Exploring the Broad-spectrum Activity of Carbohydrate-based Iberin Analogues: From Anticancer Effect to Antioxidant Properties

Research paper Exploring the broad-spectrum activity of carbohydrate-based Iberin analogues: From anticancer effect to antioxidant properties L.A. Prieto a , N. Khiar-Fern´ andez b , J.M. Calder´ on-Monta˜ no c , M. L´ opez-L´ azaro c , J. Lucía-Tamudo d , J.J. Nogueira e,f , R. Le´ on g , N. Moreno a , V. Valdivia a,* , R. Recio a , I. Fern´ andez a,** a Departamento de Química Org´ anica y Farmac´ eutica, Facultad de Farmacia, Universidad de Sevilla, 41012, Sevilla, Spain b Department of Organic Chemistry, School of Chemistry, Universidad Complutense de Madrid, Plaza de las Ciencias s/n, 28040, Madrid, Spain c Department of Pharmacology, Faculty of Pharmacy, University of Seville, 41012, Seville, Spain d Faculty of Chemistry and Pharmacy, Institute of Physical and Theoretical Chemistry, University of Regensburg, 93040, Regensburg, Germany e Department of Chemistry, Universidad Aut´ onoma de Madrid, Calle Francisco Tom´ as y Valiente, 7, 28049, Madrid, Spain f Institute for Advanced Research in Chemistry (IAdChem), Universidad Aut´ onoma de Madrid, Calle Francisco Tom´ as y Valiente, 7, 28049, Madrid, Spain g Instituto de Química M´ edica, Consejo Superior de Investigaciones Científicas (IQM-CSIC), C/Juan de la Cierva 3, Madrid, 28006, Spain ABSTRACT Iberin is a lower homologue of sulforaphane (SFN) which has shown effectiveness in addressing various pathologies, including its anti-inflammatory properties, antitumor activity against various cancers, and antimicrobial effects. Building on this activity, a series of carbohydrate-based analogues of the natural isothiocyanate (ITC) iberin were synthesized, and their anticancer and antioxidant activities were evaluated. Cytotoxicity studies on three cancer cell lines using Resazurin assay demonstrated significant cytotoxic activity, particularly against bladder cancer. The sulfonyl derivatives exhibited the most potent effects, with IC 50 values comparable to those of reference natural isothiocyanates (from 10 to 20 μ M). Computational simulations support the hypothesis that carbohydrate-based ITCs can interact with STAT3’s SH2 domain in a manner similar to SFN, laying the groundwork for their potential development as STAT3-targeted anticancer agents. The antioxidant potential of these compounds was assessed by their ability to activate the Nrf2 factor, yielding CD values (concentration required to double luciferase activity compared to basal conditions) between 1.55 and 10.36 μ M, without cytotoxicity at these concentrations. Notably, the phenylsulfone derivative 22βdisplayed slightly higher or comparable antioxidant activity to that of natural isothiocyanates. Based on these findings, this phenylsulfone analogue was selected as the optimal compound due to its dual anticancer and antioxidant activities. An additional advantage of this carbohydrate-based ITC is that it is a solid compound, making it easier to handle than natural isothiocyanates, which are typically liquids. 1. Introduction Iberin (IBE), 1-isothiocyanato-3-(methylsulfinyl)-propane (Fig. 1), a structurally simple chiral sulfoxide akin to sulforaphane (SFN), was first identified in cabbage extracts and shared similarities with SFN in its multitarget pharmacological profile [1]. Since its discovery, IBE has been recognized for its potential in targeting various pathways, including Toll-like receptors, GPX-1 enzyme, HDAC, and Nrf2, mirroring the diverse action spectrum of SFN. Despite its promising therapeutic potential, IBE has garnered comparatively less attention from the scientific community. However, recent studies have highlighted its efficacy against a range of pathologies, such as anti-inflammatory effects, antitumor activity in various cancer types, including glioblastoma and melanoma, and antimicrobial properties [2–10]. The similarities between SFN and IBE in their pharmacological actions underscore the potential of IBE as a lead compound for drug development. Nonetheless, challenges persist, including issues related to stability, particularly in aqueous solutions at room temperature, which have hindered the commercialization of medications containing SFN as an active ingredient [11–13]. In light of these considerations, the exploration of novel IBE-based analogues and formulations shows great potential for addressing unmet medical needs across diverse therapeutic areas. Given the advantages of incorporating a carbohydrate moiety into the structure of bioactive molecules, such as potentially enhancing stability, bioavailability, and pharmacological activity [14–16], in this work we have focused on the development of a small library of N-glycosyl isothiocyanates (ITCs) as novel iberin analogues (Fig. 1). Utilization of carbohydrates in drug design offers the advantage that * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (V. Valdivia), [email protected] (I. Fern´ andez). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: www.elsevier.com/locate/ejmech https://doi.org/10.1016/j.ejmech.2025.117469 Received 28 November 2024; Received in revised form 20 February 2025; Accepted 27 February 2025 European Journal of Medicinal Chemistry 289 (2025) 117469 Available online 28 February 2025 0223-5234/© 2025 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). most of their derivatives are solid compounds, which can be easily manipulated [17–19]. This characteristic simplifies various stages of drug development, from synthesis to formulation, enabling straightforward handling and processing in pharmaceutical manufacturing. Moreover, using carbohydrates as a scaffold for synthesizing iberin analogues could facilitate recognition by active transporters, leading to more efficient crossing of certain membranes and thus improving bioavailability. Monosaccharides also offer rigid molecular structures that serve as templates for positioning the pharmacophore with precise spatial orientations [20,21]. This pre-organization reduces entropic penalties associated with binding and has proven effective in enhancing binding affinity in various glycomimetics [22,23]. Our primary goal is to explore the therapeutic potential of the synthesized carbohydrate-based iberin analogues, with a particular focus on their anticancer and antioxidant activities. Several studies attribute the anticancer activity of natural isothiocyanates to their ability to interact with STAT3 and related pathways, which are critical in tumor progression [24,25]. Thus, computational studies are conducted to determine whether carbohydrate-based ITCs can interact with the STAT3’s SH2 domain in a similar manner to SFN. Concurrently, their antioxidant properties can be linked to their capacity to activate the phase II antioxidant response by activating Nrf2, the master regulator of cellular defense mechanisms [26–29]. Upregulation of the Nrf2-ARE pathway increases the availability of multiple antioxidants, and a significant volume of preclinical research has explored the capacity of natural isothiocyanates to safeguard the nervous system from numerous diseases and toxins [30–34]. This dual mechanism of action exemplifies a typical multitarget approach, underscoring the potential of isothiocyanates as versatile therapeutic agents in cancer prevention and treatment. As sulfur derivatives, both the influence of sulfur’s oxidation state (thioether, sulfoxide or sulfone) and its chirality in the case of sulfoxides have been studied to determine the activity differences between the two sulfur epimers. 2. Results and discussion 2.1. Synthesis Although the new carbohydrate-based analogues appear structurally more complex than iberin, they can be readily synthesized from diacetone-D-glucose (DAG) as unique starting material using the well-known carbohydrate chemistry as indicated in the retrosynthetic scheme shown in Fig. 2. The synthetic route comprises two key stages: (i) introducing the sulfenyl group at position 3 of the sugar and (ii) incorporating the azide (as a precursor of the isothiocyanate group) at the anomeric carbon. The alkylsulfinyl group was introduced via nucleophilic displacement of the 3-O-mesylated derivative 2in the allo configuration (Scheme 1). The alcohol precursor 1, is readily obtained through oxidation of diacetone-D-glucose and subsequent stereoselective reduction [35,36]. The alkyl and phenyl thioderivatives, 3–5[37], were obtained in high yields from the O-mesylate derivative (Scheme 1). Hydrolysis of both isopropylidene acetals of 3–5, followed by acetylation of hydroxyl groups, yields a 1:1 mixture of both anomers, α and β, of the corresponding tetraacetylated derivatives, which are used directly in the next step without separation. The subsequent azidoglycosylation reaction, performed by treating with trimethylsilyl azide (TMSA) in the presence of SnCl 4 as a Lewis acid, produced both anomeric azides, with the βanomer being predominant. Column chromatography enabled the isolation of diastereomerically pure α and β anomers of the phenyl and methylthio derivatives. However, for ethylthio derivatives, only the βanomer could be isolated as a single diastereomer (Scheme 1). The conversion of azide to isothiocyanate proceeded in two steps: first, reduction with triphenylphosphine, followed by treatment with carbon disulfide under microwave irradiation. In all cases, β-configured isothiocyanates were obtained. This outcome highlights the feasibility of conducting this transformation on a mixture of epimeric azides without the necessity for chromatographic separation, thereby streamlining the experimental process. To confirm that the observed epimerization is unaffected by substitution at carbon 3, the reaction was performed on both anomers (9 α and 9β) [38] of the peracetylated 1-azido-D-glucose derivatives as a model substrate. It was confirmed that in both cases, the same βisothiocyanate 13βwas obtained (Scheme 3). This result aligns with the findings of Gy¨ orgyde´ ak et al., [39] who, in their synthesis of aminoglycoside series from azides, demonstrated that the Staudinger reduction of glycosyl azides generates aza-ylide intermediates (or iminophosphoranes), that undergo anomeric isomerization, favoring the formation of β-anomers. The oxidation of thioether to sulfoxide (Scheme 2) with m-CPBA requires a gradual rise in temperature to reach room temperature for the reaction to complete. Under these conditions, the reaction proceeds without stereoselectivity, yielding an approximately equimolar mixture of both epimers at the sulfur atom [40]. Chromatographic separation of the diastereomeric alkylsulfoxides was not achievable, thus the activity of the mixed sulfur epimers, (R/S)-17β(R=Me) and (R/S)-18β(R=Et), was studied. The phenylsulfinylglycosides (R)-19βand (S)-19βwere also obtained in a substantial mixed ratio and they could be separated by column chromatography. This separation enables the individual study of each diastereomer’s activity and allows for determining the influence of sulfur chirality on activity. The sulfur configuration of both phenylsulfoxides was unequivocally Fig. 1. Structure of natural isothiocyanates sulforaphane and iberin (A), general structure of N-glycosyl ITCs of this work (B). L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 2 assigned through X-ray crystallography (Fig. 3) of the crystals obtained for the less polar diastereomer, which exhibited the Rconfiguration. The 1 H NMR spectrum of this diastereomer, (R)-19β, shows the H5 proton to be more shielded and H1 less shielded compared to the corresponding protons in the (S)-19βdiastereomer. The configuration of the other alkylsulfoxides was assigned by analogy to the spectroscopic pattern observed in the spectra of the epimeric sulfoxides mixtures, (R/S)-17β and (R/S)-18β. Fig. 2. Retrosynthetic pathway for the synthesis of N-glycosyl isothiocyanates analogues of iberin. Scheme 1. Synthesis of α and βN-azidoglycosyl-3-alkyl(aryl)thio derivatives of glucopyranose, (10–12). Scheme 2. Synthesis of 1-isothiocyanate derivatives of 3-alkyl(aryl)thio-β-d-glucopyranosides (14–16), along with their corresponding sulfinyl (17–19) and sulfonyl analogues (20–22). Scheme 3. Synthesis of isothiocyanate 13βfrom both anomers of the peracetylated 1-azido-D-glucose derivatives, 9 α and 9β. L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 3 Treatment of alkyl and arylthioglycosyl derivatives (14β16β) with two equivalents of m-CPBA as the oxidizing agent in a CH 2 Cl 2 /CHCl 3 mixture at room temperature yielded the corresponding sulfones 20β21βin moderate to high yields (Scheme 2). Finally, the natural ITC-sulfoxide iberin, a lower homologue of sulforaphane, along with its analogues iberverin (thioether) and cheirolin (sulfone) [7,41], were also synthesized to serve as reference compounds, and the detailed routes are found in the Supporting Information. The water solubility and bioavailability of all final isothiocyanates were assessed using the SwissADME software [42], with the results provided in the Supplementary Material. For all synthesized ITCs derivatives, water solubility was estimated based on the ESOL method [43], while good bioavailability for oral administration was inferred according to Lipinski’s rules [44]. 2.2. Evaluation of selective cytotoxic activity of 1-isothiocyanate derivatives of 3-alkyl(aryl)thio-β-D-glucopyranoside Sulforaphane and other natural isothiocyanates have demonstrated anticancer activity against various cancer types, including breast, bladder, prostate, pancreatic, renal, ovarian, colorectal, and gastric cancers, among others [45–47]. This inspired us to conduct a focused screening in our study of the anticancer activity of the synthesized products on three different cancer cell lines. Specifically, we selected lung adenocarcinoma (A549), melanoma (MeWo), and bladder cancer (T24) cell lines to evaluate their potential cytotoxic activity, as these cancers are among the most prevalent and have high mortality rates in Fig. 3. X-ray analysis of the less polar diastereomer of 1-isothiocyanato-3phenylsulfinyl-β-D-glucopyranoside derivative (R)-19β. Table 1 IC 50 Values of carbohydrate-based iberin analogues, 13β-22β, natural isothyociantes, 26–28, and gemcitabine on three different human cancer cell lines and non-malignant keratinocyte. The selectivity index is the mean of the selectivity indexes calculated in each individual experiment. The selectivity index is calculated by dividing the IC 50 value obtained in the HaCat cell line (Non-malignant keratinocytes) by that in the cancer cell lines (A549, MeWo and T24). The most active compounds are highlighted in gray, and the IC 50 of the most selective compounds are indicated in bold. L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 4 advanced stages. To evaluate the anticancer selective activity of our compounds, they were also studied against a human non-malignant (HaCaT) cell line. These cells were originated from normal adult tissue and exhibit a division rate comparable to cancer cells [48]. Most anticancer drugs lack adequate selectivity, often targeting both cancerous and normal cells with similar rates of division. The cell lines were exposed to varying concentrations of the synthesized carbohydrate-based iberin analogues, during 72 h before quantifying the cell viability with the Resazurin assay. The half-maximal inhibitory concentration (IC 50 ) value of each compound and the selectivity index (SI) were calculated [49](Table 1). Gemcitabine, a well-known anticancer standard drug, served as a positive control. The three known natural isothyocianates iberin, iberberin and chierolin were used as reference controls to compare with the activity of the new carbohydrate-based derivatives. The anticancer activity of these compounds has been previously reported against various types of cancer [3, 10,50]. Wang et al. reported that iberverin and iberin were cytotoxic against A549 lung cancer cells [10], with IC 50 values of 48 μ M and 30.2 μ M, respectively. Our findings demonstrate similar cytotoxic effects, with IC 50 values of 31 μ M for iberverin and 20.7 μ M for iberin against A549 cells. Most of the synthesized glycosyl isothiocyanates exhibit cytotoxic effects against the three cancer cell lines examined. Apart from the alkyl sulfoxides, 17βand 18β(entries 5 and 6, Table 1), the other derivatives display IC 50 values below 100 μ M. In all cases, the highest cytotoxicity was observed against the bladder cancer cell line. The anticancer activity of natural isothiocyanates, such as sulforaphane and iberin, against bladder cancer has been documented [51]. Accordingly, the natural alkyl isothiocyanates 26–28, used as reference compounds, also demonstrated higher activity against bladder cancer, with IC 50 values ranging from 10.7 to 14.4 μ M. Regarding the influence of sulfur oxidation state on activity, thioethers 14β¡16β(entries 2–4, Table 1) and sulfones 20β-22β(entries 9–11, Table 1) exhibit grater activity than sulfoxides, with no significant impact of sulfur configuration on the activity of sulfoxides (compare entries 7 and 8, Table 1). Regarding the nature of the sulfur substituent, phenyl derivatives exhibit greater activity than their alkyl (methyl and ethyl) analogues for both thioethers and sulfoxides, while demostrating similar activity to alkyl derivatives in the case of sulfones. Focusing on bladder cancer (T24 cell line) and