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Article

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

by
Mónica Martínez-Montiel
1,2,
Laura L. Romero-Hernández
1,
Simone Giovannuzzi
3,
Paloma Begines
3,
Adrián Puerta
4,
Ana I. Ahuja-Casarín
1,
Miguel X. Fernandes
4,
Penélope Merino-Montiel
1,
Sara Montiel-Smith
1,
Alessio Nocentini
3,
José M. Padrón
4,
Claudiu T. Supuran
3,
José G. Fernández-Bolaños
2 and
Óscar López
2,*
1
Facultad de Ciencias Químicas, Ciudad Universitaria, Benemérita Universidad Autónoma de Puebla, Puebla 72570, PUE, Mexico
2
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Seville, Spain
3
NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, University of Florence, 50019 Florence, Italy
4
BioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Universidad de La Laguna, c/Astrofísico Francisco Sánchez 2, E-38206 La Laguna, Spain
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(11), 9401; https://doi.org/10.3390/ijms24119401
Submission received: 18 April 2023 / Revised: 14 May 2023 / Accepted: 16 May 2023 / Published: 28 May 2023

Abstract

:
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 14 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.

Graphical Abstract

1. 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 CO2, to furnish HCO3 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 106 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 membrane-anchored 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 (14, 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 14 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.

2. Results and Discussion

2.1. 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 5-hydroxy-imidazolidine-2-thione (Scheme 1). Such cyclization takes place through the nucleophilic attack of N-3 on the latent aldehyde moiety of the reducing sugar via a 5-exo-trig pathway according to Baldwin rules [33]. Such a compound was obtained in high stereoselectivity towards epimer 5R (98:2 5R:5S in DMSO-d6) starting from d-glucosamine [29] and towards 5S (4:96 5R:5S in CD3OD) starting from d-mannosamine [32]. Subsequent acid-catalysed cyclodehydration furnished (Scheme 1) the corresponding glucofurano-imidazolidine-2-thione [29] through a stabilized carbocation. As expected, the cyclization of the C-5 hydroxyl group on the carbocation to furnish a 6,5-bicyclic system was not observed, according to previous analogous compounds [29,32].
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 6ac with d-glucosamine hydrochloride in the presence of NaHCO3 afforded thioureas 3ac. The preparation of targeted bicyclic derivatives 8ac 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).
Compounds 8ac showed an E4 conformation in the carbohydrate residue, as evidenced by vicinal coupling constants (e.g., J1,2 = 6.0 Hz, J2,3~0 Hz, J3,4 = 2.2 Hz for 8a). Accordingly, H-1 and H-2 adopt a relative cis arrangement, giving a J1,2 significantly higher than expected for an α-anomer. Moreover, H-2 and H-3 are arranged with a dihedral angle close to 90°, and the exocyclic dihydroxyethyl chain exhibits conformational flexibility. 13C-NMR resonances at roughly 180 (CS) and 95 (C-1) ppm further confirmed the proposed structures. These data are in agreement with analogous pseudonucleosides [29].
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 appendage of coumarins was accomplished on the C-7 position.
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 N-deprotection (NaOH). Alternatively, the coupling of 11 and d-glucosamine hydrochloride in the presence of NaHCO3, 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.
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, CH3, 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,ω-dibromoalkanes under basic conditions (K2CO3) to furnish ω-bromoalkyl derivatives 17ai. Subsequent nucleophilic displacement with NaN3, Pd/C-catalysed hydrogenolysis, and isothiocyanation reaction with thiophosgene afforded isothiocyanate derivatives 20ai in good overall yields (Scheme 5).
Finally, isothiocyanates 20ai were transformed with excellent yields into both linear thioureas 21e,f, and into bicyclic counterparts 24ai (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 bicyclic scaffold of imidazolidines 24 was again supported by 1H-NMR data; as an example, compound 24b exhibited J1,2 = 6.1 Hz, J2,3 ~ 0 Hz, J3,4 = 2.5 Hz.
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.

2.2. Biological Assessments

In Vitro Carbonic Anhydrase Inhibition

The panel of CA-directed compounds prepared herein were assessed in vitro against a series of isoforms of human CA with relevant therapeutic interest. Such compounds were: arylsulfonamido-derived imidazolidine-2-thiones 8ac, 9a,b, coumarin-derived imidazolidine-2-thiones 16, 24, 26, and C-2 glyco-thioureas 13, 21e,f. Activities were measured using the stopped-flow CO2 hydration assay, and were compared with previously reported [26,27] β-d-glycopyranosyl thioureas 1, 2, glyco-thioureas 3, 4, and the drug acetazolamide (AAZ).
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 tumour-associated 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).
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 (Ki = 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 (14). 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.
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:
  • For compounds lacking linkers, almost no difference in activities can be found for non-cyclic (13) and bicyclic (16) thioureas.
  • The insertion of a linker between the carbohydrate and the coumarin residues of byclicic structures proved to be benefitial for the inhibition of both membrane-bound enzymes (16 vs. 24ac).
  • 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.
  • Considering the effect of the substituents (n = 4), the observed order of activity is:
    • CA IX: R1 = Me, R2 = Cl (24h) > R1 = Me, R2 = H (24b) > R1 = Ph, R2 = H (24e). Indeed, compound 24h provided the strongest CA IX inhibitor of the series (Ki = 6.8 nM), roughly 3.8-fold stronger than AAZ.
    • CA XII: R1 = Me, R2 = H (24b) > R1 = Me, R2 = Cl (24h) > R1 = Ph, R2 = H (24e).
  • 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 Ki = 10.1 nM
  • Little differences in activity were found by changing the carbohydrate configuration (24c vs. 26).

2.3. 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 (GI50 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 (GI50 > 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 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.

2.4. 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 Zn2+ 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/OH- coordinated 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 Zn2+ 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, respectively.
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 Zn2+ (E form). Moreover, the Z form only interacts with the Thr 200 and the prosthetic Zn2+ 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.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Methods

