Synthesis of sp2 -Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties

molecules Article Synthesis of sp2-Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties Elena M. Sánchez-Fernández 1,* , Raquel García-Hernández 2, Francisco Gamarro 2, Ana I. Arroba 3, Manuel Aguilar-Diosdado 3, JoséM. Padrón4, JoséM. García Fernández 5and Carmen Ortiz Mellet 1   Citation: Sánchez-Fernández, E.M.; García-Hernández, R.; Gamarro, F.; Arroba, A.I.; Aguilar-Diosdado, M.; Padrón, J.M.; García Fernández, J.M.; Ortiz Mellet, C. Synthesis of sp2Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties. Molecules 2021,26, 7501. https:// doi.org/10.3390/molecules26247501 Academic Editor: RenéCsuk Received: 5 November 2021 Accepted: 8 December 2021 Published: 11 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Organic Chemistry, Faculty of Chemistry, University of Seville, C/Profesor García González 1, 41012 Seville, Spain; [email protected] 2Instituto de Parasitología y Biomedicina “López-Neyra”, Parque Tecnológico de Ciencias de la Salud, 18016 Granada, Spain; [email protected] (R.G.-H.); [email protected] (F.G.) 3Research Unit, Biomedical Research and Innovation Institute of Cádiz, Puerta del Mar University Hospital, Av/Ana de Viya 21, 11009 Cádiz, Spain; anaarr[email protected] (A.I.A.); [email protected] (M.A.-D.) 4 BioLab, Instituto Universitario de Bio-Orgánica Antonio González, Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain; [email protected] 5 Instituto de Investigaciones Químicas, CSIC-University of Seville, Américo Vespucio 49, 41092 Sevilla, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-954-559-997 Abstract: sp 2 -Iminosugar glycolipids (sp 2 -IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp 2 -hybridized N-atom imparts chemical and enzymatic stability to sp 2 -IGLs and warrants total α -anomeric stereoselectivity in the key glycoconjugation step. A variety of O-, N-, Cand S-pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp 2 -IGLs by reporting the synthesis of α -D-gluco-configured analogs with a bicyclic (5N,6O-oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure–activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp 2 -IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C 12 vs. C 8 ), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate. Keywords: seleno-sp 2 -iminoglycolipids; multitarget; immunomodulation; cancer; Leishmania; inflammation 1. Introduction The outstanding beneficial effects of selenium-containing molecules in a broad spectrum of pathologies have boosted research in this area over the last two decades [ 1 – 4 ]. Thus, a large variety of organoselenium compounds encompassing selenide, diselenide, selenocyanate, selenoester, selenocarbamate, selenazo or selenourea derivatives have shown remarkable antitumor [ 5 , 6 ], antiinfective [ 7 ], anti-inflammatory [ 8 ] and antioxidant [ 9 ] properties. In the field of carbohydrates, several selenium-containing products with interesting biological properties have been reported. Some representative examples are depicted in Figure 1. The antimetastatic N-glycoside 1 and its analogue 2 , both incorporating the known Ebselen heterocycle in their structures [ 10 ], target multiple protein Molecules 2021,26, 7501. https://doi.org/10.3390/molecules26247501 https://www.mdpi.com/journal/molecules Molecules 2021,26, 7501 2 of 15 kinase-signalling cascades involved in cancer progression and inflammation [ 11 – 14 ]. The symmetric selenodigalactoside 3 behaves as a relevant ligand for human galectins [ 15 ], whereas α -Se-galactosyl ceramide 4 [ 16 ], a mimetic of the powerful immunostimulant glycolipid α -galactosyl ceramide (KRN7000) [ 17 ], shows interest as a potential adjuvant. In general terms, substitution of the glycosidic oxygen atom by Se results in conjugates that are more robust against enzymatic and chemical degradation, as is also the case with other heteroatoms, such as N or S, empowering the synthesis and biological evaluation of valuable glycomimetics [18–22]. Figure 1. Chemical structures of biologically relevant sugar-based molecules incorporating nitrogen, carbon, sulfur and selenium as glycosidic linkages (1–7). The majority of available methods for the synthesis of X-glycosides (X= heteroatom other than oxygen) mainly afford the β -anomer or α , β -anomeric mixtures [ 23 – 26 ], the stereoselective synthesis of α -anomers being consistently more demanding [ 27 – 29 ]. sp 2 - Iminosugars remain a unique exception to the rule [ 30 – 33 ]. The distinctive architecture of these sugar analogues, which bear a pseudoamide-type nitrogen, with substantial sp 2 - hybridation in place of the endocyclic oxygen of monosaccharides, provokes a strong reinforcement of the anomeric effect that compels the axial orientation of pseudoanomeric substituents. This property warrants total α -stereochemical control in glycosylation reactions and imparts chemical and enzymatic stability to the resulting α -pseudoglycosides [ 34 , 35 ], a scenario sharply different to that encountered with classical iminosugars. It has been already capitalized in the design of specific glycosidase inhibitors and effectors [ 36 , 37 ], lectin ligands [ 38 – 41 ] and tumor-associated carbohydrate antigen (TACA) mimics [ 42 , 43 ], including a sp 2 -iminosugar glycopeptide-based anticancer vaccine [ 44 ]. Conjugates conjoining a sp 2 -minosugar glycone and an α -oriented lipid aglycone, generically termed sp 2 -iminosugar glycolipids (sp 2 -IGLs), have also shown promising abilities as innate immune system regulators with anti-inflammatory [ 45 – 47 ], anticancer [ 48 , 49 ] and antiparasitic [ 50 , 51 ] behaviors. A variety of sp 2 -IGLs with C-, N-, Oand S-pseudoglycosidic bonds are already on record [ 52 – 54 ]. Structure–activity relationship studies have shown that (i) the lipid aglycone plays an essential role in the therapeutic effect of the sp 2 -IGLs; and (ii) the nature of the glycosidic functionality bridging the aglycone and lipid moieties is also critical for activity. The configurational profile of the glycomimetic component is comparatively less influencing. For instance, epimeric compounds displaying hydroxylation patterns of stereochemical complementarity with D-glucopyranose or D-galactopyranose behave similarly in different settings [ 50 , 53 ]. Investigations into the molecular mechanism at play revealed that the sp 2 -IGLs interfere with kinase signaling pathways. Thus, in breast cancer cells, the C-octyl nojirimycin-related derivative 5 (Figure 1) promoted a reduction in the phosphorylation levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2) [ 48 ], a member of the mitogen-activated protein Molecules 2021,26, 7501 3 of 15 kinase (MAPK) group. The sulfone derivative 6 and the N-glycoside 7 (Figure 1) triggered activation of p38 α -MAPK, a master regulator of inflammation. Indeed, computational experiments suggested that sp 2 -IGLs can bind to the lipid-binding pocket of p38 and induce its autophosphorylation [50,55]. Given the therapeutic potential of selenium-containing compounds, the synthesis of seleno-sp 2 -IGLs seemed very appealing in this context. Here we present the stereoselective preparation of the first members of this category, namely compounds 8 – 10 (Figure 2). Their in vitro antiproliferative, leishmanicidal and anti-inflammatory activities, in comparison with N-(11,12) and S-(13,14) analogues, are also reported. Figure 2. Chemical structures of the sp 2 -IGLs derived from nojirimycin evaluated in this study (8–14). 