considering IC 50 values, phenylthioglycoside 16βand the sulfones—both alkyl (20β, 21β) and aryl-substituted (22β)—emerge as the most active compounds. Notably, the sulfones and the natural alkyl isothiocyanates (26–28) were also the most selective compounds, exhibiting approximately twoand three-fold greater cytotoxicity against bladder cancer cells compared to non-malignant cells, respectively (Fig. 4). Although ITCs exhibited lower selectivity than gemcitabine, a chemotherapeutic agent commonly used in the treatment of bladder cancer, this does not preclude their therapeutic potential. Treatment options for bladder cancer have significantly expanded in recent years; however, the combination of cisplatin and gemcitabine remains the standard treatment. Rapid development of resistance to these drugs is often encountered. Sulforaphane has demonstrated activity against a panel of gemcitabineand cisplatin-resistant bladder cancer cell lines [52]. Therefore, the combination of isothiocyanate derivatives with standard therapies may represent a novel approach in the fight against cancer. 2.2.1. Computational studies: docking, molecular dynamics simulations and binding energy predictions The SH2 domain of the STAT3 protein is a target of interest due to its role in signaling pathways implicated in cancer progression. Accordingly to existing literature, natural isothiocyanate have demonstrated binding affinity for STAT3’s SH2 domain [25,53], suggesting that related compounds may exhibit similar interactions and potentially modulate STAT3’s activity in cancer cells. Based on this premise, a docking study was conducted to identify the primary binding pockets relevant to the interaction between dephosphorylated human STAT3 and SFN, along with several target iberin derivative. This study aimed to provide evidence that these novel compounds could indeed exert their anticancer activity through interaction with STAT3. The docking results revealed three primary binding pockets within Fig. 4. Evaluation of selective cytotoxic activity of sulfones 20β,21β,22βand 28 on human non-malignant cells (HaCaT) and human bladder cancer cells (T24). Cells were exposed to several concentrations of compounds for 72 h and cell viability was measured using the resazurin assay. Data represent mean ±SEM from at least three independent experiments. *p <0.05, **p˂0.01. L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 5 the SH2 domain where SFN could potentially bind. The pocket with the highest binding energy, shown on the left in Fig. 5, was also the site of the most robust interactions for the majority of carbohydrate-based iberin analogues. Each derivative predominantly localized within this pocket, demonstrating a similar binding preference and suggesting that this pocket may serve as a critical site for interaction with isothiocyanate-containing compounds. Additionally, SFN and the iberin analogues shared an additional secondary binding pocket, with all derivatives exhibiting at least one binding pose with large binding free energy in this site. In the third identified pocket, most iberin derivatives displayed binding interactions, further reinforcing the likelihood of shared interaction mechanisms between SFN and these novel N-glycosyl ITCs. In order to characterize the primary binding pocket, the ten closest amino acids of the protein, common to all iberin derivatives, were identified (see lower right panel in Fig. 5). Subsequently, the contribution of the interaction between the compounds and each of these amino acids to the binding free energy obtained from the molecular dynamics (MD) simulations was determined (see lower left panel in Fig. 5). After thoroughly analyzing these results, it can be observed that the amino acid Met95 has a significant influence on the binding free energy of most iberin derivatives in this binding pocket. Other relevant amino acids in the STAT3-iberin derivative interaction include Arg27, Gly92, Tyr183, and Pro184. It can also be observed that, in the case of anomer 15βand (R)-19β, these amino acids do not significantly contribute to the binding free energy. This is because, in the MD simulations, these compounds slightly shifted away from the binding pocket, although they remained interacting with the protein in a region very close to it. These findings underscore the potential of iberin derivatives to mimic the biological role of SFN by binding to the SH2 domain of STAT3, potentially modulating STAT3-related pathways. Following the docking analysis, the binding pose with the highest binding free energy for each iberin analogue-protein complex in the primary binding pocket was selected for running MD simulations to Fig. 5. Schematic representation of the predicted binding pockets within the SH2 domain (circled region) by the docking study (upper panel). Relevant amino acids involved in the interaction with the carbohydrate-based iberin analogues (lower right panel) and their individual contribution to the binding energy (lower left panel) for each of the compounds. Table 2 Binding energies for the iberin derivatives at the MM-PBSA level of theory. Entry X Compound Binding Energy (kcal/mol) 1–Sulforaphane −21.61 2β-MeS 14β−14.04 3β-EtS 15β−6.69 4β-PhS 16β−24.80 5 α -PhS 16 α −14.12 6β-MeSO 17β−17.16 7β-EtSO 18β−10.79 8β-(R)-PhSO (R)-19β−14.50 9β-(S)-PhSO (S)-19β−15.54 10 β-MeSO 2 20β−9.29 11 β-EtSO 2 21β−8.52 12 β-PhSO 2 22β−8.93 L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 6 compute the binding free energies in a more accurate way. Table 2 summarizes the results, indicating favorable interaction energies for all the iberin derivatives within this pocket. Notably, the beta anomer of the phenylthio derivative 16βexhibited a binding affinity stronger than that of SFN, suggesting that 16βand similar compounds may achieve robust stabilization within the SH2 domain of STAT3. The interaction with the alpha anomer 16 α proves to be weaker. This result is significant considering that our synthetic approach only allows for the production of βanomers. In the case of sulfoxides, there is no significant influence of the sulfinyl sulfur configuration on the strength of the interaction with STAT3 (see entries 8 and 9, Table 2). The results of this study reinforce the idea that iberin derivatives can interact with the SH2 domain of STAT3 similarly to SFN. This insight paves the way for developing these compounds as targeted therapies against STAT3 in cancer treatment. 