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

3.1.2. General Procedure for the Preparation of Imidazolidine-2-Thiones 8ac, 9a,b

A mixture of commercially available d-glucosamine/galactosamine hydrochloride (1.0 equiv.), NaHCO3 (1.0 equiv.), and the corresponding isothiocyanate 6ac (1.2 equiv.) in 2:1 H2O–EtOH (6 mL) was heated at 75 °C. When the starting material was consumed as evidenced by TLC, AcOH was added (1.5 mL) and the corresponding mixture was refluxed for 2 h. Then, the crude reaction mixture was concentrated to dryness, and the residue was purified by column chromatography (CH2Cl2 → 10:1 CH2Cl2–MeOH).
1-(4′-Sulfonamidophenyl)-(1″,2″-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (8a). Isothiocyanate 6a (117.8 mg, 0.55 mmol, 1.2 equiv.), d-glucosamine hydrochloride (100.0 mg, 0.46 mmol, 1.0 equiv.), and NaHCO3 (38.6 mg, 0.46 mmol, 1.0 equiv.) were used. Compound 8a was obtained as a yellow oil. Yield: 118 mg (68%); [ α ] D 23 +54 (c 0.11, MeOH); 1H-NMR (300 MHz, CD3OD) δ 7.92 (m, 2H, Ar-H), 7.79 (m, 2H, Ar-H), 6.10 (d, 1H, J1″,2″ = 6.2 Hz, H-1″), 4.35 (d, 1H, J2″,3″ = 0, H-2″), 4.34 (d, 1H, J3″,4″ = 2.2 Hz, H-3″), 3.98 (ddd, 1H, J4″,5″ = 8.3 Hz, J5″,6a″ = 2.8 Hz, J5″,6b″ = 5.6 Hz, H-5″), 3.92 (dd, 1H, H-4″), 3.80 (dd, 1H, J6a″,6b″ = 11.4 Hz, H-6a″), 3.64 (dd, 1H, H-6b″) ppm (Figure S1); 13C-NMR (75.5 MHz, CD3OD) δ 183.1 (CS), 143.6, 142.6 (C-1′, C-4′), 127.8, 127.5 (C-2′/C-6′, C-3′/C-5′), 96.4 (C-1″), 81.0 (C-4″), 75.9 (C-3″), 70.1 (C-5″), 66.9 (C-2″), 65.1 (C-6″) ppm (Figure S2); HRESI-MS m/z calcd. for C13H17N3NaO6S2 ([M+Na]+): 398.0451, found: 398.0450.
1-(3′-Sulfonamidophenyl)-(1″,2″-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (8b). Isothiocyanate 6b (119 mg, 0.56 mmol, 1.2 equiv.), d-glucosamine hydrochloride (100.0 mg, 0.46 mmol, 1.0 equiv.), and NaHCO3 (38.6 mg, 0.46 mmol, 1.0 equiv.) were used. Compound 8b was obtained as a yellow oil. Yield: 106 mg (61%); [ α ] D 23   +35 (c 0.15, MeOH); 1H-NMR (300 MHz, CD3OD) δ 8.11 (t, 1H, J2′,4′=J2′,6′= 1.7 Hz, H-2′), 7.84 (ddd, 1H, JH,H = 1.1 Hz, JH,H = 1.4 Hz, J4′,5′ = 7.8 Hz, H-4′), 7.76 (ddd, 1H, JH,H = 1.1 Hz, JH,H = 1.9 Hz, J5′,6′ = 8.1 Hz, H-6′), 7.57 (t, 1H, H-5′), 6.06 (d, 1H, J1″,2″ = 6.3 Hz, H-1″), 4.38 (d, 1H, J2″,3″ = 0, H-2″), 4.36 (d, 1H, J3″,4″ = 2.3 Hz, H-3″), 4.02–3.93 (m, 2H, H-4″, H-5″), 3.83 (dd, 1H, J5″,6″a = 2.5 Hz, J6a″,6b″ = 11.4 Hz, H-6a″), 3.67 (dd, 1H, J5″,6b″ = 4.9 Hz, H-6″b) ppm (Figure S3); 13C-NMR (75.5 MHz, CD3OD) δ 183.1 (CS), 144.8, 140.3 (C-1′, C-3′), 132.2 (C-5′), 130.4 (C-6′), 126.0 (C-4′) 125.5 (C-2′), 96.2 (C-1″), 80.5 (C-4″), 75.8 (C-3″), 69.8 (C-5″), 66.9 (C-2″), 64.9 (C-6″) ppm (Figure S4); HRESI-MS m/z calcd. for C13H17N3NaO6S2 ([M+Na]+): 398.0451, found: 398.0447.
1-[2′-(4″-Sulfonamidophenyl)]ethyl-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (8c). Isothiocyanate 6c (200 mg, 0.83 mmol, 1.2 equiv.), d-glucosamine hydrochloride (148.0 mg, 0.69 mmol, 1.0 equiv.), and NaHCO3 (58.0 mg, 0.68 mmol, 1.0 equiv.) were used. Compound 8c was obtained as a white solid. Yield: 160 mg (57%); [ α ] D 23   +12 (c 0.62, MeOH); 1H-NMR (300 MHz, CD3OD) δ 7.84 (m, 2H, Ar-H), 7.47 (m, 2H, Ar-H), 5.69 (d, 1H, J1‴,2″″ = 6.6 Hz, H-1‴), 4.18 (d, 1H, J2‴,3‴ = 0, J3‴,4‴ = 2.5 Hz, H-3‴), 4.07 (d, 1H, H-2‴), 3.91 (ddd, 1H, J5‴,6a‴ = 3.1 Hz, J5‴,6‴b = 5.9 Hz, J4‴,5‴ = 8.7 Hz, H-5‴), 3.85–3.45 (m, 5H, H-4‴, H-6‴a, H-6‴b, CH2), 3.15 (m, 1H, CHA), 3.01 (m, 1H, CHB) ppm (Figure S5); 13C-NMR (75.5 MHz, CD3OD) δ 183.9 (CS), 145.2, 143.0 (C-1″, C-4″), 130.6, 127.3 (C-2″/C-6″, C-3″/C-5″), 94.7 (C-1‴), 80.8 (C-4‴), 76.2 (C-3‴), 70.2 (C-5‴), 66.4 (C-2‴), 65.1 (C-6″), 46.6 (N-CH2), 35.2 (CH2-Ar) ppm (Figure S6); ESI-MS m/z calcd. for C15H21N3NaO6S2 ([M+Na]+): 426.0764, found: 426.0760.
1-(4′-Sulfonamidophenyl)-(1″,2″-dideoxy-α-d-galactofurano)[2,1-d]imidazolidine-2-thione (9a). Isothiocyanate 6a (117.8 mg, 0.55 mmol, 1.2 equiv.), d-galactosamine hydrochloride (100.0 mg, 0.46 mmol, 1.0 equiv.), and NaHCO3 (38.6 mg, 0.46 mmol, 1.0 equiv.) were used. Compound 9a was obtained as a yellow oil. Yield: 115 mg (67%); [ α ] D 23 +87 (c 0.88, DMSO); 1H-NMR (300 MHz, CD3OD) δ 7.92 (m, 4H, Ar-H), 6.10 (d, 1H, J1″,2″ = 6.7 Hz, H-1″), 4.40 (brdd, 1H, J2″,3‴ = 1.3 Hz, J3″,4‴ = 2.8 Hz, H-3″), 4.32 (dd, 1H, H-2″), 4.03 (m, 1H, H-5″), 4.06–4.00 (m, 3H, H-4″, H-6a′′, H-6b″) ppm (Figure S7); 13C-NMR (125.7 MHz, CD3OD) δ 182.0 (CS), 143.7, 142.3 (C-1′, C-4′), 127.5, 127.4 (C-2′/C-6′, C-3′/C-5′), 96.8 (C-1″) 89.0 (C-4″), 77.7 (C-3″), 72.5 (C-5″), 68.1 (C-2″), 64.8 (C-6″) ppm (Figure S8).
1-(3′-Sulfonamidophenyl)-(1″,2″-dideoxy-α-d-galactofurano)[2,1-d]imidazolidine-2-thione (9b). Isothiocyanate 6b (117.8 mg, 0.55 mmol, 1.2 equiv.), d-galactosamine hydrochloride (100.0 mg, 0.46 mmol, 1.0 equiv.), and NaHCO3 (38.6 mg, 0.46 mmol, 1.0 equiv.) were used. Compound 9b was obtained as a yellow oil. Yield: 101 mg (58%); [ α ] D 23 +85 (c 0.65, MeOH); 1H-NMR (300 MHz, CD3OD) δ 8.25 (t, 1H, J2′,4′= J2′,6′= 1.9 Hz, H-2′), 7.92 (ddd, 1H, JH,H = 1.0 Hz, JH,H = 2.1 Hz, J4′,5′= 7.9 Hz, H-4′), 7.81 (ddd, 1H, JH,H = 1.2 Hz, JH,H = 1.9 Hz, J5′,6′= 8.0 Hz, H-6′), 7.56 (t, 1H, H-5′), 6.08 (d, 1H, J1″,2″ = 6.5 Hz, H-1″), 4.41 (m, 1, H-3″), 4.26 (dd, 1H, H-2″), 4.41 (dd, 1H, J2″,3‴ = 1.3 Hz, J3″,4‴= 2.8 Hz, H-3″), 4.36 (dd, 1H, J2″,3″ = 1.0 Hz, H-2″), 4.05 (m, 1H, H-5″), 3.70–3.58 (m, 3H, H-4″, H-6a″, H-6b″) ppm (Figure S9); 13C-NMR (125.7 MHz, CD3OD) δ 182.3 (CS), 145.1, 140.8 (C-1′, C-3′), 131.7 (C-5′), 130.2 (C-6′), 125.5, 125.2 (C-4′, C-2′), 96.9 (C-1″) 89.0 (C-4″), 77.8 (C-3″), 72.5 (C-5″), 68.1 (C-2″), 64.8 (C-6″) ppm (Figure S10); HRESI-MS m/z calcd. for C13H17N3NaO6S2 ([M+Na]+): 398.0451, found: 398.0448.

3.1.3. N-(Methyl 2-deoxy-α-d-Glucopyranosid-2-yl)-N′-(4-methyl-2′-oxo-2′H-chromen-7′-yl)thiourea (13)

A mixture of coumarin-derived isothiocyanate 11 (159.0 mg, 0.73 mmol, 1.0 equiv.) and methyl 2-amino-2-deoxy-α-d-glucopyranoside 12 (141.0 mg, 0.73 mmol, 1.0 equiv.) in 2:1 EtOH–H2O (5 mL) was heated at 60 °C for 24 h. Then, the crude reaction mixture was concentrated to dryness and the residue was purified by column chromatography (CH2Cl2 → 10:1 CH2Cl2–MeOH) to give 13. Yield: 214 mg (71%). 1H-NMR (500 MHz, DMSO-d6) δ 10.08 (s, 1H, NH′), 8.01 (d, 1H, J6′,8′ = 1.8 Hz, H-8′), 7.92 (d, 1H, JNH,2-Glc = 7.9 Hz, NH) 7.69 (d, 1H, J5′,6′ = 8.7 Hz, H-5′), 7.44 (dd, 1H, H-6′), 6.26 (s, 1H, H-3′), 5.12 (d, 1H, JOH,4 = 5.7 Hz, OH 4-Glc), 5.06 (d, 1H, JOH,3 = 5.8 Hz, OH 3-Glc), 4.84 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.60 (t, 1H, JOH,6 = 5.9 Hz, OH 6-Glc), 4.33 (m, 1H, H-2), 3.66 (ddd, 1H, J6a,6b = 11.8 Hz, J6a,OH = 5.7 Hz, J5,6a = 1.9 Hz, H-6a), 3.54 (m, 1H, H-3), 3.51 (dt, 1H, J6b,OH = J5,6b = 5.8 Hz, H-6b), 3.36 (m, 1H, H-5), 3.29 (s, 3H, OCH3), 3.22 (m, 1H, H-4), 2.40 (s, 3H, coumarin-CH3) ppm (Figure S11); 13C-NMR (125.7 MHz, DMSO-d6) δ 180.1 (CS), 160.1 (C-2′), 153.3 (C-9′), 153.2 (C-4′), 143.3 (C-7′), 125.5 (C-5′), 117.2 (C-6′), 115.0 (C-10′), 112.2 (C-3′), 107.3 (C-8′), 97.1 (C-1), 73.0 (C-5), 71.0 (C-3), 70.6 (C-4), 60.7 (C-6), 58.1 (C-2), 54.4 (OCH3), 18.1 (coumarin-CH3) ppm (Figure S12); HRESI-MS m/z calcd. for C18H22N2NaO7S ([M+Na]+): 433.1040, found: 433.1034.