2. Results and Discussion 2.1. Design and Synthesis The ensemble of data collected on the immunomodulatory behavior of sp 2 -IGLs [45–55] instils that linear aliphatic tails with a length equal or higher than C 8 are required to elicit significant anticancer, antiparasitic and/or anti-inflammatory responses. Accordingly, we focused on n-octyl and n-docecyl aglycone lipid tails in this study. As the glycone moiety, the bicyclic 5N,6O-oxamethylidenenojirimycin (ONJ) core was chosen since ONJ-based conjugates have already demonstrated high promise as drug candidates. In order to evaluate the impact of the nature of the glycosidic atom on the pharmacological properties, we settled on synthesizing the new seleno-sp 2 -IGLs 9 and 10 , and the homologous aminoand thio-glycosides 11 , 12 and 13 , 14 , respectively. The preparation of the phenyl selenoglycoside 8 , expectedly a negative control, was additionally undertook to test the robustness of the selenoglycosylation reaction. The α -phenyl(octyl)(dodecyl) ONJ pseudoanomeric selenides 8 – 10 were synthesized from (1R)-1,2,3,4-tetra-O-acetyl-5N,6O-oxomethylidenenojirimycin ( 16 ), accessible through an efficient synthetic scheme from D-glucuronolactone [ 56 ], by reaction with phenyl, octyl or docecyl selenol, respectively, in the presence of boron trifluoride etherate (BF 3· Et 2 O) as glycosidation promotor ( →17 – 19 ) (Scheme 1). The aliphatic selenides were prepared from the corresponding commercially available bromoalkanes, elemental selenium and sodium borohydride [ 57 ]. Final conventional de-O-acetylation under Zemplén conditions afforded the target fully unprotected α -pseudoglycosyl selenides 8 – 10 (Scheme 1). This reaction scheme parallels that previously optimized for the synthesis of the S-linked analogues 13 [ 34 ] and 14 [ 51 ]. The N-dodecyl derivative 12 on its side was straightforward prepared following the reported experimental procedure used for the synthesis of the N-octyl sp 2 - IGL 11 [ 34 ]. A solution of reducing ONJ ( 15 ) and commercially available dodecylamine in methanol heated at 65 ◦ C for 24 h led to the gem-diamine-type conjugate 12 in 67% yield after purification by column chromatography (Scheme 1). Only the α -anomer, with the lipid aglycone in axial orientation, was detected in the reaction mixture, even in this later case, which is amazing considering the overwhelming tendency of classical glycosylamines to enforce the equatorial disposition of the anomeric substituent by virtue of the reverse anomeric effect [58]. The structure of all compounds was confirmed by 1 H, 13 C and 77 Se (for Se-derivatives), NMR, MS and combustion analysis. Note that all the Se-, Sand N-linked sp 2 -IGLs synthesized adopt a 4 C 1 chair type conformation with vicinal 3 J 1,2 coupling constants ranging between 4.8–6.0 Hz, characteristic of the α-anomeric configuration. Molecules 2021,26, 7501 4 of 15 Scheme 1. Synthesis of the novel pseudoα -N-dodecyl ( 12 ) and Se-phenyl(octyl)(dodecyl) glycosides (8–10). 