a property was strongly dependent on the linker size, as the longer imidazolidine-2-thione derivative 24f (n = 6) exhibited GI50 values > 100 µM for all the tested cell lines. Two of the tested compounds (21f, 24e) exhibited strong activity on the SW1573 cell line (GI50 = 9.7, 5.7 µM, respectively). Thiourea 21f also showed the best profile for the other five lines, with good GI50 values ranging from 23 to 36 µM. Table 3. Antiproliferative activity (GI50, µM) of selected compounds (mean ± SD).

Docking Simulations
Docking studies shed light on the molecular interactions that could take place between compounds and the different hCA isoforms. Arylsulfonamide 9b and coumarin derivative 24h were selected for such studies.
D-Galacto-configured sulfonamide 9b was predicted to act as a zinc-chelating agent through its sulfonamido moiety (Figure 3). The deprotonated form of the sulfonamide interacts, through the NH moiety, with the Zn 2+ cation of CA XII. A hydrogen bond interaction is also established between Thr 198, Thr 199, and the sulfonamido scaffold. In the active form of the CA, Thr 198 is hydrogen bonded with the H2O/OHcoordinated with the zinc ion [50]. Although docking techniques do not allow the simulation of the displacement of water molecules, the interaction of 9b directly with Zn 2+ and the Thr 198 residue could explain the inhibitory effect towards the catalytic activity of the enzyme. The 2D-and 3D-predicted interactions of 9b and the active site of CA XII are depicted in Figure 3A and Figure 3B,  counterparts (21f and 24e), provided an increase in the antiproliferative potency, probably by improving the hydrophilic/hydrophobic balance for cell penetration. Interestingly, such a property was strongly dependent on the linker size, as the longer imidazolidine-2-thione derivative 24f (n = 6) exhibited GI50 values > 100 µM for all the tested cell lines. Two of the tested compounds (21f, 24e) exhibited strong activity on the SW1573 cell line (GI50 = 9.7, 5.7 µM, respectively). Thiourea 21f also showed the best profile for the other five lines, with good GI50 values ranging from 23 to 36 µM.

Docking Simulations
Docking studies shed light on the molecular interactions that could take place between compounds and the different hCA isoforms. Arylsulfonamide 9b and coumarin derivative 24h were selected for such studies.
D-Galacto-configured sulfonamide 9b was predicted to act as a zinc-chelating agent through its sulfonamido moiety (Figure 3). The deprotonated form of the sulfonamide interacts, through the NH moiety, with the Zn 2+ cation of CA XII. A hydrogen bond interaction is also established between Thr 198, Thr 199, and the sulfonamido scaffold. In the active form of the CA, Thr 198 is hydrogen bonded with the H2O/OHcoordinated with the zinc ion [50]. Although docking techniques do not allow the simulation of the displacement of water molecules, the interaction of 9b directly with Zn 2+ and the Thr 198 residue could explain the inhibitory effect towards the catalytic activity of the enzyme. The 2D-and 3D-predicted interactions of 9b and the active site of CA XII are depicted in Figure 3A and Figure 3B,  bly by improving the hydrophilic/hydrophobic balance for cell penetration. Interestingly, such a property was strongly dependent on the linker size, as the longer imidazolidine-2-thione derivative 24f (n = 6) exhibited GI50 values > 100 µM for all the tested cell lines. Two of the tested compounds (21f, 24e) exhibited strong activity on the SW1573 cell line (GI50 = 9.7, 5.7 µM, respectively). Thiourea 21f also showed the best profile for the other five lines, with good GI50 values ranging from 23 to 36 µM.

Docking Simulations
Docking studies shed light on the molecular interactions that could take place between compounds and the different hCA isoforms. Arylsulfonamide 9b and coumarin derivative 24h were selected for such studies.
D-Galacto-configured sulfonamide 9b was predicted to act as a zinc-chelating agent through its sulfonamido moiety (Figure 3). The deprotonated form of the sulfonamide interacts, through the NH moiety, with the Zn 2+ cation of CA XII. A hydrogen bond interaction is also established between Thr 198, Thr 199, and the sulfonamido scaffold. In the active form of the CA, Thr 198 is hydrogen bonded with the H2O/OHcoordinated with the zinc ion [50]. Although docking techniques do not allow the simulation of the displacement of water molecules, the interaction of 9b directly with Zn 2+ and the Thr 198 residue could explain the inhibitory effect towards the catalytic activity of the enzyme. The 2D-and 3D-predicted interactions of 9b and the active site of CA XII are depicted in Figure 3A and Figure 3B,   It has been widely reported that coumarins undergo hydrolysis at the entrance of the CA active site. For that reason, both open structures (E-and Z-configured) of the coumarin derivative 24h were considered in docking simulations [51,52].
As depicted in Table 4, the binding energy scores showed the enhanced interaction of the hydroxycinnamic forms compared to the coumarin one ("closed form"). Docking simulations (Figure 4) predict that the hydrolysed product of 24h is located inside of the binding pocket of CA IX, with the hydroxyl group interacting with Thr 201 and the carboxylate moiety interacting with Zn 2+ (E form). Moreover, the Z form only interacts with the Thr 200 and the prosthetic Zn 2+ cation through the carboxylate group. Similar interactions were seen in the CA XII isoform ( Figure 5).
It is worth noting the specific interaction of the Z stereoisomer with Thr 200 and Thr 198 residues in the CA IX and CAXII, respectively, compared to the E counterpart, which interacts with Thr 201 and Thr 199. Moreover, the position of the tail protrudes from the active site, suggesting a possible occlusion of the entrance of the enzyme and, therefore, reducing its catalytic activity.  It has been widely reported that coumarins undergo hydrolysis at the entrance of the CA active site. For that reason, both open structures (E-and Z-configured) of the coumarin derivative 24h were considered in docking simulations [51,52].
As depicted in Table 4, the binding energy scores showed the enhanced interaction of the hydroxycinnamic forms compared to the coumarin one ("closed form"). Docking simulations (Figure 4) predict that the hydrolysed product of 24h is located inside of the binding pocket of CA IX, with the hydroxyl group interacting with Thr 201 and the carboxylate moiety interacting with Zn 2+ (E form). Moreover, the Z form only interacts with the Thr 200 and the prosthetic Zn 2+ cation through the carboxylate group. Similar interactions were seen in the CA XII isoform ( Figure 5).
It is worth noting the specific interaction of the Z stereoisomer with Thr 200 and Thr 198 residues in the CA IX and CAXII, respectively, compared to the E counterpart, which interacts with Thr 201 and Thr 199. Moreover, the position of the tail protrudes from the active site, suggesting a possible occlusion of the entrance of the enzyme and, therefore, reducing its catalytic activity.

General Methods
The same general procedures concerning chromatography, NMR spectroscopy, and MS spectrometry as reported previously [53] were used.

General Methods
The same general procedures concerning chromatography, NMR spectroscopy, and MS spectrometry as reported previously [53] were used.

General Procedure for the Preparation of 7-Hidroxycoumarins via Pechmann Condensation
A mixture of 60% H 2 SO 4 (23 mL) and resorcinol (1.0 g, 9.08 mmol, 1.0 equiv.) was stirred at 0 • C for 5 min; then, the corresponding β-ketoester (1.1 equiv.) was slowly added at that temperature. After the addition was completed, the mixture was stirred at rt for 4 h; then, it was poured over a water/ice mixture and the resulting precipitate was filtrated and washed with cold H 2 O. Coumarins were purified by column chromatography (7:3 hexane-EtOAc).