2.3. Antioxidant activity as Nrf2 inductors As mentioned, the N-glycosyl isothiocyanates were designed to act also as Nrf2 inducers, featuring the isothiocyanate function in the anomeric position. This strategic placement allows them to react with crucial cysteine residues in KEAP1, mirroring the mechanisms previously described for iberin, SFN, and other natural isothiocyanates [54, 55]. Therefore, we examined the Nrf2 induction potential of our novel derivatives using the stable human mammary MCF7-derived reporter cell line AREc32 [56]. The corresponding CD values (concentration of compound required to double luciferase expression compared to basal conditions), derived from the dose–response curves, are summarized in Table 3. Overall, there is a clear influence of the sulfur substituent at position 3 of the sugar ring, as well as the oxidation state and nature of the X group, on the ability to induce the Nrf2 factor. In the case of natural isothiocyanates, no significant differences in CD values were noted based on the state of oxidation of sulfur. However, the thioether iberverin (entry 12, Table 3) demonstrates slightly higher activity compared to the sulfoxide iberin and sulfone cheirolin (entries 13 and 14, Table 3). This result contrasts with the findings from our derivatives, where thioethers exhibited no significant Nrf2 induction activity. The thioethers were inactive regardless of the nature of the substituent on sulfur, whether alkyl (14βand 15β, entries 2 and 3, Table 3) or aryl (16β, entry 3). In these instances, calculating the CD values is not feasible, as concentrations exceeding 30 μ M led to a reduction in luciferase expression below the basal level. A similar trend is observed in the case of the tetraacetylated derivative 13β(Table 3, entry 1). In contrast, the N-glycosyl isothiocyanates containing sulfur in higher oxidation states, specifically sulfoxides and sulfones, displayed significant Nrf2 induction activities, with CD values in the low micromolar range. These findings highlight intriguing structure-activity relationships. The alkyl (methyl and ethyl) sulfoxides were examined as mixtures of both sulfur epimers, (R/S)-17βand (R/S)-18β, due to the inability to separate them using chromatographic techniques. The results indicated no significant differences between them (entries 5 and 6). Increasing the length of the alkylsulfinyl chain (ethyl v.s. methyl) also fails to enhance activity in the alkyl sulfones 20βand 21β, which exhibit similar CD values of 6.21 ±0.46 μ M and 10.36 ±2.53 μ M, respectively (entries 9 and 10). As previously noted, phenyl sulfoxides were successfully separated chromatographically, and the CD values for each diastereomer, (S)-19β and (R)-19β, were determined to be 2.09 ±1.35 μ M and 1.55 ±0.33 μ M, respectively (entries 7 and 8), demonstrating greater activity than their alkyl sulfoxide counterparts. The CD value for phenyl sulfone 22β(2.09 +0.13 μ M, entry 12) was found to be comparable to that of phenyl sulfoxides, demostrating a greater activation capacity than the alkyl sulfones (compare entries 11 with entries 9 and 10). In summary, phenylthio derivatives demonstrate greater efficacy as activating agents compared to their alkylthio counterparts. For synthetic simplicity, phenyl sulfone 22βemerges as the most active compound, as it can be directly synthesized through the oxidation of precursor thioether 16β. This method circumvents the need to separate diastereoisomers of sulfoxides via chromatography, which can lead to lower yields and complicate the assessment of biological activity in mixtures. Additionally, the final phenyl sulfone 22βis obtained as a solid, which is preferable for further applications. 3. Conclusions In this work, a straightforward and efficient synthesis of carbohydrate-based analogues of the natural isothiocyanate iberin is presented, along with an evaluation of their anticancer and antioxidant properties. Cytotoxicity assays conducted on three different cancer cell lines revealed good cytotoxicity, particularly against bladder cancer. The sulfonyl derivatives demonstrated the highest potency, with IC 50 values comparable to those of reference natural isothiocyanates. Computational studies provide compelling evidence that carbohydratebased iberin derivatives can interact with the SH2 domain of STAT3 similarly to sulforaphane (SFN). This discovery highlights the potential of these compounds as targeted therapies for cancer treatment by specifically modulating STAT3 activity. Considering their antioxidant activity, it was assessed by examining their ability to activate the Nrf2 factor, resulting in CD values between 1.55 and 10.36 μ M, without cytotoxicity at these concentrations. Notably, the phenylsulfone derivative 22βshowed superior antioxidant capacity compared to natural isothiocyanates. This compound was selected as the most promising, based on both its anticancer and antioxidant activities. Furthermore, its solid form provides a practical advantage over the natural isothiocyanates, which are liquids, making it easier to handle and apply. Table 3 Nrf2 induction activity of N-glycosyl isothiocyanates 13β-22βand natural ITCs 