2.2. Antiproliferative and Antiparasitic Properties of the sp2-IGLs In order to assess whether the anticancer potential and the capabilities to inhibit parasite growth of this family of sp 2 -glycoconjugates are interrelated, in vitro evaluation of the antiproliferative and antiparasitic properties was conducted in parallel. Highthroughput screening (HTS) against a panel of different human solid tumor cell lines including lung (A549, SW1573), breast (HBL-100, T-47D), cervix (HeLa) and colon (WiDr) cancer linages, helped us determine the antiproliferative potential of these sp 2 -IGLs. As for the antiparasitic activity, the growth inhibition of intracellular amastigotes of Leishmania donovani HU3 species, responsible for visceral Leishmaniasis (VL), was investigated. The results, expressed as the concentration to achieve 50% growth inhibition of tumor cells (GI 50 ) and the concentration of compound that reduces cell growth by 50% versus untreated control cells (EC 50 ) for arresting parasite development, are collected in Figure 3and Table 1 , respectively. Figure 3. Comparison of the antiproliferative activity of sp 2 -IGLs 8 – 14 after evaluation by HTS. GI 50 values and standard deviation (SD) from three independent experiments are depicted. The critical effect of the lipid chain length on the antiproliferative activity was evident in the gem-diamine derivatives. The N-glycoside-type sp 2 -IGL 12 , bearing a dodecyl chain, Molecules 2021,26, 7501 5 of 15 showed GI 50 values in the 18–30 µ M range against all the cell lines tested, while the N-octyl derivative 11 was over the threshold in this assay (GI 50 > 100 µ M) in all cases. Although to a lesser extent, the same trend was encountered for Sand Se-sp 2 -IGLs. Thus, the dodecyl derivatives 14 and 10 displayed GI 50 values of 16–28 µ M, whereas the Sand Se-octyl counterparts, 13 and 9 , afforded GI 50 values from 36 µ M to >100 µ M (Figure 3). The antiproliferative activity of the N-, Sand Se-dodecyl sp 2 -IGLs ( 12 , 14 and 10 ) is similar to that of the antineoplastic agent 5-fluorouracyl (5-FU) in the same assay (GI 50 5–50 µ M) [ 9 ]. The utmost importance of the aliphatic lipid chain was further highlighted by the lack of activity of the aromatic Se-pseudoglycoside derivative 8 . Bearing in mind the variation of pH in tumor cells with respect to normal cells, specifically the acidity of the extracellular environment of a tumor tissue [ 59 ], stability experiments of selenoglycosides 8 and 9 at pH~4 were performed. The 1 H-NMR spectra obtained after monitoring for 12 h in formate buffer evidenced their stability (See Supplementary Information). Whilst in the dodecyl series the atom of the glycosidic linkage was essentially irrelevant for the antiproliferative potency, it was found to be a major determinant regarding the antiparasitic activity. Only the α -dodecylselenide 10 showed an EC 50 value ( 13.42 ±1.63 µM ) for annihilation of the intracellular form of the protozoan parasite (amastigote forms) below the threshold (20 µ M) in this assay (Table 1). Although this still implies an over one-order-of-magnitude lower efficacy as compared with the reference drug miltefosine (EC 50 0.44 ± 0.08 µ M), it represents an interesting hit susceptible of optimization, given the versatility of the synthetic approach. Moreover, the toxicity profile against the monocytic and lung cell lines THP-1 and MRC-5, respectively, was not significantly different from that of miltefosine. Altogether, the data underline the α -dodecyl selenoglycoside 10 as a promising leishmanicidal agent in terms of potency and safety. Table 1. EC 50 ( µ M) values of sp 2 -IGLs 8–14 against intracellular amastigote forms of Leishmania donovani HU3 and cytotoxicity profile in THP-1 and MRC-5 cells 1. Compound Intracellular Amastigotes HU3 THP-1 Cell Line MRC-5 Cell Line 8(Se-Ph) >20 >200 172.16 ±27.02 9(Se-C8) >20 >200 94.31 ±4.95 10 (Se-C12) 13.42 ±1.63 84.25 ±0.06 31.22 ±1.61 11 (NH-C8) >20 208.69 ±10.32 154.52 ±5.30 12 (NH-C12) >20 122.50 ±13.30 49.60 ±4.20 13 (S-C8) >20 122.33 ±5.79 58.14 ±12.62 14 (S-C12) >20 70.46 ±3.75 43.07 ±9.83 Miltefosine 0.44 ±0.08 26.86 ±3.08 57.58 ±6.38 1Data are means of EC50 ±SD from three independent experiments. 