3.1.4. 1-(4′-Methyl-2′-oxo-2′H-chromen-7′-yl)-(1″,2″-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (16)

A solution of coumarin-derived isothiocyanate 11 (215.1 mg, 0.99 mmol, 1.1 equiv.) in EtOH (5 mL) was added to a solution of d-glucosamine hydrochloride (195.0 mg, 0.90 mmol, 1.0 equiv.) and NaHCO3 (75.6 mg, 0.90 mmol, 1.0 equiv.) in a 3:1 EtOH-H2O mixture (5 mL). The resulting mixture was heated at 60 °C for 4 h; then, AcOH (154 µL, 2.7 mmol, 3.0 equiv.) was added, and refluxed for 2 h. After that, the crude reaction was concentrated to dryness and the residue was purified by column chromatography (CH2Cl2 → 10:1 CH2Cl2–MeOH) to give 16. Yield: 323 mg (95%). 1H-NMR (500 MHz, DMSO-d6) δ 9.47 (s, 1H, NH), 7.78 (d, 1H, J5′,6′ = 8.6 Hz, H-5′), 7.69 (d, 1H, J6′,8′ = 2.0 Hz, H-8′), 7.64 (dd, 1H, H-6′), 6.87 (brq, 1H, JCH3,H = 1.1 Hz, H-3′), 6.14 (d, 1H, J1,2 = 6.2 Hz, H-1″), 5.47 (d, JOH,3 = 4.9 Hz, 1H, OH 3-Glc), 4.80 (d, JOH,5 = 6.1 Hz, 1H, OH 5-Glc), 4.56 (t, JOH,6″ = 5.6 Hz, 1H, OH 6-Glc), 4.21 (d, 1H, J2″,3″ = 0 Hz, H-2″), 4.14 (dd, J3,OH = 4.8 Hz, J3,4 = 2.2 Hz, 1H, H-3″), 3.74 (m, 1H, H-5″), 3.68 (dd, 1H, J4″,5″ = 8.6 Hz, J3″,4″ = 2.2 Hz, H-4″), 3.58 (ddd, 1H, J6a″,6b″ = 11.3 Hz, J5″,6a″ = 2.6 Hz, H-6a″), 3.41 (ddd, 1H, J6b″,OH = J5″,6″b = 5.6 Hz, H-6″b), 2.43 (d, 3H, CH3) ppm (Figure S13); 13C-NMR (125.7 MHz, DMSO-d6) δ 180.3 (CS), 159.9 (C-2′), 153.1 (C-4′), 152.7 (C-9′), 142.2 (C-7′), 125.2 (C-5′), 121.5 (C-6′), 117.3 (C-10′), 113.7 (C-3′), 112.7 (C-8′), 94.3 (C-1″), 79.7 (C-4″), 73.6 (C-3″), 68.1 (C-5″), 65.3 (C-2″), 63.7 (C-6″), 18.1 (CH3) ppm (Figure S14); HRESI-MS m/z calcd. for C17H18N2NaO6S ([M+Na]+): 401.0778, found: 401.0774.

3.1.5. General Procedure for the Preparation of 7-Hidroxycoumarins via Pechmann Condensation

A mixture of 60% H2SO4 (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 H2O. Coumarins were purified by column chromatography (7:3 hexane–EtOAc).

3.1.6. General Procedure for the O-Alkylation of 7-Hydroxycoumarins with α,ω-Dibromoalkanes (17ai)

To a solution of 7-hydroxycoumarins (1.0 equiv.) in dry MeCN (7 mL) was added anhydrous K2CO3 (1.5 equiv.) and the corresponding α,ω-dibromoalkanes (10.0 equiv.); the reaction mixture was heated under Ar at 65 °C for 2 h. Then, it was concentrated to dryness and the residue was purified by column chromatography (hexane→9:1 hexane–EtOAc).

3.1.7. General Procedure for the Preparation of Azides 18ai

To a solution of bromoderivatives 17ai (500 mg, 1.0 equiv.) in DMF (15 mL) was added NaN3 (5.0 equiv.) and the corresponding mixture was stirred at rt for 2 h. Then, it was diluted with EtOAc (20 mL) and washed with brine (4 × 15 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. The residue was purified by column chromatography (2:1 hexane-EtOAc) to give azidoderivatives 18ai.
7-(3′Azidopropoxy)-4-phenylcoumarin (18d). Bromoderivative 17d (500 mg, 1.39 mmol) and NaN3 (453 mg, 6.97 mmol) were used. Yield: 400 mg (90%, solid). Mp: 67 °C; 1H-NMR (500 MHz, CDCl3) δ 7.43 (m, 3H, Ar-H), 7.36 (m, 2H, Ar-H), 7.31 (d, 1H, J5,6 = 8.9 Hz, H-5), 6.80 (s, 1H, H-8), 6.71 (d, 1H, H-6), 6.14 (s, 1H, H-3), 4.04 (m, 2H, CH2), 3.72 (m, 2H, CH2), 1.98 (m, 2H, CH2) ppm (Figure S15); 13C-NMR (125.7 MHz, CDCl3) δ 160.7 (C-7), 160.2 (C-2), 154.8 (C-9), 154.7 (C-4), 134.41 (Ar-Cipso), 128.6 (Ar-Cp), 127.8 (x2) (Ar-Cm), 127.3 (x2) (Ar-Co), 127.0 (C-5), 111.6 (C-6), 111.4 (C-10), 110.6 (C-3), 100.6 (C-8), 64.0 (C-1′), 46.9 (C-3′), 27.4 (C-2′) (Figure S16); HRESI-MS m/z calcd. for C18H15N3NaO3 ([M+Na]+): 344.1006, found: 344.1003.
7-(4′Azidobutoxy)-4-phenylcoumarin (18e). Bromoderivative 17e (500 mg, 1.34 mmol) and NaN3 (435.6 mg, 6.70 mmol) were used. Yield: 435 mg (97%, solid). Mp: 35–36 °C; 1H-NMR (500 MHz, CDCl3) δ 7.52 (m, 3H, Ar-H), 7.44 (m, 2H, Ar-H), 7.38 (d, 1H, J5,6 = 8.9 Hz, H-5), 6.87 (d, 1H, J6,8 = 2.5 Hz, H-8), 6.79 (dd, 1H, H-6), 6.21 (s, 1H, H-3), 4.07 (t, 2H, JH,H = 6.1 Hz, H-1′), 3.39 (t, 2H, JH,H = 6.7 Hz, H-4′), 1.93 (m, 2H, H-2′), 1.81 (m, 2H, H-3′) ppm (Figure S17); 13C-NMR (125.7 MHz, CDCl3) δ 162.1 (C-7), 161.3 (C-2), 156.1 (C-9), 155.9 (C-4), 135.6 (Ar-Cipso), 129.7 (Ar-Cp), 128.9 (x2) (Ar-Cm), 128.5 (x2) (Ar-Co), 128.1 (C-5), 112.7 (C-6), 112.6 (C-10), 111.9 (C-3), 101.6 (C-8), 67.9 (C-1′), 51.2 (C-4′), 26.4 (C-2′), 25.7 (C-3′) ppm (Figure S18).
7-(3′-Azidopropoxy)-3-chloro-4-methylcoumarin (18g). Bromoderivative 17g (500 mg, 1.51 mmol) and NaN3 (490.8 mg, 7.55 mmol) were used. Yield: 390 mg (88%, solid). Mp: 62 °C; 1H NMR (300 MHz, CDCl3) δ 7.55 (d, 1H, J5,6 = 8.9 Hz, H-5), 6.92 (dd, 1H, J6,8 = 2.5 Hz, H-6), 6.85 (d, 1H, H-8), 4.14 (t, 2H, JH,H = 6.1 Hz, H-1′), 3.55 (t, 2H, H-1′), 2.12 (s, 3H, CH3), 1.93 (s, 1H, H-3′), 1.82 (s, 1H, H-2′) ppm (Figure S19); 13C-NMR (125.7 MHz, CDCl3) δ 161.5 (C-7), 157.3 (C-2), 153.1 (C-9), 147.8 (C-4), 125.9 (C-5), 117.9 (C-3), 113.5 (C-10), 113.1 (C-6), 101.4 (C-8), 65.2 (C-1′), 48.0 (C-3′) 30.1 (C-2′), 16.7 (CH3) ppm (Figure S20); HRESI-MS m/z calcd. for C13H12ClN3NaO3 ([M+Na]+): 316.0459, found: 316.0456.
7-(4′-Azidobutoxy)-3-chloro-4-methylcoumarin (18h). Bromoderivative 17h (500 mg, 1.45 mmol) and NaN3 (471.3 mg, 7.25 mmol) were used. Yield: 387 mg (87%, solid). Mp: 50–51 °C; 1H-NMR (500 MHz, CDCl3) δ 7.52 (d, 1H, J5,6 = 8.9 Hz, H-5), 6.90 (dd, 1H, J6,8 = 2.4 Hz, H-6), 6.80 (d, 1H, H-8), 4.06 (t, 2H, JH,H = 6.1 Hz, H-1′), 3.39 (t, 2H, J4′,3′ = 6.7 Hz, H-4′), 2.55 (s, 3H, CH3), 2.12 (s, 3H, CH3), 1.93 (m, 2H, H-2′), 1.82 (m, 2H, H-3′) ppm (Figure S21); 13C-NMR (125.7 MHz, CDCl3) δ 161.9 (C-7), 157.6 (C-2), 153.2 (C-9), 148.1 (C-4), 126.0 (C-5), 117.9 (C-3), 113.4 (C-10), 113.4 (C-6), 101.3 (C-8), 68.0 (C-1′), 51.2 (C-4′), 26.4 (C-2′), 25.7 (C-3′), 16.3 (CH3) ppm (Figure S22).

3.1.8. General Procedure for the Preparation of Amines 19ai

A mixture of the corresponding azide 18ai (500 mg) and Pd/C (50 mg) in 1:1 THF–MeOH (10 mL) was hydrogenated at 1 atm and rt for 2 h. Then, it was filtrated over a Celite® pad, the filtrate was concentrated to dryness, and the residue was used directly for the next step without any further purification.