26–28, which served as reference compound, in the AREc32 cell line model. Entry Compound X CD ( μ M) a,b 113βOAc n.a. 214βMeS n.a. 315βEtS n.a. 416βPhS n.a. 5(R/S)-17βMeS(O) 5.75 ±2.03 6(R/S)-18βEtS(O) 6.32 ±0.02 7(S)-19βPhS(O) 2.09 ±1.35 8(R)-19βPhS(O) 1.55 ±0.33 920βMeSO 2 6.21 ±0.46 10 21βEtSO 2 10.36 ±2.53 11 22βPhSO 2 2.09 ±0.13 12 26 (Iberverin) MeS 2.94 ±1.51 13 rac-27 (Iberin) MeSO 3.12 ±0.64 14 28 (Cheirolin) MeSO 2 3.22 ±1.14 15 TBHQ c –1.68 ±0.30 a The calculated cell viability, expressed as EC 50 , was found to be greater than 30 μ M in all cases. b n.a.: non active. c tert-Butylhydroquinone (TBHQ) was used as positive control of Nrf2 induction L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 7 4. Experimental section 4.1. Chemical synthesis 4.1.1. Materials and methods In the cases in which it has been indicated that the reaction has taken place under an inert atmosphere, glassware previously dried in an oven and anhydrous solvents have been used. The solvents have been dried with a 4 Å molecular sieve, previously activated in microwaves and under vacuum. TLC was performed on Silica Gel GF254 (Merck) with detection by charring with phosphomolybdic acid/EtOH. For flash chromatography, silica Gel (Merck 230–400 mesh) was used. Columns were eluted with positive air pressure. Chromatographic eluents are given as volume to volume ratios (v/v). NMR spectra were recorded on the Bruker DRX-500 devices of the Nuclear Magnetic Resonance Service of the University of Seville. Chemical shifts are reported in ppm, and coupling constants are reported in Hz. Routine spectra were referenced to the residual proton or carbon signals of the solvent. High-resolution mass spectra were recorded on a Kratos EM-80RFA 241 MC spectrometer using electronic impact or FAB techniques, by the Mass Spectrometry service of the University of Seville. Optical rotations were determined with a PerkinElmer 341 polarimeter using a sodium lamp (λ =589 nm) with a 10 cm cell length. 4.1.2. 1,2,5,6-O-Isopropylideneα -D-allofuranose (1) To 25 mL of DMSO under argon atmosphere and at 18–20 ◦C, P 2 O 5 (5.45 g, 38.42 mmol) is slowly added. After stirring for 15 min at 18–20 ◦C, a solution of DAG (10.00 g, 38.42 mmol) in 50 mL of DMSO is added. The mixture is then heated at 50 ◦C until the starting product is consumed (3 h). Next, after adding TBME (60 mL) to the mixture, both phases are separate, and the aqueous phase is extracted with TBME (40 mL). The combined organic phases are evaporated under reduced pressure to a volume of 75 mL and added, at 0–10 ◦C, to a solution of NaBH 4 (0.92 g, 24.20 mmol) in distilled H 2 O (38 mL). After stirring for 30 min, the reaction mixture is diluted with distilled H 2 O (40 mL), both phases are separated, the aqueous phase is extracted with CH 2 Cl 2 (3 × 40 mL) and the combined organic phases are evaporated under reduced pressure. The obtained crude is dissolved in TBME (75 mL) and extracted with distilled H 2 O (3 ×30 mL). The combined aqueous phases are extracted with CH 2 Cl 2 (5 ×40 mL) and the organic extracts are dried over anhydrous Na 2 SO 4 . The solvent is evaporated under reduced pressure to obtain 1(9.92 g, 38.11 mmol, quant yield) as a white solid; 1 H NMR (500 MHz, CDCl 3 ): 5.81 (d, J=3.8 Hz, 1H, H 1 ), 4.61 (dd, J= 3.9 and 5.1 Hz, 1H, H 4 ), 4.30 (td, J=4.8 and 6.6 Hz, 1H, H 5 ), 4.10–3.99 (m, 3H, H 2 , H 6 , H 6’ ), 3.82 (dd, J=4.8 and 8.5 Hz, 1H, H 3 ), 2.52 (d, J= 8.4 Hz, 1H, OH), 1.58 (s, 3H, CH 3 ), 1.46 (s, 3H, CH 3 ), 1.38 (s, 3H, CH 3 ), 1.37 (s, 3H, CH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ113.0, 110.0, 104.1, 80.0, 79.2, 75.8, 72.7, 66.1, 26.7 (2C), 26.5, 25.4 ppm; HRMS: Calculated for C 12 H 20 O 6 Na [M+Na] + : 283.1152; found 283.1156 (0.4 ppm). 4.1.3. 3-Deoxy-1,2,5,6-O-isopropylideneα -D-allofuranose 3-methanesulfonate (2) To a solution of 1(200 mg, 0.77 mmol) in THF (2 mL), under an argon atmosphere and at 0 ◦C, triethylamine (0.13 mL, 0.92 mmol) and methanesulfonyl chloride (0.07 mL, 0.92 mmol) are consecutively added. Once the starting product is consumed, after stirring for 4 h, a saturated NH 4 Cl solution (20 mL) is added. The aqueous phase is extracted with CH 2 Cl 2 (3 ×20 mL) and the combined organic phases are washed with saturated NaHCO 3 (1 ×20 mL) and saturated NaCl (1 ×20 mL), dried over anhydrous Na 2 SO 4 and the solvent evaporated under reduced pressure. Thus, compound 2(248 mg, 0.73 mmol, 95 % yield)) is obtained as a white solid with a good degree of purity to be used in the next step without further purification. 1 H NMR (500 MHz, CDCl 3 ): δ5.82 (d, J=3.4 Hz, 1H, H 1 ), 4.82–4.78 (m, 2H, H 2 and H 4 ), 4.34–4.30 (m, 1H, H 5 ), 3.14 (dd, J=7.7 and 4.5 Hz, 1H, H 3 ), 4.10 (dd, J=8.7 and 6.9 Hz, 1H, H 6 ), 3.94 (dd, J=8.7 and 5.8 Hz, 1H, H 6’ ), 3.14 (s, 3H, -SCH 3 ), 1.58 (s, 3H, CH 3 ), 1 0.48 (s, 3H, CH 3 ), 1.37 (s, 3H, CH 3 ), 1.36 (s, 3H, CH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ 113.9, 110.4, 104.1, 78.1, 75.0 65.8, 39.0, 26.9, 26.8, 26.4, 25.1 ppm. HRMS: Calculated for C 13 H 22 O 8 SNa [M+Na] + : 361.0928; found 361.0920 (−2.1 ppm). 4.1.4. General procedure for the synthesis of 3-alkylthioα -D-glucofuranose derivatives To a solution of the thiolate (400 mol%), either as a commercial reagent or one formed in situ by treating the thiol (500 mol%) in DMF, under argon atmosphere, with NaH (400 mol%) (for 10 min), a solution of compound 2(100 mol%) in DMF is added. Once the starting product is consumed, a saturated NaCl solution is added. The aqueous phase is extracted with EtOAc (x 3). The combined organic phases are dried over anhydrous Na 2 SO 4 and the solvent is evaporated under reduced pressure. 4.1.5. 