2.3. Anti-inflammatory Properties of the sp2-IGLs The most active sp 2 -IGL in the tumor and parasite growth inhibition trials, namely the Se-dodecyl sp 2 -IGL 10 , was further assessed as an anti-inflammatory agent. Towards this end, its effect on nitrite production and on the expression of induced nitric oxide synthase (iNOS), major mediators of inflammation [ 60 , 61 ], under an inflammatory context has been investigated in comparison with the S-glycoside analogue 14. Firstly, cell viability assays using a murine microglia Bv.2 cell line were performed after treatment with different concentrations of 14 and 10 for 24 h (Figure 4). As observed in Figure 4A,B, none of the concentrations used induced a negative effect on the cellular viability. Molecules 2021,26, 7501 6 of 15 Figure 4. ( A ) Effect of Se-glycoside 10 and ( B )S-glycoside 14 on the cellular viability (Bv.2 cells) determined by crystal violet staining. ( C ) Effect of 10 and ( D ) 14 on nitrite production. Bv.2 microglial cells were treated for 24 h with LPS (200 ng/mL) or LPS plus 10 (1–10 µ M) and 14 (1–50 µ M), respectively. Nitrite production was analyzed and related to the basal levels. Colorimetric quantification was performed. ( E ) Effect of 10 at 10 µ M and ( F ) 14 at 50 µ M on the expression of the LPS-induced iNOs protein. ( G ) Effect of 10 at 10 µ M and ( H ) 14 at 50 µ M on the levels of the anti-inflammatory marker arginase-1 in the absence or in the presence of LPS in Bv.2 microglial cells. In all cases, data represent mean ± S.E.M of fold changes relative to the basal values. * p ≤ 0.05 vs. Basal condition; # p ≤ 0.05 vs. LPS stimuli; and p ≤ 0.05 vs. L + C 10 (1 µ M) or L + C 14 (10 µ M) (two-way ANOVA followed by Bonferroni t-test). Molecules 2021,26, 7501 7 of 15 To gauge the effect of these compounds on nitrite production, Bv.2 cells were stimulated with bacterial lipopolysaccharide (LPS; 200 ng/mL), a pro-inflammatory stimulus, in the absence and in the presence of the Sand Se-sp 2 -IGLs ( 14 and 10 ), respectively, for 24 h. Both compounds exerted a dose-dependent reduction on nitrite production ( Figure 4C,D ), which was significantly more pronounced in the case of the selenide derivative 10 (basal level reached at 10 µ M) as compared with the sulfide analogue 14 (basal level reached at 50 µ M). Additionally, treatment with 10 at 10 µ M fully abrogated the enhancement of iNOS elicited by LPS, whereas 14 at a five-fold higher concentration only reduced iNOS increase from 20-fold to about 8-fold (Figure 4E,F). Compound 10 at 10 µ M also showed a direct effect on the induction of arginase-1, a marker of the classical antiinflammatory response (M2) in microglia, either in the absence or in the presence of LPS (200 ng/mL; Figure 4G ). However, treatment with 14 did not induce arginase-1 expression (Figure 4H). Taken together, these data accentuate the strong anti-inflammatory potential of selenium-linked sp2-IGLs. 3. Materials and Methods 3.1. General Methods Reagents and solvents were purchased from commercial sources. 1 H, 13 C and 77 Se NMR experiments were performed at 300, 75.5 and 95.4 MHz, respectively. 2-D COSY and HMQC experiments were carried out to assist on signal assignment. 