3.1.9. General Procedure for the Preparation of Isothiocyanates 20ai

To a solution of aminocoumarins 19ai (500 mg, 1.0 equiv.) in CH2Cl2 (6 mL) was added Et3N (2.0 equiv.) and heated at 35 °C for 10 min. Then, it was cooled down to rt, and thiophosgene (3.5 equiv.) was dropwise added and heated at 35 °C for further 20 min. The crude reaction medium was concentrated to roughly half volume, furnishing an orange precipitate, which was filtrated through a Celite® pad and washed with CH2Cl2. The filtrate was concentrated to dryness and the residue was purified by column chromatography (4:1 hexane–EtOAc).
4-Phenyl-7-(3′-isothiocyanatopropoxy)coumarin (20d). Aminocoumarin 19d (500 mg, 1.69 mmol), Et3N (0.47 mL, 3.38 mmol, 2.0 equiv.), thiophosgene (0.45 mL, 5.92 mmol, 3.5 equiv.) were used. Compound 20d was obtained as a white solid. Yield: 372 mg (65%). Mp: 127 °C; 1H-NMR (300 MHz, CDCl3) δ 7.45 (m, 3H, Ar-H), 7.38 (m, 2H, Ar-H), 7.34 (d, 1H, J5,6 = 9.0 Hz, H-5), 6.81 (d, 1H, J6,8 = 2.5 Hz, H-8), 6.75 (dd, 1H, H-6), 6.12 (s, 1H, H-3), 4.09 (m, 2H, H-1′), 3.73 (m 2H, H-3′), 2.13 (m, 2H, H-2′) ppm (Figure S23); 13C-NMR (125.7 MHz, CDCl3) δ 161.9 (C-7), 161.1 (C-2), 160.1 (C-9), 154.6 (C-4), 152.3 (Ar-Cipso), 130.6 (NCS), 129.8 (Ar-Cp), 128.3 (x2) (Ar-Cm), 128.1 (x2) (Ar-Co), 127.6 (C-5), 113.2 (C-10), 111.8 (C-6), 111.2 (C-3), 100.9 (C-8), 64.5 (C-1′), 41.6 (C-3′), 35.8 (C-2′) ppm (Figure S24); HRESI-MS m/z calcd. for C19H15NNaO3S ([M+Na]+): 360.0665, found: 360.0661.
4-Phenyl-7-(4′-isothiocyanatobutoxy)coumarin (20e). Aminocoumarin 19e (500 mg, 1.62 mmol), Et3N (0.45 mL, 3.24 mmol, 2.0 equiv.), thiophosgene (0.43 mL, 5.67 mmol, 3.5 equiv.) were used. Compound 20e was obtained as a white solid. Yield: 400 mg (70%). Mp: 141 °C; 1H-NMR (500 MHz, CDCl3) δ 7.52 (m, 3H, Ar-H), 7.44 (m, 2H, Ar-H), 7.39 (d, 1H, J5,6 = 8.9 Hz, H-5), 6.87 (d, 1H, J6,8 = 2.5 Hz, H-8), 6.79 (dd, 1H, H-6), 6.21 (s, 1H, H-3), 4.09 (t, 2H, JH,H = 5.5 Hz, H-1′), 3.64 (t, 2H, JH,H = 6.1 Hz, H-4′), 1.98 (m, 2H, H-2′), 1.94 (m, 2H, H-3′) ppm (Figure S25); 13C-NMR (125.7 MHz, CDCl3) δ 162.0 (C-7), 161.3 (C-2), 156.1 (C-9), 155.9 (C-4), 135.6 (Ar-Cipso), 130.6 (NCS), 129.7 (Ar-Cp), 128.9 (x2) (Ar-Cm), 128.5 (x2) (Ar-Co), 128.2 (C-5), 112.7 (C-10), 112.6 (C-6), 112.0 (C-3), 101.7 (C-8), 67.6 (C-1′), 44.9 (C-4′), 27.0 (C-3′), 26.3 (C-2′) ppm (Figure S26).
3-Chloro-7-(4′-isothiocyanatobutoxy)-4-methylcoumarin (20h). Aminocoumarin 19h (500 mg, 1.77 mmol), Et3N (0.49 mL, 3.54 mmol, 2.0 equiv.), thiophosgene (0.48 mL, 6.20 mmol, 3.5 equiv.) were used. Compound 20h was obtained as a white solid. Yield: 373 mg (65%). Mp: 62 °C; 1H-NMR (300 MHz, CDCl3) δ 7.45 (d, 1H, J5,6 = 8.9 Hz, H-5), 6.83 (dd, 1H, J6,8 = 2.4 Hz, H-6), 6.73 (d, 1H, H-8), 4.01 (t, 2H, JH,H = 6.1 Hz, H-1′), 3.59 (t, 2H, JH,H = 6.7 Hz, H-4′), 2.10 (s, 3H, CH3), 1.89 (m, 2H, H-2′), 1.22 (m, 2H, H-3′) ppm (Figure S27); 13C-NMR (125.7 MHz, CDCl3) δ 161.6 (C-7), 157.3 (C-2), 153.0 (C-9), 147.9 (C-4), 125.9 (C-5), 117.9 (C-3), 113.4 (C-10), 113.1 (C-6), 101.3 (C-8), 67.6 (C-1′), 44.8 (C-4′), 26.8 (C-2′), 26.1 (C-3′), 16.1 (CH3) ppm (Figure S28); HRESI-MS m/z calcd. for C15H14ClNNaO3S ([M+Na]+): 346.0275, found: 346.0272.

3.1.10. General Procedure for the Preparation of Coumarin-Derived Glycol-Thioureas 21e,f

To a solution of coumarin isothiocyanate 20e,f (1.0 equiv.) in EtOH at 60 °C was added a solution of methyl glycoside 12 (1.0 equiv.) and Et3N (0.50 mL, 3.60 mmol) in H2O. The resulting mixture was heated at 60 °C during the time indicated in each case. Then, the crude reaction was concentrated to dryness and the residue was purified by column chromatography (40:1 CH2Cl2–MeOH) to give thioureas 21e,f as white solids.
N-(Methyl 2-deoxy-α-d-glucopyranosid-2-yl)-N′-{4′-[7″-(4″-phenyl-2″-oxo-2″H-chromen-7″-yl)oxy]butyl}thiourea (21e). Isothiocyanate 20e (100 mg, 0.28 mmol) in EtOH (15 mL), methyl glycoside 12 (54.1 mg, 0.28 mmol, 1.0 equiv.), and Et3N (0.50 mL, 3.6 mmol, 12.9 equiv.) in H2O (5 mL) were used. Reaction proceeded for 8.5 h. Yield: 120 mg (79%). [ α ] D 23 +66 (c 0.15, DMSO); mp: 165 °C; 1H-NMR (300 MHz, DMSO-d6–(CD3)2CO) δ 7.94 (m, 1H, NH), 7.63–7.46 (m, 5H, Ar-H Ph), 7.35 (d, 1H, J5″,6″ = 8.9 Hz, H-5″), 7.16 (brs, 1H, NH), 7.09 (d, 1H, J6″,8″ = 2.4 Hz, H-8″), 6.94 (dd, 1H, H-6″), 6.23 (s, 1H, H-3″), 5.01 (brs, 1H, OH), 4.87 (brs, 1H, OH), 4.75 (brd, 1H, J1,2 = 3.4 Hz, H-1), 4.52 (brt, 1H, JOH,6 = 5.6 Hz, OH 6-Glc), 4.21 (brs, 1H, H-2), 4.13 (t, 1H, JH,H = 6.4 Hz, CH2), 3.65 (dd, 1H, J5,6a = 4.6 Hz, J6a,6b = 11.3 Hz, H-6a), 3.51–3.46 (m, 2H, H-6b, H-3), 3.28 (m, 1H, H-5), 3.24 (s, 3H, OMe), 3.17 (m, 1H, H-4), 1.77 (quint, 2H, JH,H = 6.8 Hz, CH2), 1.64 (quint, 2H, JH,H = 6.5 Hz, CH2) ppm (Figure S29); 13C-NMR (75.5 MHz, DMSO-d6) δ 161.9, 160.0 (C-2″, C-7″), 155.5, 155.2 (C-4″, C-9″), 135.0 (Ar-Cipso, Ph) 129.6, 129.2, 128.8, 128.5, 128.4, 127.8 (Ar-C), 112.7, 111.7, 111.2, (C-3″, C-6″, C-10″), 101.6 (C-8″), 97.6 (C-1), 72.8 (C-5), 70.7 (C-3, C-4), 68.1 (CH2O), 60.8 (C-6), 54.3 (OMe), 25.9, 25.4 (CH2) ppm (Figure S30); HRESI-MS m/z calcd. for C27H32N2NaO8S ([M+Na]+): 567.1772, found: 567.1769.
N′-(Methyl 2-deoxy-α-d-glucopyranosid-2-yl)-N′-{6′-[7″-(4″-phenyl-2″-oxo-2″H-chromen-7″-yl)oxy]hexyl}thiourea (21f). Isothiocyanate 20f (120 mg, 0.32 mmol) in EtOH (20 mL), methyl glycoside 12 (61.8 mg, 0.32 mmol, 1.0 equiv.), and Et3N (0.50 mL, 3.6 mmol, 11.3 equiv.) in H2O (7 mL) were used. Reaction proceeded for 17.5 h. Yield: 160 mg (87%). [ α ] D 23 +58 (c 0.11, DMSO); mp: 148 °C; 1H-NMR (300 MHz, DMSO-d6) δ 7.55 (m, 6H, Ar-H, NH), 7.34 (d, 1H, J5″,6″ = 8.9 Hz, H-5″), 7.12 (brs, 1H, NH), 7.08 (d, 1H, J6″,8″= 2.4 Hz, H-8″), 6.93 (dd, 1H, H-6″), 6.23 (s, 1H, H-3″), 5.01 (d, 1H, JOH,4 = 5.5 Hz, OH 4-Glc), 4.85 (brs, OH 3-Glc), 4.74 (d, 1H, J1,2 = 3.4 Hz, H-1), 4.54 (t, 1H, JOH,6 = 5.8 Hz, OH 6-Glc), 4.19 (brs, 1H, H-2), 4.09 (t, 2H, JH,H= 6.6 Hz, OCH2), 3.64 (dd, 1H, J5,6a = 5.2 Hz, J6a,6b= 11.3 Hz, H-6a), 3.49 (dd, 1H, J5,6b = 5.5 Hz, H-6b), 3.44 (m, 1H, H-3), 3.29 (m, 1H, H-5), 3.17 (m, 1H, H-4), 3.23 (s, 3H, OCH3), 1.75 (quint, 2H, JH,H= 6.8 Hz, CH2), 1.54–1.34 (m, 6H, 3CH2) ppm (Figure S31); 13C-NMR (75.5 MHz, DMSO-d6) δ 182.7 (CS), 162.0 (C-2″), 160.0 (C-7″), 155.5 (C-9″), 155.2 (C-4″), 135.0 (Ar-Cipso, Ph), 129.7 (Ar-Cp, Ph), 128.9 (Ar-C, Ph), 128.4 (Ar-C, Ph), 127.8 (C-5″), 112.8 (C-6″) 111.7 (C-10″), 111.2 (C-3″), 101.6 (C-8″), 97.7 (C-1), 72.8 (C-5), 71.3 (C-3), 70.8 (C-4), 68.3 (OCH2), 60.8 (C-6), 58.0 (C-2), 54.3 (OCH3), 43.7 (N-CH2), 28.7, 28.4, 26.2, 25.2 (CH2) ppm (Figure S32); HRESI-MS m/z calcd. for C29H36N2NaO8S ([M+Na]+): 595.2085, found: 595.2079.