3-Deoxy-1,2,5,6-O-isopropylidene-3-methylthioα -D-glucofuranose (3) It is obtained following the general procedure, starting from CH 3 SNa (165 mg, 2.36 mmol) and mesilate 2(200 mg, 0.59 mmol) in DMF (6 mL). The crude is purified by silica gel column chromatography (hexane/EtOAc 7:1), obtaining 3(135 mg, 0.46 mmol, 79 % yield) as a yellow syrup. 1 H NMR (500 MHz, CDCl 3 ): δ5.84 (d, J=3.5 Hz, 1H, H 1 ), 4.69 (d, J=3.6 Hz, 1H, H 4 ), 4.33–4.29 (m, 1H, H 5 ), 4.21 (dd, J=8.8 and 3.9 Hz, 1H, H 2 ), 4.11 (dd, J=8.7 and 6.1 Hz, 1H, H 6 ), 4.00 (dd, J= 8.7 and 4.9 Hz, 1H, H 6’ ), 3.29 (d, J=3.9 Hz, 1H, H 3 ), 2.21 (s, 3H, -SCH 3 ), 1.52 (s, 3H, CH 3 ), 1.43 (s, 3H, CH 3 ), 1.37 (s, 3H, CH 3 ), 1.32 (s, 3H, CH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ112.0, 109.7, 105.1, 85.4, 80.6, 74.1, 67.8, 53.6, 27.1, 26, 8, 26.4, 25.4, 15.4 ppm; HRMS: Calculated for C 13 H 22 O 5 NaS [M+Na] + : 313.1080; found 313,1080 (−0.7 ppm). 4.1.6. 3-Deoxy-1,2,5,6-O-isopropylidene-3-ethylthioα -D-glucofuranose (4) It is obtained following the general procedure, method A, starting from ethanethiol (3.41 mL, 47.25 mmol) in DMF (40 mL), NaH (0.91 g, 37.80 mmol) in DMF (40 mL) and compound 2(3.19 g, 9.45 mmol) in DMF (40 mL). The crude obtained is purified by column chromatography on silica gel (hexane/EtOAc 10:1), obtaining 4(2.46 g, 8.08 mmol, 86 % yield) as a yellow syrup. 1 H NMR (500 MHz, CDCl 3 ): δ5.83 (d, J=3.5 Hz, 1H, H 1 ), 4.69 (d, J=3.5 Hz, 1H, H 4 ), 4.36–4.32 (m, 1H, H 5 ), 4.22 (dd, J=8.6 and 3.8 Hz, 1H, H 2 ), 4.11 (dd, J=8.6 and 6.1 Hz, 1H, H 6 ), 3.99 (dd, J=8.7 and 5.1 Hz, 1H, H 6’ ), 3.36 (d, J=3.8 Hz, 1H, H 3 ), 2.79–2.62 (m, 2H, -SCH 2 CH 3 ), 1.51 (s, 3H, CH 3 ), 1.42 (s, 3H, CH 3 ), 1.36 (s, 3H, CH 3 ), 1.32 (s, 3H, CH 3 ), 1.29 (t, J=7.4 Hz, 3H, -SCH 2 CH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ112.0, 109.5, 105.0, 86.1, 80.4, 74.2, 67.8, 51.6, 27.0, 26, 8, 26.4, 26.2, 25.4, 14.6 ppm. HRMS: Calcd for C 14 H 24 O 5 N 3 NaS [M+Na] + : 327.1248; found 327.1240 (−2.5 ppm). 4.1.7. 3-Deoxy-1,2,5,6-O-isopropylidene-3-phenylthioα -D-glucofuranose (5) It is carried out following the general, method A, procedure starting from thiophenol (2.42 mL, 23.65 mmol) in DMF (20 mL), NaH (0.45 g, 18.92 mmol) in DMF (20 mL) and compound 2(1.60 g, 4.73 mmol) in DMF (20 mL). The crude obtained is purified by column chromatography on silica gel (hexane/EtOAc 10:1), obtaining compound 5(1.59 g, 4.51 mmol, 95 % yield) as a yellow syrup. 1 H NMR (500 MHz, CDCl 3 ): δ 7.44–7.42 (m, 2H, -SC 6 H 5 ), 7.34–7.30 (m, 2H, -SC 6 H 5 ), 7.26–7, 23 (m, 1H, -SC 6 H 5 ), 5.88 (d, J=3.5 Hz, 1H, H 1 ), 4.63 (d, J=3.6 Hz, 1H, H 4 ), 4.42–4.38 (m, 1H, H 5 ), 4.34 (dd, J=8.6 and 3.8 Hz, 1H, H 2 ), 4.16 (dd, J =8.6 and 6.0 Hz, 1H, H 6 ), 4.03 (dd, J=8.7 and 4.2 Hz, 1H, H 6’ ), 3.89 (d, J=3.8 Hz, 1H, H 3 ), 1.52 (s, 3H, CH 3 ), 1.45 (s, 3H, CH 3 ), 1.36 (s, 3H, CH 3 ), 1.27 (s, 3H, CH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ134.1, 130.5 (2), 129.3 (2), 127.0, 112.2, 109.8, 105.2, 85.5, 80.1, 73.9, 67.9, 53.6, L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 8 27.1, 26.8, 26.5, 25.4 ppm; HRMS: Calcd for C 18 H 24 O 5 NaS [M+Na] + : 375.1236; found 375.1236 (−1.8 ppm). 4.1.8. General procedure for the synthesis of 3-alkylthio α ,β-Dglucopyranose derivatives The corresponding furanose derivative (100 mol%) is dissolved in a CF 3 COOH (27000 mol%):H 2 O (20:3) mixture at 0 ◦C. After the starting product is consumed, after stirring for 1 h, the solvent is evaporated under reduced pressure. The obtained crude is dissolved in pyridine and acetic anhydride (4900 mol%) is added at 0 ◦C under an argon atmosphere. Subsequently, the temperature is allowed to rise to room temperature. After stirring for 18 h, an ice-water mixture is added and extracted with CH 2 Cl 2 (x3). Next, the organic phase is washed successively with 2 M H 2 SO 4 solution (x 3), saturated NaHCO 3 solution (x 3) and saturated NaCl solution, dried over anhydrous Na 2 SO 4 and the solvent is evaporated under reduced pressure. 4.1.9. 3-Methylthio-3-deoxyα ,β-D-glucopyranose tetraacetate (6 α ,β) It is synthesized following the general procedure starting from compound 3(0.64 g, 2.21 mmol) in CF 3 COOH (4.56 mL, 59.60 mmol): H 2 O (0.68 mL), pyridine (22.43 mL) and acetic anhydride (10.22 mL, 108.14 mmol). The crude obtained is purified by column chromatography on silica gel (hexane/EtOAc 5:1), obtaining compound 6 α ,β(0.50 g, 1.32 mmol, 78 % yield) as a white syrup like a mixture of the two possible α and βanomers in a ratio of 1:0.9. 1 H NMR (500 MHz, CDCl 3 ): δ 6.27 (d, J=3.6 Hz, 1H α , H 1 ), 5.68 (d, J=8.1 Hz, 1Hβ, H 1 ), 5.09 (dd, J= 11.6 and 3.6 Hz, 1H α , H 2 ), 5.06–4.99 (m, 1H α , H 4 , 2Hβ, H 2 , H 4 ), 4.27 (dd, J=12 .4 and 4.9 Hz, 1Hβ, H 6 ), 4.24 (dd, J=12.5 and 4.6 Hz, 1H α , H 6 ), 4.12–4.05 (m, 2H α , H 5 , H 6’ , 1Hβ, H 6’ ), 3.80 (ddd, J=9.7, 4.9 and 2.2 Hz 1Hβ, H 5 ), 3.00 (t, J=11.2 Hz, 1H α , H 3 ), 2.70 (t, J=10.9 Hz, 1Hβ, H 3 ), 2.17 (s, 3H α , -OCOCH 3 ), 2.13 (s, 3H α , -OCOCH 3 ), 2.12 (s, 3Hβ, -OCOCH 3 ), 2.11 (s, 3Hβ, 3H α , -SCH 3 ), 2.09 (s, 3Hβ, 3H α , -OCOCH 3 ), 2.08 (s, 3Hβ, -OCOCH 3 ), 2.03 (s, 3Hβ, -OCOCH 3 ), 2.02 (s, 3H α , -OCOCH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ170.9, 170.8, 169.7, 169.5 (2), 169.5, 169.3, 168.9, 93.4, 89.2, 75.4, 71.0, 67.5, 66.9, 65.1, 64.8, 62.3, 62.3, 50.3, 46.5, 21.5, 21.0, 20 .9 (2), 20.8 (2), 20.7, 20.7, 10.9, 10.7 ppm. HRMS: Calcd for C 15 H 22 O 9 NaS [M+Na] + : 401.0877; found 401.0872 (−1.1 ppm). 4.1.10. 3-Ethylthio-3-deoxyα ,β-D-glucopyranose tetraacetate (7 α ,β) It is synthesize following the general procedure starting from compound 4(2.46 g, 8.08 mmol) in CF 3 COOH (16.7 mL, 218.3 mmol):H 2 O (3.73 mL), pyridine (82.17 mL) and acetic anhydride (37.45 mL, 396.16 mmol). The crude obtained is purified by column chromatography on silica gel (hexane/EtOAc 5:1), obtaining compound 7 α ,β(1.71 g, 4.36 mmol, 54 % yield) as a white syrup like a mixture of the two possible α and βanomers in a ratio of 1:0.8. 