1 H-NMR-monitored kinetic evaluation of the stability of selenoglycosides ( 8 and 9 ) were performed at 500 MHz. For ESI mass spectra, 0.1 pM sample concentrations were used, mobile phase 50% aq MeCN at 0.1 mL min −1 . Thin-layer chromatography was performed on precoated TLC plates, silica gel 30F-245, with visualization by UV light and by carrying with 0.2% w/v cerium (IV) sulphate-5% ammonium molybdate in 2 M H 2 SO 4 or 0.1% ninhydrin in EtOH. Column chromatography was performed on Chromagel (silice 60 AC.C 70–200 µ m). Optical rotations were measured with a JASCO P-2000 polarimeter, using a sodium lamp ( λ= 589 nm ) at 22 ◦ C in 1 cm tube. All compounds were purified to ≥ 95% purity as determined by elemental microanalysis results obtained on a CHNSTruSpect Micro elemental analyzer (Instituto de Investigaciones Químicas de Sevilla, Spain) from vacuum-dried samples. The analytical results for C, H, N, and S were within ± 0.4 of the theoretical values. Deacetylation reactions were carried out by using a Zemplén procedure [ 62 ]. Addition of NaOMe (0.1 equiv/Ac mol) in MeOH at room temperature, followed by neutralization with solid CO 2 , evaporation of the solvent and purification by column chromatography. (1R)-5N,6O-Oxomethylidenenojirimycin ( 15 ) [ 56 ], (1R)-1,2,3,4-tetra-O-acetyl5N,6O-oxomethylidenenojirimycin ( 16 ) [ 56 ], (1S)-1-amino-N-octyl-5N,6O-oxomethylidene1-deoxynojirimycin ( 11 ) [ 34 ], (1R)-1-octylthio-5N,6O-oxomethylidenenojirimycin ( 13 ) [ 34 ], (1R)-1-dodecylthio-5N,6O-oxomethylidenenojirimycin ( 14 ) [ 51 ] were prepared according to previously reported procedures. Octyl selenol and dodecyl selenol were prepared from octyl bromide and dodecyl bromide, respectively, according to the literature procedure [ 57 ]. 3.2. Synthesis of the sp2-IGLs 3.2.1. Procedure for the Synthesis of Pseudo-N-Glycoside (12) A solution of (1R)-5N,6O-oxomethylidenenojirimycin ( 15 ) (0.24 mmol) and the ndodecylamine (0.24 mmol) in MeOH (3 mL) was heated at 65 ◦ C for 24 h under Ar atmosphere. The solvent was eliminated under reduced pressure and the resulting residue was purified by column chromatography to afford the corresponding α-glycosylamine (12). (1S)-1-Amino-N-dodecyl-5N,6O-oxomethylidene-1-deoxynojirimycin ( 12 ). Column chromatography (20:1 DCM-MeOH). Yield: 60 mg (67%). R f 0.69 (50:10:1 DCM-MeOHH 2 O). [ α ] D + 59.8 (c0.9 in MeOH). 1 H NMR (300 MHz, CD 3 OD): δ 4.63 (d, 1 H, J 1,2 = 5.1 Hz, H-1), 4.48 (t, 1 H, J 6a,6b =J 5,6a = 8.5 Hz, H-6a), 4.27 (dd, 1 H, J 5,6b = 4.8 Hz, H-6b), 3.82 (ddd, 1 H, J 4,5 = 9.5 Hz, H-5), 3.61 (t, 1 H, J 2,3 =J 3,4 = 9.5 Hz, H-3), 3.47 (dd, 1 H, H-2), 3.26 (t, 1 H, H-4), 2.63–2.53 (m, 2 H, NHCH 2 ), 1.58–1.20 (m, 20 H, CH 2 ), 0.90 (t, 3 H, 3 J H,H = 6.9 Hz, CH 3 ). 13 C NMR (75.5 MHz, CD 3 OD): δ 159.2 (CO), 75.6 (C-4), 74.5 (C-3), 72.3 (C-2), Molecules 2021,26, 7501 8 of 15 69.6 (C-1) , 67.9 (C-6), 54.6 (C-5), 47.5 (CH 2 NH), 33.1–23.7 (CH 2 ), 14.4 (CH 3 ). ESIMS: m/z 395.4 [M + Na] + . Anal. Calcd for C 19 H 36 N 2 O 5 : C 61.26, H 9.74, N 7.52. Found: C 61.14, H 9.58, N 7.33. 3.2.2. General Procedure for the Synthesis of Pseudo-Se-Glycosides (17–19) To a stirred solution of (1R)-1,2,3,4-tetra-O-acetyl-5N,6O-oxomethylidenenojirimycin ( 16 ) (150 mg, 0.40 mmol) in anhydrous DCM (8 mL) under Ar atmosphere, the corresponding selenol (0.84 mmol, 2.1 equiv.) and BF 3 .Et 2 O (0.18 mL, 1.41 mmol, 3.5 equiv.) were added at 0 ◦ C. The mixture was stirred for 15 min, diluted with DCM (30 mL), washed with water (10 mL), saturated aqueous solution of NaHCO 3 (2 × 10 mL) and water (10 mL), dried (MgSO 4 ), filtered and concentrated to afford the corresponding per-O-acetylated selenoglycosides. The pure α -anomer O-acetylated intermediates ( 17 – 19 ) were obtained after purification by column chromatography using the solvents indicated in each case. (1R)-2,3,4-Tri-O-acetyl-1-phenylseleno-5N,6O-oxomethylidenenojirimycin ( 17 ). Column chromatography (1:2 EtOAc-cyclohexane). Yield: 53 mg (84%). R f 0.78 (2:1 EtOAccyclohexane). [ α ] D + 142.7 (c1.1 in DCM). 