3.1.11. General Procedure for the Preparation of Coumarin-Derived Imidazolidine-2-Thiones 24ai, 26

The same procedure indicated for compound 16 was used (Section 3.1.4.) with isothiocyanates 20ai and d-glucosamine/galactosamine hydrochlorides.
1-{3′-[7″-(4″-Methyl-2″-oxo-2″H-chromen-7″-yl)oxy]propyl}-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (24a). d-Glucosamine hydrochloride (142 mg, 0.66 mmol, 1.0 equiv.), isothiocyanate 20a (200 mg, 0.73 mmol, 1.1 equiv.), NaHCO3 (55 mg, 0.66 mmol, 1.0 equiv.), and AcOH (114 µL, 1.99 mmol, 3.0 equiv.) were used. Compound 24a was obtained as a white solid. Yield: 256 mg (89%). [ α ] D 23 +25 (c 0.52, DMSO); mp: 151 °C; 1H-NMR (300 MHz, DMSO-d6) δ 8.69 (s, 1H, NH), 7.68 (d, J5″,6″ = 8.6 Hz, 1H, H-5″), 6.97 (m, 2H, H-6″, H-8″), 6.20 (brq, 1H, J3″,CH3 = 1.1 Hz, H-3″), 5.80 (d, 1H, J1‴,2‴ = 6.5 Hz, H-1‴), 5.34 (d, 1H, JH,H= 4.5 Hz, OH), 4.75 (m, 1H, OH), 4.45 (brt, 1H, JH,H= 5.6 Hz, OH 6-Glc), 4.39 (m, 1H), 4.11 (t, 2H, JH,H= 6.7 Hz, OCH2), 4.02–4.00 (m, 1H, H-3‴), 3.99 (d, 1H, H-2‴), 3.72–3.62 (m, 3H, H-6a‴, CH2), 3.60 (brdd, 1H, J5‴,6‴b= 4.2 Hz, J6a‴,6b‴= 11.7 Hz, H-6b‴), 3.38 (m, 1H), 2.39 (d, 3H, CH3), 2.08 (m, 2H, CH2) ppm (Figure S33); 13C-NMR (75.5 MHz, DMSO-d6) δ 181.7 (CS), 161.7, 160.2 (C-2″, C-7″), 154.7, 153.4 (C-4″, C-9″), 126.4 (C-5″), 113.1, 112.5, 111.1 (C-3″, C-6″, C-10″), 101.2 (C-8″), 92.6 (C-1‴), 79.3 (C-4‴), 73.9 (C-3‴), 68.1 (C-5‴), 66.2, 64.6 (C-2‴, OCH2), 63.8 (C-6‴), 41.1 (N-CH2), 29.0 (CH2), 18.1 (CH3) ppm (Figure S34); HRESI-MS m/z calcd. for C20H24N2NaO7S ([M+Na]+): 459.1196, found: 459.1194.
1-{4′-[7″-(4″-Methyl-2″-oxo-2″H-chromen-7″-yl)oxy]butyl}-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (24b). d-Glucosamine hydrochloride (112 mg, 0.52 mmol, 1.0 equiv.), isothiocyanate 20b (200 mg, 0.69 mmol, 1.3 equiv.), NaHCO3 (44 mg, 0.52 mmol, 1.0 equiv.), and AcOH (111 µL, 1.94 mmol, 3.7 equiv.) were used. Compound 24b was obtained as a white solid. Yield: 203 mg (87%). [ α ] D 23   +47 (c 0.35, DMSO); mp: 128 °C; 1H-NMR (300 MHz, DMSO-d6) δ 8.69 (s, 1H, NH), 7.66 (d, J5″,6″ = 9.2 Hz, 1H, H-5″), 7.68 (m, 1H, NH), 6.95 (m, 2H, H-6″, H-8″), 6.20 (brq, 1H, J3″,CH3 = 1.1 Hz, H-3″), 5.78 (d, J1‴,2‴ = 6.5 Hz, 1H, H-1‴), 5.26 (d, 1H, JH,H = 5.1 Hz, OH), 4.69 (d, 1H, JH,H= 6.1 Hz, OH), 4.43 (t, 1H, JH,H = 5.6 Hz, OH 6-Glc), 4.10 (m, 2H, OCH2), 4.01 (d, 1H, J3‴,4‴= 2.5 Hz, H-3‴), 3.98 (d, 1H, J2‴,3‴= 0 Hz, H-2‴), 3.72 (m, 1H, H-5‴), 3.58 (m, 3H, H-6‴a, N-CH2), 3.39–3.35 (m, 2H, H-4‴, H-6b‴), 2.39 (d, 3H, CH3), 1.74 (m, 4H, 2CH2) ppm (Figure S35); 13C-NMR (75.5 MHz, DMSO-d6) δ 181.6 (CS), 161.7 (C-2″), 160.2 (C-7″), 154.7, 153.4 (C-4″, C-9″), 126.4 (C-5″), 113.0 (C-6″), 112.4 (C-10″), 111.0 (C-3″), 101.1 (C-8″), 92.4 (C-1‴), 79.3 (C-4‴), 74.0 (C-3‴), 68.2, 68.0 (C-5‴, OCH2), 64.6 (C-2‴), 63.8 (C-6‴), 43.5 (N-CH2), 25.8, 24.3 (CH2), 18.1 (CH3) ppm (Figure S36); HRESI-MS m/z calcd. for C21H26N2NaO7S ([M+Na]+): 473.1353, found: 473.1354.
1-{6′-[7″-(4″-Methyl-2″-oxo-2″H-chromen-7″-yl)oxy]hexyl}-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (24c). d-Glucosamine hydrochloride (123 mg, 0.57 mmol, 1.0 equiv.), isothiocyanate 20c (200 mg, 0.63 mmol, 1.1 equiv.), NaHCO3 (48 mg, 0.57 mmol, 1.0 equiv.), and AcOH (102 µL, 1.78 mmol, 3.1 equiv.) were used. Compound 24c was obtained as a white solid. Yield: 250 mg (91%). [ α ] D 23   +77 (c 0.15, DMSO); mp: 87 °C; 1H-NMR (300 MHz, DMSO-d6) δ 8.67 (s, 1H, NH), 7.58 (m, 1H, H-5″), 6.96 (m, 2H, H-6″, H-8″), 6.21 (brq, 1H, J3″,CH3 = 1.1 Hz, H-3″), 5.79 (d, 1H, J1‴,2‴ = 6.5 Hz, H-1‴), 5.24 (d, 1H, JH,H = 4.7 Hz, OH), 4.68 (d, 1H, JH,H= 6.5 Hz, OH), 4.43 (t, 1H, JH,H = 5.8 Hz, OH 6-Glc), 4.01 (d, 1H, J3‴,4‴ = 2.4 Hz, H-3‴), 3.98 (d, 1H, J2‴,3‴= 0 Hz, H-2‴), 3.72 (m, 1H, H-5‴), 3.54 (m, 3H, H-6‴a, N-CH2), 3.37 (m, 2H, H-4‴, H-6‴b), 2.40 (d, 3H, CH3), 1.66 (m, 6H, 3CH2) ppm (Figure S37); 13C-NMR (75.5 MHz, DMSO-d6) δ 181.6 (CS), 161.8 (C-2″), 160.1 (C-7″), 154.7, 153.4 (C-4″, C-9″), 126.4 (C-5″), 113.0 (C-6″), 112.4 (C-10″), 111.0 (C-3″), 101.2 (C-8″), 92.3 (C-1‴), 79.3 (C-4‴), 74.0 (C-3‴), 68.2, 68.0 (C-5‴, OCH2), 64.6 (C-2‴), 63.8 (C-6‴), 30.7, 25.8, 24.2 (3CH2), 18.1 (CH3) ppm (Figure S38); HRESI-MS m/z calcd. for C23H30N2NaO7S ([M+Na]+): 501.1666, found: 501.1658.
1-{3′-[7″-(4″-Phenyl-2″-oxo-2″H-chromen-7″-yl)oxy]propyl}-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (24d). d-Glucosamine hydrochloride (116 mg, 0.54 mmol, 1.0 equiv.), isothiocyanate 20d (200 mg, 0.59 mmol, 1.1 equiv.), NaHCO3 (45 mg, 0.54 mmol, 1.0 equiv.), and AcOH (97 µL, 1.70 mmol, 3.1 equiv.) were used. Compound 24d was obtained as a white solid. Yield: 142 mg (53%). [ α ] D 23   +53 (c 0.13, DMSO); mp: 136 °C; 1H-NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H, NH), 7.57 (m, 5H, Ar-H) 7.35 (d, 1H, J5″,6″ = 8.7 Hz, H-5″), 7.08 (d, 1H, J6″,8″ = 2.2 Hz, H-8″), 6.94 (dd, 1H, H-6″), 6.23 (s, 1H, H-3″), 5.79 (d, 1H, J1‴,2‴ = 6.7 Hz, H-1‴), 5.27 (d, 1H, JH,H= 4.8 Hz, OH), 4.68 (d, 1H, JH,H = 6.2 Hz, OH), 4.43 (m, 1H, OH 6-Glc), 4.13 (m, 2H, OCH2), 4.00 (m, 2H, H-2‴, H-3‴), 3.67 (m, 4H, H-5‴, H-6‴a, CH2), 3.38 (m, 2H, H-6b‴, H-4‴) ppm (Figure S39); HRESI-MS m/z calcd. for C25H26N2NaO7S ([M+Na]+): 521.1353, found: 521.1345.
1-{4′-[7″-(4″-Phenyl-2″-oxo-2″H-chromen-7″-yl)oxy]butyl}-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (24e). d-Glucosamine hydrochloride (111 mg, 0.51 mmol, 1.0 equiv.), isothiocyanate 20e (200 mg, 0.57 mmol, 1.1 equiv.), NaHCO3 (42.8 mg, 0.51 mmol, 1.0 equiv.), and AcOH (93 µL, 1.63 mmol, 3.2 equiv.) were used. Compound 24e was obtained as a white solid. Yield: 244 mg (93%). [ α ] D 23   +70 (c 0.21, DMSO); 1H-NMR (300 MHz, DMSO-d6) δ 8.69 (s, 1H, NH), 7.55 (m, 5H, Ar-H, Ph) 7.34 (d, 1H, J5″,6″ = 8.6 Hz, H-5″), 7.08 (d, 1H, J6″,8″ = 2.4 Hz, H-8″), 6.94 (dd, 1H, H-6″), 6.23 (s, 1H, H-3″), 5.78 (d, 1H, J1‴,2‴ = 6.5 Hz, H-1‴), 5.24 (d, 1H, JH,H = 5.0 Hz, OH), 4.68 (d, 1H, JH,H = 6.1 Hz, OH), 4.43 (t, 1H, JH,H= 5.7 Hz, OH 6-Glc), 4.13 (brt, 2H, JH,H = 5.4 Hz, OCH2) 4.01 (d, 1H, J3‴,4‴= 2.3 Hz, H-3‴), 3.98 (d, J2‴,3‴ = 0 Hz, H-2‴), 3.72 (m, 1H, H-5‴), 3.59 (m, 3H, H-6‴a, N-CH2), 3.38 (m, 2H, H-6‴b, H-4‴), 1.75 (m, 4H, 2CH2) ppm (Figure S40); 13C-NMR (75.5 MHz, DMSO-d6) δ 181.7 (CS), 161.9, 160.0 (C-2″, C-7″), 155.5, 155.2 (C-4″, C-9″), 135.0 (Ar-Cipso, Ph), 129.6 (Ar-Cp, Ph), 128.6, 128.4 (Ar-Co, Ar-Cm, Ph), 127.8 (C-5″), 112.8, 111.7, 111.2 (C-3″, C-6″, C-10″), 101.6 (C-8″), 92.3 (C-1‴), 79.3 (C-4‴), 74.0 (C-3‴), 68.2, 68.1 (OCH2, C-5‴), 64.6 (C-2‴), 63.8 (C-6‴), 43.5 (C-4´) 25.8 (C-2´), 24.3 (C-3´) ppm (Figure S41); HRESI-MS m/z calcd. for C26H28N2NaO7S ([M+Na]+): 535.1509, found: 535.1509.
1-{6′-[7″-(4″-Phenyl-2″-oxo-2″H-chromen-7″-yl)oxy]hexyl}-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (24f). d-Glucosamine hydrochloride (100 mg, 0.46 mmol, 1.0 equiv.), isothiocyanate 20f (200 mg, 0.53 mmol, 1.2 equiv.), NaHCO3 (38.6 mg, 0.46 mmol, 1.0 equiv.), and AcOH (83 µL, 1.45 mmol, 2.6 equiv.) were used. Compound 24f was obtained as a white solid. Yield: 216 mg (87%). [ α ] D 23   +70 (c 0.21, DMSO); 1H-NMR (300 MHz, DMSO-d6) δ 8.63 (s, 1H, NH), 7.56 (m, 5H, Ar-H, Ph), 7.34 (d, 1H, J5″,6″ = 8.9 Hz, H-5″), 7.08 (d, 1H, J6″,8″ = 2.4 Hz, H-8″), 6.94 (dd, 1H, H-6″), 6.22 (s, 1H, H-3″), 5.76 (d, 1H, J1‴,2‴ = 6.5 Hz, H-1‴), 5.25 (d, 1H, JH,H = 4.9 Hz, OH), 4.69 (d, 1H, JH,H = 6.2 Hz, OH), 4.43 (t, 1H, JH,H = 5.7 Hz, OH 6-Glc), 4.08 (brt, 2H, JH,H= 6.4 Hz, OCH2), 4.00 (d, 1H, J3‴,4‴= 2.4 Hz, H-3‴), 3.97 (d, 1H, J2‴,3‴ = 0 Hz, H-2‴), 3.72 (m, 1H, H-5‴), 3.57 (m, 1H, H-6‴a), 3.45 (brt, JH,H = 7.1 Hz, N-CH2), 3.35 (m, 2H, H4‴, H-6‴b), 1.75 (quint, 2H, JH,H = 6.6 Hz, CH2), 1.66–1.29 (m, 6H, 3CH2) ppm (Figure S42); 13C-NMR (75.5 MHz, DMSO-d6) δ 181.5 (CS), 161.9, 160.0 (C-2″, C-7″), 155.4, 155.1 (C-4″, C-9″), 135.0 (Ar-Cipso, Ph), 129.5 (Ar-Cp, Ph), 128.8, 128.3 (Ar-Co, Ar-Cm, Ph), 127.7 (C-5″), 112.7, 111.6, 111.1 (C-3″, C-6″, C-10″), 101.6 (C-8″), 92.3 (C-1‴), 79.2 (C-4‴), 73.9 (C-3‴), 68.3, 68.2 (OCH2, C5‴), 64.4 (C-2‴), 63.8 (C-6‴), 43.7 (N-CH2), 28.3, 27.4, 25.9, 25.1 (4CH2) ppm (Figure S43); HRESI-MS m/z calcd. para C28H32N2NaO7S ([M+Na]+): 563.1822, found: 563.1811.
1-{4′-[7″-(3″-Chloro-4″-methyl-2″-oxo-2″H-chromen-7″-yl)oxy]butyl}-(1‴,2‴-dideoxy-α-d-glucofurano)[2,1-d]imidazolidine-2-thione (24h). d-Glucosamine hydrochloride (126 mg, 0.58 mmol, 1.0 equiv.), isothiocyanate 20h (200 mg, 0.62 mmol, 1.1 equiv.), NaHCO3 (48.7 mg, 0.58 mmol, 1.0 equiv.), and AcOH (105 µL, 1.84 mmol, 3.2 equiv.) were used. Compound 24h was obtained as a white solid. Yield: 214 mg (76%). [ α ] D 23   +45 (c 0.30, DMSO); mp: 127 °C; 1H-NMR (300 MHz, DMSO-d6) δ 8.58 (s, 1H, NH), 7.73 (m, 1H, H-5″), 7.00 (m, 2H, H-6″, H-8″), 5.78 (d, 1H, J1‴,2‴ = 6.8 Hz, H-1‴), 5.26 (d, 1H, JH,H= 4.4 Hz, OH), 4.69 (d, 1H, JH,H= 6.8 Hz, OH), 4.43 (t, 1H, JH,H= 5.7 Hz, OH 6-Glc), 4.11 (m, 2H, OCH2), 4.00 (d, 1H, J3‴,4‴= 2.3 Hz, H-3‴), 3.98 (d, 1H, J2‴,3‴= 0 Hz, H-2‴), 3.72 (m, 1H, H-5‴), 3.61–3.52 (m, 2H, H-6‴a, CH2), 3.39 (m, 2H, H-4‴, H-6‴b), 2.52 (s, 3H, CH3), 1.75 (m, 4H, 2CH2) ppm (Figure S44); 13C-NMR (125.7 MHz, DMSO-d6) δ 181.6 (CS), 161.7 (C-7″), 156.4 (C-2″), 152.6 (C-9″), 148.8 (C-4″), 126.9 (C-5″), 116.0 (C-3″), 113.0 (C-6″), 112.6 (C-10″), 101.1 (C-8″), 92.4 (C-1‴), 79.3 (C-4‴), 74.0 (C-3‴), 68.2 (x2) (C-5‴, OCH2), 64.6 (C-2‴), 63.8 (C-6‴), 43.5 (N-CH2), 25.8, 24.2 (CH2), 16.0 (CH3) ppm (Figure S45); HRESI-MS m/z calcd. for C21H25ClN2NaO7S ([M+Na]+): 507.0963, found: 507.0958.
1-{4′-[7″-(4″-Methyl-2″-oxo-2″H-chromen-7″-yl)oxy]hexyl}-(1‴,2‴-dideoxy-α-d-galactofurano)[2,1-d]imidazolidine-2-thione (26). d-Galactosamine hydrochloride (123 mg, 0.57 mmol, 1.0 equiv.), isothiocyanate 20c (200 mg, 0.63 mmol, 1.1 equiv.), NaHCO3 (47.9 mg, 0.57 mmol, 1.0 equiv.), and AcOH (102 µL, 1.78 mmol, 3.1 equiv.) were used. Compound 26 was obtained as a white solid. Yield: 223 mg (82%). [ α ] D 23   +46 (c 0.22, DMSO); 1H-NMR (500 MHz, DMSO-d6) δ 8.72 (s, 1H, NH), 7.67 (d, 1H, J5″,6″= 8.4 Hz, H-5″), 6.97 (m, 2H, H-6″, H-8″) 6.18 (br2, 1H, H-3″), 5.72 (d, 1H, J1‴,2‴ = 6.9 Hz, H-1‴), 4.06 (m, 4H, OCH2, H-2‴, H-3‴), 2.3 (brs, 3H, CH3), 1.74 (quint, 2H, JH,H= 6.9 Hz, CH2), 1.59, (m, 2H, CH2), 1.44 (m, 2H, CH2), 1.31 (m, 2H, CH2) ppm (Figure S46); 13C-NMR (125.7 MHz, DMSO-d6) δ 180.8 (CS), 162.0, 160.5 (C-2″, C-7″), 154.9, 153.8 (C-4″, C-9″), 126.7 (C-5″), 113.2, 112.7, 111.2 (C-6″, C-10″, C-3″), 101.3 (C-8″), 92.2 (C-1‴), 87.4 (C-4‴), 76.4 (C-3‴), 70.6 (C-5‴), 68.6 (OCH2). 66.0, 63.5 (C-2‴, C-6‴), 43.6 (N-CH2) 28.6, 27.1, 26.2, 25.4 (CH2), 18.1 (CH3) ppm (Figure S47); HRESI-MS m/z calcd. for C23H30N2NaO7S ([M+Na]+): 501.1666, found: 501.1661.

3.2. CA Inhibition Assays

The inhibitory properties of title compounds against CAs were determined using the stopped-flow CO2 hydrase assay, as previously reported [46]. All enzymes employed were recombinant and obtained in-house as reported, with concentrations in the assay ranging from 5 to 12 nM.

3.3. Antiproliferative Assays

Minor modifications of the US National Cancer Institute (NCI) protocol were used [49].

3.4. Docking Simulations

Structures for all proteins (CA IX: PDBid 5FL4; CA XII: PDBid 4HT2) were retrieved from the Protein DataBank. Crystal structures were optimized using QuickPrep protocol from MOE (Chemical Computing Group). All ligands were drawn, hydrogens added, and geometry optimized with MOE. To simulate conditions in the enzymatic environment, sulfonamide and open form of coumarin were deprotonated. For the docking calculations, ligands were placed in the area of co-crystalized ligand from pdb file. In the placement stage, we used the Triangle Matcher algorithm with the London dG scoring scheme. In the refinement stage, we kept the receptor rigid and used the GBVI/WSA dG scoring scheme.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24119401/s1.

Author Contributions

Conceptualization, Ó.L.; molecular docking and modelling, A.P., M.X.F. and J.M.P.; data analysis, A.N., M.X.F., J.M.P. and C.T.S.; synthesis and characterization: M.M.-M., L.L.R.-H. and A.I.A.-C.; biological assays, A.P., S.G., P.B. and J.M.P.; writing—original draft preparation, Ó.L.; writing—review and editing, A.I.A.-C., P.M.-M., S.M.-S., A.N., J.M.P., C.T.S., J.G.F.-B. and Ó.L.; supervision, P.M.-M., S.M.-S., A.N., M.X.F., J.M.P., C.T.S., J.G.F.-B. and Ó.L.; funding acquisition, J.M.P., C.T.S., J.G.F.-B. and Ó.L. All authors have read and agreed to the published version of the manuscript.

Funding

J.G.F.-B. and Ó.L. thank the Spanish Government (project PID2020-116460RB-I00 funded by MCIN/AEI/10.13039/501100011033) and Junta de Andalucía (FQM134) for financial support. A.P. and J.M.P. also thank the Spanish Government (Project PID2021-123059OB-I00 funded by MCIN/AEI /10.13039/501100011033/FEDER, UE) for financial support. This research was also funded by the Italian Ministry for University and Research (MIUR), grant PRIN: prot. 2017XYBP2R (CTS).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

P.B. thanks the Spanish University Ministry for the award of a Margarita Salas grant, funded by NextGenerationEU. A.P. thanks the EU Social Fund (FSE) and the Canary Islands ACIISI for a predoctoral grant TESIS2020010055. M.X.F. thanks FCT-Fundação para a Ciência e a Tecnologia support via UIDB/00674/2020 and UIDP/00674/2020 programs. We would also like to thank the Servicio de Resonancia Magnética Nuclear, CITIUS (University of Seville) for the performance of NMR experiments.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Reported flexible thiourea-containing glyco-sulfonamides.
Figure 1. Reported flexible thiourea-containing glyco-sulfonamides.
Ijms 24 09401 g001
Figure 2. General design of the novel conformationally restricted CA glycoconjugates.
Figure 2. General design of the novel conformationally restricted CA glycoconjugates.
Ijms 24 09401 g002
Scheme 1. Transformation of reducing sugar thioureas on C-2 into bicyclic-imidazolidine-2-thiones.
Scheme 1. Transformation of reducing sugar thioureas on C-2 into bicyclic-imidazolidine-2-thiones.
Ijms 24 09401 sch001
Scheme 2. Preparation of gluco-imidazolidine-2-thiones 8a–c derived from arylsulfonamides. Reactions and conditions: (a) aq. HCl, thiophosgene, rt (for 5a, 5b); DCC, CS2, Py, rt (for 5c); (b) d-Glucosamine·HCl, NaHCO3, 2:1 EtOH–H2O, 75 °C; (c) AcOH, 2:1 EtOH–H2O, 90 °C.
Scheme 2. Preparation of gluco-imidazolidine-2-thiones 8a–c derived from arylsulfonamides. Reactions and conditions: (a) aq. HCl, thiophosgene, rt (for 5a, 5b); DCC, CS2, Py, rt (for 5c); (b) d-Glucosamine·HCl, NaHCO3, 2:1 EtOH–H2O, 75 °C; (c) AcOH, 2:1 EtOH–H2O, 90 °C.
Ijms 24 09401 sch002
Scheme 3. Preparation of galacto-imidazolidine-2-thiones 9a,b derived from arylsulfonamides. Reactions and conditions: (a) d-Galactosamine·HCl, NaHCO3, 2:1 EtOH–H2O, 75 °C; (b) AcOH, 2:1 EtOH–H2O, 90 °C.
Scheme 3. Preparation of galacto-imidazolidine-2-thiones 9a,b derived from arylsulfonamides. Reactions and conditions: (a) d-Galactosamine·HCl, NaHCO3, 2:1 EtOH–H2O, 75 °C; (b) AcOH, 2:1 EtOH–H2O, 90 °C.
Ijms 24 09401 sch003
Scheme 4. Preparation of thiourea 13 and glucofurano-imidazolidine-2-thione 16 derived from coumarins. Reactions and conditions: (a) CSCl2, CH2Cl2, Et3N, 35 °C; (b) 12, 2:1 EtOH–H2O; (c) d-Glucosamine, NaHCO3, 3:1 EtOH–H2O, 60 °C; (d) AcOH, 3:1 EtOH–H2O, reflux.
Scheme 4. Preparation of thiourea 13 and glucofurano-imidazolidine-2-thione 16 derived from coumarins. Reactions and conditions: (a) CSCl2, CH2Cl2, Et3N, 35 °C; (b) 12, 2:1 EtOH–H2O; (c) d-Glucosamine, NaHCO3, 3:1 EtOH–H2O, 60 °C; (d) AcOH, 3:1 EtOH–H2O, reflux.
Ijms 24 09401 sch004
Scheme 5. Preparation of coumarin isothiocyanates 20ai. Reactions and conditions: (a) β-Ketoesters, 60% H2SO4, 0 °C; (b) α,ω-Dibromoalkanes, K2CO3, anh. CH3CN, 65 °C; (c) NaN3, DMF, rt; (d) H2, Pd/C, MeOH-THF, rt; (e) Et3N, CSCl2, CH2Cl2, 35 °C.
Scheme 5. Preparation of coumarin isothiocyanates 20ai. Reactions and conditions: (a) β-Ketoesters, 60% H2SO4, 0 °C; (b) α,ω-Dibromoalkanes, K2CO3, anh. CH3CN, 65 °C; (c) NaN3, DMF, rt; (d) H2, Pd/C, MeOH-THF, rt; (e) Et3N, CSCl2, CH2Cl2, 35 °C.
Ijms 24 09401 sch005
Scheme 6. Preparation of thioureas 21e,f and gluco-imidazolidine-2-thiones 24ai derived from coumarins. Reactions and conditions: (a) 2:1 EtOH–H2O, 60 °C; (b) NaHCO3, 2:1 EtOH–H2O, 60 °C; (c) AcOH, 2:1 EtOH–H2O, 60 °C.
Scheme 6. Preparation of thioureas 21e,f and gluco-imidazolidine-2-thiones 24ai derived from coumarins. Reactions and conditions: (a) 2:1 EtOH–H2O, 60 °C; (b) NaHCO3, 2:1 EtOH–H2O, 60 °C; (c) AcOH, 2:1 EtOH–H2O, 60 °C.
Ijms 24 09401 sch006
Scheme 7. Preparation of galacto-imidazolidine-2-thione 26 derived from coumarin. Reactions and conditions: (a) NaHCO3, EtOH, 60 °C; (b) AcOH, EtOH, 60 °C.
Scheme 7. Preparation of galacto-imidazolidine-2-thione 26 derived from coumarin. Reactions and conditions: (a) NaHCO3, EtOH, 60 °C; (b) AcOH, EtOH, 60 °C.
Ijms 24 09401 sch007
Figure 3. Predicted binding mode of 9b and CA XII. (A) Two-dimensional view of main residues involved in the ligand–protein interactions. (B) Three-dimensional structure of CA XII showing the binding site.
Figure 3. Predicted binding mode of 9b and CA XII. (A) Two-dimensional view of main residues involved in the ligand–protein interactions. (B) Three-dimensional structure of CA XII showing the binding site.
Ijms 24 09401 g003
Figure 4. Predicted binding mode of the hydrolysed form of 24h and CA IX. (A,C) Two-dimensional view of main residues involved in the ligand–protein interactions corresponding to closed, open E, and open Z forms, respectively. (B,D) Three-dimensional structure of CA IX showing the binding site corresponding to closed, open E, and open Z forms, respectively.
Figure 4. Predicted binding mode of the hydrolysed form of 24h and CA IX. (A,C) Two-dimensional view of main residues involved in the ligand–protein interactions corresponding to closed, open E, and open Z forms, respectively. (B,D) Three-dimensional structure of CA IX showing the binding site corresponding to closed, open E, and open Z forms, respectively.
Ijms 24 09401 g004
Figure 5. Predicted binding mode of the hydrolysed form of 24h and CA XII. (A,C) Two-dimensional view of main residues involved in the ligand–protein interactions corresponding to closed, open E, and open Z forms, respectively. (B,D) Three-dimensional structure of CA XII showing the binding site corresponding to closed, open E, and open Z forms, respectively.
Figure 5. Predicted binding mode of the hydrolysed form of 24h and CA XII. (A,C) Two-dimensional view of main residues involved in the ligand–protein interactions corresponding to closed, open E, and open Z forms, respectively. (B,D) Three-dimensional structure of CA XII showing the binding site corresponding to closed, open E, and open Z forms, respectively.
Ijms 24 09401 g005
Table 1. Inhibition constants and selectivity indexes of sulfonamido-containing imidazolidine-2-thiones 8, 9 against hCAs I, II, IV, IX, and XII compared with thioureas 14 and AAZ a.
Table 1. Inhibition constants and selectivity indexes of sulfonamido-containing imidazolidine-2-thiones 8, 9 against hCAs I, II, IV, IX, and XII compared with thioureas 14 and AAZ a.
Ijms 24 09401 i001
CompoundKi (nM)S.I. b
hCA IhCA
II
hCA
IV
hCA
IX
hCA XIII/IXI/XIIII/IXII/XII
d-Gluco8a
(n = 0,
R = p-SO2NH2)
765.560.0347.6175.0502.54.41.50.30.1
8b
(n = 0,
R = m-SO2NH2)
4578505345.1252.6216.018.121.220.023.4
8c
(n = 2,
R = p-SO2NH2)
84.78.9 d105061.451.41.41.60.10.1
d-Galacto9a
(n = 0,
R = p-SO2NH2)
90.3116.0457256572.90.0231.10.0240.0
9b
(n = 0,
R = m-SO2NH2)
7807927.627,25096275.10.815310.1181.9
Ijms 24 09401 i002
1a [26]
76807.0--- c2828.227.29370.020.9
Ijms 24 09401 i003
1c [26]
9.0108---8.79.71.00.912.411.1
Ijms 24 09401 i004
2a [26]
6840222---7.020.1977.134031.711.0
Ijms 24 09401 i005
2c [26]
57909.3---2.810.22067.95683.30.9
Ijms 24 09401 i006
3a [27]
27009700---777.935.13421261228
Ijms 24 09401 i007
3b [27]
1008600---9.020711.10.595641.5
Ijms 24 09401 i008
4a [27]
36007700---7410448.634.610474.0
Ijms 24 09401 i009
4b [27]
4300940---4214102.4307.122.467.1
AAZ25012.174.025.85.79.743.90.52.1
a Mean from three different assays, by a stopped-flow technique (errors were in the range of ±5–10% of the reported values); b S.I. = Ki (CA I or II)/Ki (CA XI or XII); c Not tested; d Bold values indicate strong inhibition (Ki < 10.5 nM).
Table 2. Inhibition constants and selectivity indexes of glyco-derived imidazolidine-2-thiones 16, 24, 26 against hCAs I, II, IX, and XII a.
Table 2. Inhibition constants and selectivity indexes of glyco-derived imidazolidine-2-thiones 16, 24, 26 against hCAs I, II, IX, and XII a.
Ijms 24 09401 i010
CompoundKi (nM)S.I. b
hCA
I
hCA
II
hCA IXhCA
XII
I/IXI/XIIII/IXII/XII
Ijms 24 09401 i011>100,000>100,00073.956.1>1353>1783>1353>1783
Ijms 24 09401 i012>100,000>100,00089.191.0>1122>1099>1122>1099
Ijms 24 09401 i013>100,000>100,000105.8227.8>945>439>945>439
Ijms 24 09401 i014>100,00017,300116.5163.9>858>610148106
24a
R1 = Me, R2 = H, n = 3
>100,000>100,00045.310.1 c>2208>9901>2208>9901
24b
R1 = Me, R2 = H, n = 4
>100,000>100,00070.725.7>1414>3891>1414>3891
24c
R1 = Me, R2 = H, n = 6
>100,000>100,00022.472.1>4464>1387>4464>1387
24d
R1 = Ph, R2 = H, n = 3
>100,000>100,00091.4260.3>1094>384>1094>384
24e
R1 = Ph, R2 = H, n = 4
>100,000>100,000177.3140.4>564>712>564>712
24h
R1 = Me, R2 = Cl, n = 4
>100,000>100,0006.837.5>14,706>2667>14,706>2667
Ijms 24 09401 i015>100,000>100,00028.661.4>3947>1629>3947>1629
a Mean from three different assays, by a stopped-flow technique (errors were in the range of ±5–10% of the reported values); b S.I. = Ki (CA I or II)/Ki (CA XI or XII); c Bold values indicate strong inhibition (Ki < 10.5 nM).
Table 3. Antiproliferative activity (GI50, µM) of selected compounds (mean ± SD).
Table 3. Antiproliferative activity (GI50, µM) of selected compounds (mean ± SD).
CompoundDrug-Sensitive Cell LinesMultidrug-Resistant Cell Lines
A549
(Lung, Non-Small)
HBL-100
(Breast)
HeLa
(Cervix)
SW1573
(Lung, Non-Small)
T-47D
(Breast)
WiDr
(Colon)
Ijms 24 09401 i01679 ± 3686 ± 2583 ± 3079 ± 37>100>100
Ijms 24 09401 i01734 ± 4.023 ± 3.225 ± 8.09.7 a30 ± 7.436 ± 16
Ijms 24 09401 i01864 ± 3123 ± 0.831 ± 0.25.7 ± 1.870 ± 2347 ± 11
a Bold values indicate strong inhibition (Ki < 10.5 nM).
Table 4. Docking predicted binding energies for coumarin 24h (closed and open forms) with CAIX and CAXII (kcal/mol).
Table 4. Docking predicted binding energies for coumarin 24h (closed and open forms) with CAIX and CAXII (kcal/mol).
Enzyme/Compound24h (Closed)24h (Open E)24h (Open Z)
hCA IX−8.4781−10.1987−9.6243
hCA XII−7.1633−9.3104−9.8885
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Martínez-Montiel, M.; Romero-Hernández, L.L.; Giovannuzzi, S.; Begines, P.; Puerta, A.; Ahuja-Casarín, A.I.; Fernandes, M.X.; Merino-Montiel, P.; Montiel-Smith, S.; Nocentini, A.; et al. Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors. Int. J. Mol. Sci. 2023, 24, 9401. https://doi.org/10.3390/ijms24119401

AMA Style

Martínez-Montiel M, Romero-Hernández LL, Giovannuzzi S, Begines P, Puerta A, Ahuja-Casarín AI, Fernandes MX, Merino-Montiel P, Montiel-Smith S, Nocentini A, et al. Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors. International Journal of Molecular Sciences. 2023; 24(11):9401. https://doi.org/10.3390/ijms24119401

Chicago/Turabian Style

Martínez-Montiel, Mónica, Laura L. Romero-Hernández, Simone Giovannuzzi, Paloma Begines, Adrián Puerta, Ana I. Ahuja-Casarín, Miguel X. Fernandes, Penélope Merino-Montiel, Sara Montiel-Smith, Alessio Nocentini, and et al. 2023. "Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors" International Journal of Molecular Sciences 24, no. 11: 9401. https://doi.org/10.3390/ijms24119401

APA Style

Martínez-Montiel, M., Romero-Hernández, L. L., Giovannuzzi, S., Begines, P., Puerta, A., Ahuja-Casarín, A. I., Fernandes, M. X., Merino-Montiel, P., Montiel-Smith, S., Nocentini, A., Padrón, J. M., Supuran, C. T., Fernández-Bolaños, J. G., & López, Ó. (2023). Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors. International Journal of Molecular Sciences, 24(11), 9401. https://doi.org/10.3390/ijms24119401

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