1 H NMR (500 MHz, CDCl 3 ): δ6.26 (d, J =3.5 Hz, 1H α , H 1 ), 5.65 (d, J=8.1 Hz, 1Hβ, H 1 ), 5.05–4.98 (m, 2H α , H 2 , H 4 , 2Hβ, H 2 , H 4 ), 4.24 (dd, J=12.4 and 4.9 Hz, 1Hβ, H 6 ), 4.21 (dd, J =12.4 and 4.5 Hz, 1H α , H 6 ), 4.10 (dd, J=12.6 and 2.3 Hz, 1H α , H 6’ ), 4.07 (dd, J=12.5 and 2.4 Hz, 1Hβ, H 6’ ), 4.03 (ddd, J=9.9, 4.4 and 2.3 Hz 1H α , H 5 ), 3.77 (ddd, J=9.7, 4.9 and 2.3 Hz 1Hβ, H 5 ), 3.01 (t, J= 11.3 Hz, 1H α , H 3 ), 2.71 (t, J=10.9 Hz, 1Hβ, H 3 ), 2.62–2.51 (m, 2H α , -SCH 2 CH 3 , 2Hβ, -SCH 2 CH 3 ), 2.17 (s, 3H α , -OCOCH 3 ), 2.12 (s, 3H α , -OCOCH 3 ), 2.11 (s, 3Hβ, -OCOCH 3 ), 2.10 (s, 3H α , -OCOCH 3 , 3Hβ, -OCOCH 3 ), 2.08 (s, 3H α , -OCOCH 3 , 6Hβ, -OCOCH 3 ), 1.18 (t, J=7.4 Hz, 3H α , -SCH 2 CH 3 ), 1.15 (t, J=7.4 Hz, 3Hβ, -SCH 2 CH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ170.9, 170.8, 169.7, 169.5, 169.4, 169.3, 169.3, 168.9, 93.4, 89.2, 75.5, 71.1, 69.0, 68.8, 66.6, 66.5, 62.3, 62.3, 50.6, 46.7, 24.0, 23.6, 21.0, 21.0, 20.9, 20.8, 20.8, 20.7, 14.5, 14.1 ppm; HRMS: Calculated for C 16 H 24 O 9 NaS [M+Na] + : 415.1014; found 415.1044 (−4.1 ppm). 4.1.11. 3-phenylthio-3-deoxyα ,β-D-glucopyranose tetraacetate (8 α ,β) It is ynthesize following the general procedure starting from compound 5(2.57 g, 7.29 mmol) in CF 3 COOH (15.08 mL, 196.97 mmol): H 2 O (2.26 mL), pyridine (74.14 mL) and acetic anhydride (33.79 mL, 357.46 mmol). The crude obtained is purified by column chromatography on silica gel (hexane/EtOAc 3:1), obtaining compound 8 α ,β(2.04 g, 4.64 mmol, 64 % yield) as a yellow syrup like a mixture of the two possible α and βanomers in a ratio of 1:0.8. 1 H NMR (500 MHz, CDCl 3 ): δ 7.47–7.43 (m, 2H α , -SC 6 H 5 , 2Hβ, -SC 6 H 5 ), 7.35–7.27 (m, 3H α , -SC 6 H 5 , 3Hβ, -SC 6 H 5 ), 6.26 (d, J=3.6 Hz, 1H α , H 1 ), 5.67 (d, J=8.1 Hz, 1Hβ, H 1 ), 5.09–5.00 (m, 1H α , H 4 , 2Hβ, H 2 , H 4 ), 4.86 (dd, J=11.7 and 3.6 Hz, 1H α , H 2 ), 4.20–4.17 (m, 1H α , H 6 , 1Hβ, H 6 ), 4.10–4.02 (m, 2H α , H 5 , H 6’ , 1Hβ, H 6’ ), 3.79 (ddd, J=9.7, 4.9 and 2.4 Hz, 1Hβ, H 5 ), 3.51 (t, J=11.3 Hz, 1H α , H 3 ), 3.36 (t, J=10.9 Hz, 1Hβ, H 3 ), 2.16 (s, 3H α , -OCOCH 3 ), 2.09 (s, 3Hβ, -OCOCH 3 ), 2.08 (s, 3H α , -OCOCH 3 ), 2.07 (s, 3Hβ, -OCOCH 3 ), 2.03 (s, 3H α , -OCOCH 3 ,), 2.00 (s, 3H α , -OCOCH3), 1.98 (s, 6Hβ, -OCOCH 3 ) ppm; 13 C NMR (125 MHz, CDCl 3 ): δ170.9, 170.8, 169.6, 169.5 (2), 169.3, 169.2, 168.9, 133.7 (2), 133.1 (3), 132.4, 129.3 (4), 128.4, 128.2, 93.3, 89.1, 75.5, 71.1, 70.6, 69.3, 68.4, 68.1, 62.2, 62.2, 54.1, 49.9, 21.1, 21.0, 20.9, 20.8 (2), 20.7, 20.6 ppm; HRMS: Calculated for C 20 H 24 O 9 NaS [M+Na] + : 463.1033; found 463.1026 (−1.6 ppm). 4.1.12. 2,3,4,6-tetra-O-acetyl-1-azido-1-deoxyα -D-glucopyranoside (9 α ) To a solution of β−d-glucose pentaacetate (500 mg, 1.28 mmol) in CH 2 Cl 2 (3 mL) under an argon atmosphere, AlCl 3 (128 mg, 0.96 mmol) is added. Once the starting product is consumed, after stirring for 8 h, hexane (10 mL) is added and a white precipitate appears that is filtered over Celite. To completely eliminate the white solid, the mixture is centrifugated at 1200 rpm for 15 min. The supernatant is evaporated under reduced pressure. In this way, 399 mg of the chlorine derivative is obtained, which is used directly in the following reaction. To a solution of the chlorine derivative (399 mg, 1.09 mmol) in THF (11 mL) under an argon atmosphere, TMSA (0.20 mL, 1.53 mmol) and Bu 4 NF (1 M in THF) are added (1.53 mL, 1.53 mmol) and left stirring at 65 ◦C overnight. Once the starting product is consumed, the solvent is evaporated under reduced pressure. The crude oil obtained is purified by silica gel column chromatography (hexane/EtOAc 4:1), obtaining 9 α (193 mg, 0.52 mmol, 47 % yield) as a yellow solid. M.p.: 115–118 ◦C. 1 H NMR 500 MHz, CDCl 3 :δ5.60 (d, J=4.3 Hz, 1H, H 1 ), 5.38 (t, J=9.8 Hz, 1H, H 3 ), 5.05 (t, J=9.9 Hz, 1H, H 4 ), 4.95 (dd, J=10.2 and 4.3 Hz, 1H, H 2 ), 4.27 (dd, J=12.4 and 4.5 Hz, 1H, H 6 ), 4.18–4.12 (m, 2H, H 5 , H 6’ ), 2.10 (s, 3H, -OCOCH 3 ), 2.09 (s, 3H, -OCOCH 3 ), 2.03 (s, 3H, -OCOCH 3 ), 2.01 (s, 3H, -OCOCH 3 ) ppm. 13 C NMR 125 MHz, CDCl 3 :δ170.7, 170.1 (2), 169.6, 86.4, 70.3, 69.8, 69.7, 68.1, 61.7, 20, 8, 20.7 (3) ppm. HRMS: Calculated for C 14 H 19 O 9 N 3 Na [M+Na] + : 396.1014; found 396.1006 (−1.9 ppm). 4.1.13. General procedure for the synthesis of 1-azido derivatives To a solution of the corresponding α ,β-D-glucopyranose derivative (100 mol%) in CH 2 Cl 2 , SnCl 4 (130 mol%) is added under an argon atmosphere, followed by trimethylsilyl azide TMSA (180 mol%). Once the starting product is consumed (1–3 h), distilled H 2 O is added. The aqueous phase is extracted with CH 2 Cl 2 (x 3) and the combined organic phases are washed with 10 % HCl (x 3) and saturated NaHCO 3 solution (x 3). Dry over anhydrous Na 2 SO 4 and evaporate the solvent under reduced pressure. 4.1.14. 2,3,4,6-tetra-O-acetyl-1-azido-1-deoxy-β-D-glucopyranoside (9β) It is synthesized following the general procedure starting from β-Dglucose pentaacetate (500 mg, 1.28 mmol) in CH 2 Cl 2 (3 mL), SnCl4 (0.18 mL, 1.54 mmol) and TMSA (0.30 mL, 2.31 mmol). In this way, 9β (476 mg, 1.27 mmol, quant. yield) is obtained with a high degree of purity as a white solid; M.p.: 134–136 ◦C. 1 H NMR: (500 MHz, CDCl 3 ): δ 5.22 (t, J=9.5 Hz, 1H, H 3 ), 5.11 (t, J=9.8 Hz, 1H, H 4 ), 4.96 (t, J=9.2 Hz, 1H, H 2 ), 4.65 (d, J=8.9 Hz, 1H, H 1 ), 4.27 (dd, J=12.5 and 4.8 Hz, 1H, H 6 ), 4.17 (dd, J=12.5 and 2.3 Hz, 1H, H 6’ ), 3.80 (ddd, J=10.1, 4.8 and 2.3 Hz, 1H, H 5 ), 2.10 (s, 3H, -OCOCH 3 ), 2.07 (s, 3H, -OCOCH 3 ), 2.03 (s, 3H, -OCOCH 3 ), 2.01 (s, 3H, - OCOCH 3 ) ppm; 13 C NMR: (125 MHz, CDCl 3 ): δ170.8, 170.3, 169.5, 169.3, 88.1, 74.2, 72.8, 70.8, 68.1, 61, 8, L.A. Prieto et al. European Journal of Medicinal Chemistry 289 (2025) 117469 9