1 H NMR (300 MHz, CDCl 3 ) δ 7.70–7.25 (m, 5 H, Ph), 6.10 (d, 1 H, J 1,2 = 5.7 Hz, H-1), 5.60 (t, 1 H, J 2,3 =J 3,4 = 9.6 Hz, H-3), 5.03 (dd, 1 H, H-2), 4.95 (t, 1 H, J 4,5 = 9.6 Hz, H-4), 4.29–4.04 (m, 3 H, H-5, H-6a, H-6b), 2.09–2.06 (3 s, 9 H, MeCO). 13 C NMR (75.5 MHz, CDCl 3 ) δ 169.9–169.3 (MeCO), 154.2 (CO), 136.1–125.2 (Ph), 72.3 (C-4), 70.7 (C-2, C-3), 65.6 (C-6), 54.7 (C-1), 51.9 (C-5), 20.5 (MeCO). 77 Se NMR (95.4 MHz, CDCl 3 ) δ 347.1. ESIMS: m/z 494.1 [M + Na] + . Anal. Calcd for C 19 H 21 NO 8 Se: C 48.52, H 4.50, N 2.98. Found: C 48.61, H 4.57, N 2.84. (1R)-2,3,4-Tri-O-acetyl-1-octylseleno-5N,6O-oxomethylidenenojirimycin ( 18 ). Column chromatography (1:3 EtOAc-cyclohexane). Yield: 150 mg (74%). R f 0.85 (2:1 EtOAccyclohexane). [ α ] D + 93.3 (c1.1 in DCM). 1 H NMR (300 MHz, CDCl 3 ) δ 5.88 (d, 1 H, J 1,2 = 6.0 Hz, H-1), 5.35 (t, 1 H, J 2,3 =J 3,4 = 9.6 Hz, H-3), 4.93 (t, 1 H, J 4,5 = 9.6 Hz, H-4), 4.86 (dd, 1 H, H-2), 4.42 (t, 1 H, J 6a,6b =J 5,6a = 9.0 Hz, H-6a), 4.25 (dd, 1 H, J 5,6b = 7.0 Hz, H-6b), 4.11 (td, 1 H, H-5), 2.66 (ddd, 1 H, 2 J H,H = 12.0 Hz, 3 J H,H = 8.1 Hz, 3 J H,H = 6.6 Hz, SeCH 2 ), 2.51 (ddd, 1 H, SeCH 2 ), 2.05–2.00 (3 s, 9 H, MeCO), 1.70–1.50 (m, 2 H, SeCH 2 CH 2 ), 1.37–1.17 (m, 10 H, CH 2 ), 0.84 (t, 3 H, 3 J H,H = 7.0 Hz, CH 3 ). 13 C NMR (75.5 MHz, CDCl 3 ) δ 170.0–169.4 (MeCO), 155.0 (CO), 72.5 (C-4), 70.5 (C-2, C-3), 66.1 (C-6), 51.8 (C-5), 51.2 (C-1), 31.7–22.6 (CH 2 ), 20.7–20.5 (MeCO), 14.1 (CH 3 ). 77 Se NMR (95.4 MHz, CDCl 3 ) δ 219.9. ESIMS: m/z 530.2 [M + Na] + . Anal. Calcd for C 21 H 33 NO 8 Se: C 49.80, H 6.57, N 2.77. Found: C 49.96, H 6.69, N 2.72. (1R)-2,3,4-Tri-O-acetyl-1-dodecylseleno-5N,6O-oxomethylidenenojirimycin ( 19 ). Column chromatography (1:2 EtOAc-cyclohexane). Yield: 140 mg (62%). R f 0.57 (1:1 EtOAccyclohexane). [ α ] D + 82.8 (c1.0 in DCM). 1 H NMR (300 MHz, CDCl 3 ) δ 5.91 (d, 1 H, J 1,2 = 6.0 Hz, H-1), 5.38 (t, 1 H, J 2,3 =J 3,4 = 9.6 Hz, H-3), 4.92 (t, 1 H, J 4,5 = 9.6 Hz, H-4), 4.86 (dd, 1 H, H-2), 4.44 (t, 1 H, J 6a,6b =J 5,6a = 9.0 Hz, H-6a), 4.27 (dd, 1 H, J 5,6b = 9.0 Hz, H-6b), 4.14 (ddd, 1 H, H-5), 2.68 (ddd, 1 H, 2JH,H = 12.0 Hz, 3JH,H = 8.4 Hz, 3JH,H = 6.6 Hz, SeCH 2 ), 2.54 (ddd, 1 H, SeCH 2 ), 2.07–2.02 (3 s, 9 H, MeCO), 1.70–1.20 (m, 20 H, CH 2 ), 0.87 (t, 3 H, 3 J H,H = 7.0 Hz, CH 3 ). 13 C NMR (75.5 MHz, CDCl 3 ) δ 170.0–169.4 (MeCO), 155.0 (CO), 72.5 (C-4), 70.6–70.5 (C-2, C-3), 66.1 (C-6), 51.9 (C-5), 51.3 (C-1), 31.9–29.0 (CH 2 ), 24.0 (SeCH 2 ), 20.7–20.5 (MeCO), 14.1 (CH 3 ). 77 Se NMR (95.4 MHz, CDCl 3 ) δ 219.9. ESIMS: m/z 586.3 [M + Na] + . Anal. Calcd for C 25 H 41 NO 8 Se: C 53.38, H 7.35, N 2.49. Found: C 53.42, H 7.41, N 2.39. 3.2.3. General Procedure for the Synthesis of the Deprotected Pseudo-Se-Glycosides ( 8 – 10 ) To a stirred solution of the corresponding acetylated pseudo-Se-glycoside (0.20 mmol) in MeOH (4 mL), NaOMe 1 M (0.06 mmol) was added and the reaction mixture was stirred at room temperature for 30–60 min. Neutralization with solid CO 2 , evaporation of the solvent and purification by column chromatography using the solvents indicated in each case yielded the fully deprotected selenoglycosides 8–10. Molecules 2021,26, 7501 9 of 15 (1R)-1-Phenylseleno-5N,6O-oxomethylidenenojirimycin ( 8 ). Column chromatography (9:1 EtOAc-MeOH). Yield: 21 mg (95%). R f 0.61 (9:1 EtOAc-MeOH). [ α ] D + 228.5 (c0.8 in MeOH). 1 H NMR (300 MHz, CD 3 OD) δ 7.70–7.20 (m, 5 H, Ph), 5.80 (d, 1 H, J 1,2 = 4.8 Hz, H-1), 4.29 (t, 1 H, J 6a,6b =J 5,6a = 9.0 Hz, H-6a), 4.17 (dd, 1 H, J 5,6b = 5.7 Hz, H-6b), 3.89 (td, 1 H, J 4,5 = 9.4 Hz, H-5), 3.75–3.63 (m, 2 H, H-2, H-3), 3.38–3.31 (m, 1 H, H-4). 13 C NMR (75.5 MHz, CD 3 OD) δ 157.4 (CO), 134.6–127.7 (Ph), 76.1–72.9 (C-2, C-3, C-4), 67.6 (C-6), 60.2 (C-1), 54.8 (C-5). 77 Se NMR (95.4 MHz, CD 3 OD) δ 336.2. ESIMS: m/z 368.1 [M + Na] + . Anal. Calcd for C13H15NO5Se: C 45.36, H 4.39, N 4.07. Found: C 45.12, H 4.27, N 3.89. (1R)-1-Octylseleno-5N,6O-oxomethylidenenojirimycin ( 9 ). Column chromatography (6:1 EtOAc-MeOH). Yield: 72.4 mg (95%). R f 0.73 (1:4 MeOH-EtOAc). [ α ] D + 130.1 (c1.1 in MeOH). 1 H NMR (300 MHz, CD 3 OD) δ 5.56 (d, 1 H, J 1,2 = 4.8 Hz, H-1), 4.57 (t, 1 H, J 6a,6b =J 5,6a = 9.0 Hz, H-6a), 4.28 (dd, 1 H, J 5,6b = 6.3 Hz, H-6b), 3.92 (td, 1 H, J 4,5 = 9.0 Hz, H-5), 3.63–3.48 (m, 2 H, H-2, H-3), 3.36 (t, 1 H, J 3,4 = 9.0 Hz, H-4), 2.66–2.47 (m, 2 H, SeCH 2 ), 1.78–1.57 (m, 2 H, SeCH 2 CH 2 ), 1.45–1.24 (m, 10 H, CH 2 ), 0.90 (t, 3 H, 3 J H,H = 7.0 Hz, CH 3 ). 13 C NMR (75.5 MHz, CD 3 OD) δ 157.9 (CO), 76.0–75.4 (C-3, C-4), 72.6 (C-2), 68.1 (C-6), 56.4 (C-1), 54.9 (C-5), 32.9–23.5 (CH 2 ), 14.4 (CH 3 ). 77 Se NMR (95.4 MHz, CD 3 OD) δ 204.4. ESIMS: m/z 404.2 [M + Na] + . Anal. Calcd for C 15 H 27 NO 5 Se: C 47.37, H 7.16, N 3.68, Se 20.76. Found: C 47.17, H 7.15, N 3.53. (1R)-1-Dodecylseleno-5N,6O-oxomethylidenenojirimycin ( 10 ). Column chromatography (6:1 EtOAc-MeOH). Yield: 30 mg (97%). R f 0.61 (6:1 EtOAcMeOH). [ α ] D + 124.5 (c0.9 in MeOH). 1 H NMR (300 MHz, CD 3 OD) δ 5.55 (d, 1 H, J 1,2 = 5.1 Hz, H-1), 4.57 (t, 1 H, J 6a,6b =J 5,6a = 9.0 Hz, H-6a), 4.28 (dd, 1 H, J 5,6b = 6.0 Hz, H-6b), 3.92 (td, 1 H, J 4,5 = 9.0 Hz, H-5), 3.60–3.48 (m, 2 H, H-2, H-3), 3.40–3.31 (m, 1 H, H-4), 2.66–2.46 (m, 2 H, SeCH 2 ), 1.80–1.60 (m, 2 H, SeCH 2 CH 2 ), 1.45–1.20 (m, 18 H, CH 2 ), 0.90 (t, 3 H, 3 J H,H = 6.9 Hz, CH 3 ). 13 C NMR (75.5 MHz, CD 3 OD) δ 158.0 (CO), 76.0–75.5 (C-3, C-4), 72.7 (C-2), 68.2 (C-6), 56.5 (C-1), 54.9 (C-5), 33.0–23.5 (CH 2 ), 14.4 (CH 3 ). 77 Se NMR (95.4 MHz, CD 3 OD) δ 204.4. ESIMS: m/z 460.2 [M + Na] + . Anal. Calcd for C 19 H 35 NO 5 Se: C 52.29, H 8.08, N 3.21. Found: C 52.28, H 8.35, N 3.18. 3.3. Procedure for Antiproliferative Assays All reagents were used as purchased from commercial suppliers without further purification. The human solid tumor cell lines used in this study were: A549, SW1573 (lung), HBL-100, T-47D (breast), HeLa (cervix) and WiDr (colon). These cell lines were a kind gift from Prof. G.J. Peters (VU Medical Center, Amsterdam, The Netherlands). Chemosensitive Testing Cells were inoculated onto 96-well microtiter plates in a volume of 100 µ L per well at densities of 2500 (A549, HBL-100, HeLa, and SW1573) and 5000 (T-47D and WiDr) cells per well, based on their doubling times. Compounds were initially dissolved in DMSO at 400 times the desired final maximum test concentration. Control cells were exposed to an equivalent concentration of DMSO (0.25% v/v, negative control). Each agent was tested in triplicate at different dilutions in the range of 1–100 µ M. The drug treatment started on day 1 after plating. Drug incubation times were 48 h, after which cells were precipitated with 25 µ L ice-cold TCA (50% w/v) and fixed for 60 min at 4 ◦ C. Then the SRB assay was performed. The optical density (OD) of each well was measured at 530 nm, using BioTek’s PowerWave XS Absorbance Microplate Reader. Values were corrected for background OD from wells only containing medium. The antiproliferative activity for each compound, expressed as GI50 values, was calculated according to NCI formulas [63]. 3.4. Procedure for Antileishmanial Assays 3.4.1. Reagents For the biological assays, stock solutions of the synthesized compounds in DMSO at 10 mM were prepared. 3-(4,5-Dimethyltriazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis,