Selenoureido-iminosugars: A new family of multitarget drugs

European Journal of Medicinal Chemistry 123 (2016) 155e160

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

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Selenoureido-iminosugars: A new family of multitarget drugs

M. Padro
n c, Oscar
pez b, *, Jacob Ingemar Olsen a, b, Gabriela B. Plata c, Jose Lo a b
G. Ferna
ndez-Bolan ~ os Mikael Bols , Jose a

Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen Ø, Denmark
nica, Facultad de Química, Universidad de Sevilla, Apartado 1203, 41071 Seville, Spain Departamento de Química Orga c
nica “Antonio Gonza
lez” (IUBO-AG), Centro de Investigaciones Biom
BioLab, Instituto Universitario de Bio-Orga edicas de Canarias (CIBICAN), Universidad
nchez 2, E-38206 La Laguna, Spain de La Laguna, c/ Astrofísico Francisco Sa b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 May 2016 Received in revised form 9 July 2016 Accepted 11 July 2016 Available online 13 July 2016

Herein we report the synthesis of N-alkylated deoxynojirimycin derivatives decorated with a selenoureido motif at the hydrocarbon tether as an example of unprecedented multitarget agents. Title compounds were designed as dual drugs for tackling simultaneously the Gaucher disease (by selective inhibition of b-glucosidase, Ki ¼ 1.6e5.5 mM, with improved potency and selectivity compared to deoxynojirimycin) and its neurological complications (by inhibiting AChE, Ki up to 5.8 mM). Moreover, an excellent mimicry of the selenoenzyme glutathione peroxidase was also found for the catalytic scavenging of H2O2 (Kcat/Kuncat up to 640) using PhSH as a cofactor, with improved activity compared to known positive controls, like (PhSe)2 and ebselen; therefore, such compounds are also excellent scavengers of peroxides, an example of reactive oxygen species present at high concentrations in patients of Gaucher disease and neurological disorders. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Glycosidase inhibition Iminosugars Selenoureas AChE inhibition Antioxidant GPx mimic

1. Introduction Polyhydroxylated imino- and azasugars, that is, carbohydrate mimics where the endocyclic oxygen, or a carbon, respectively, has been replaced by a nitrogen atom, are attractive pharmacological agents due to their strong inhibitory properties against glycosidases and glycosyltransferases [1]. Although such enzymes exert pivotal functions in living beings, they also play a critical role in a series of pathologies [2], and thus, their selective inhibition has become a challenging research area [2,3]. In this context, glycosidase inhibitors have been claimed to be potentially useful in the treatment of lysosomal storage disorders (Gaucher, Fabry, Sandhoff or Tay-Sachs diseases) [4], and as anti-diabetic [5], antimicrobial [6], anti-cancer [7] and immunosuppressive [8] agents. Among the vast arsenal of glycosidase inhibitors reported so far, polyhydroxylated piperidines are particularly remarkable; a

Abbreviations: AChE, acetylcholinesterase; CCT, chemical chaperone therapy; DNJ, 1-deoxynojirimycin; ERT, enzyme replacement therapy; GBA1, b-glucosidase acid 1; GI50, growth inhibition 50%; GPx, glutathione peroxidase; ROS, reactive oxygen species; SRT, substrate-reduction therapy. * Corresponding author.
Lo
pez). E-mail address: [email protected] (O. http://dx.doi.org/10.1016/j.ejmech.2016.07.021 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

notorious example is the natural iminosugar 1-deoxynojirimycin (DNJ) 1 [9]. Some N-alkylated deoxynojirimycin derivatives, like n-BuDNJ 2 (Zavesca®, a glucosylceramide synthase inhibitor) [10], and the 2-hydroxyethyl counterpart 4 (Diastobol®, an intestinal aglucosidase inhibitor) [11] have reached the market for the treatment of Gaucher disease and diabetes-type 2, respectively. It has been demonstrated that a lengthening of the hydrocarbon chain (e.g. 3) provides better selectivity towards a-glucosidase, whereas a significant increase of the size and hydrophobicity of the N-alkyl substituent furnishes more potent glucosylceramide synthase inhibitors (e.g. 5) [1c,12]. Gaucher disease, the most frequent lysosomal storage disorder, is a recessively inherited disease caused by mutations in the glucocerebrosidase (GBA1) gene, leading to the misfolding of the bglucosidase responsible for the metabolic hydrolysis of glycoconjugates. As a result, the accumulation of partially-degraded metabolites in the lysosomes of several organs, including the brain, takes place causing severe damage to tissues and organs, and potentially, the death of the patient [13]. Currently, three possible treatments for Gaucher disease can be applied [1c]: enzyme replacement therapy (ERT), based on the administration of a recombinant glycosidase; substrate-reduction therapy (SRT), which uses glycosidase inhibitors to reduce the biosynthesis of

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glycosphingolipids; and the chemical chaperone therapy (CCT). The last approach is based on the fact that in some lysosomal storage disorders the defective glycosidase, although still keeps some of its catalytic activity, is misfolded, and thus, easily degraded by the endoplasmic reticulum quality-control system [1c]. Nevertheless, a high-affinity glycosidase inhibitor acting as a chemical chaperone can bind the mutant glycosidase at sub-inhibitory concentrations to induce its proper folding, and thus, to prevent its degradation. In this context, defective glucocerebrosidase, the enzyme responsible for the Gaucher disease, showed increased activity in cell cultures in the presence of 2 [14]. In fact, the efficiency of 2 for treating type 1 Gaucher has been claimed to be due to a combined action of inhibiting glucosylceramide synthase and a chaperone effect in misfolded glucocerebrosidase [15]. N-Bu-DNJ 2 also induced a pHdependent stabilization of imiglucerase and velaglucerase alfa, recombinant glycosidases currently used in the ERT, supporting the role of 2 as a chaperone for pharmaceutical formulations in Gaucher disease [16]. Moreover, neurological manifestations have also been found in a significant number of Gaucher disease patients [17]. Recently, it has been demonstrated that mutation in the GBA1 gene increases up to 30% the risk of developing synucleinopathies, mainly Parkinson and the Lewy body variant of Alzheimer’s disease, both in Gaucher patients and carriers [17], therefore leading also to parkinsonian symptoms. Furthermore, some of the physiological manifestations of dementia-type diseases are directly connected to acetylcholinesterase (AChE), either due to significant low levels of the neurotransmitter acetylcholine via its increased hydrolytic cleavage, or by promoting the deposit of b-amyloid plaques [18]; subsequently, AChE inhibitors have been suggested as therapeutic agents for improving the cognitive functionality in such kind of pathologies (cholinergic hypothesis) [19]. Herein our main target has been the design of a new family of dual drugs for tackling simultaneously the Gaucher disease and its neurological complications; for that purpose, we incorporated pharmacophores that afforded good b-glucosidase and acetylcholinesterase inhibition, leading to a privileged structure that might be useful for the treatment of such pathology. 2. Results and discussion 2.1. Chemistry We propose the general structure 6 (Fig. 1) as a hitherto unknown example of dual multitarget drugs designed for inhibiting simultaneously b-glucosidase, responsible for the Gaucher disease, and acetylcholinesterase, involved in the progressive loss of the

cognitive function associated to the neurological complications of Gaucher disease. We hypothesize that the incorporation of an arylalkyl fragment on the nitrogen would increase its selectivity towards b-glucosidase, in a similar way as found for the azafagomine alkylation on the same position [20]. Moreover, concerning AChE, the resolution of its crystal structure revealed [21] the presence of some critical regions located in the bottom of a gorge of roughly 20 Å; such regions must be taken into consideration when designing inhibitors. In the proximity of the catalytic triad, comprised of Ser, His and Glu, there is a region called the catalytic anionic site, responsible for binding the quaternary ammonium residue of acetylcholine, through cation-p interactions involving a Trp residue [21]. We propose that the deoxynojirimycin residue of 6, partially protonated at physiological pH values, could mimic the iminium cation of acetylcholine. Moreover, roughly 15 Å away from the catalytic anionic site there is a region known as the peripheral anionic site, capable of establishing strong interactions with planar and aromatic motifs, due to its high content of aromatic aminoacids [22]. The interaction with this domain, besides enhancing the binding of the inhibitor with the enzyme can also reduce the capacity of AChE of starting the deposits of b-amyloid plaques [23], a common feature of pathologies associated to Alzheimer's disease. For that purpose, we suggest the incorporation of an N-aryl selenoureido appendage; the selenoureido motif would also eliminate deleterious Reactive Oxygen species (ROS), present at high concentration levels in both, neurodegenerative disorders [24] and Gaucher disease; in fact, in the latter, the severity of the disease is determined by the degree of oxidative stress in the endoplasmic reticulum, and antioxidants have shown protective effects [25]. The synthesis of targeted structure 6 was accomplished following the synthetic methodology depicted in Scheme 1. The key template was O-protected DNJ 9, which can be accessed starting from methyl a-D-glucopyranoside 7 in a 5-step procedure: per-Obenzylation, glycoside hydrolysis, reduction of reducing sugar 8, Swern oxidation and cyclization of the corresponding transient and unstable hexosulose mediated by ammonium formate and sodium cyanoborohydride. Overkleeft and co-workers claimed [26] that Swern oxidation was a more reproducible procedure for preparing the key dicarbonyl compound in the synthesis of 9 than the PfitznereMoffat oxidation originally reported by Matos et al. [27]. Next, the subsequent N-alkylation of 9 via nucleophilic substitution on alkyl halides proved to be more complicated than previously anticipated; thus, attempts to accomplish such alkylation reaction with either 5-bromopentanol or monosubstitution on 1,5dibromopentane failed, as no product formation was observed.

Fig. 1. Known potent glycosidase inhibitors derived from deoxynojirimycin and proposed structure 6.

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Scheme 1. Preparation of N-alkylated deoxynojirimycin-containing selenoureas.

Probably the strong sterical hindrance exerted by the benzyl ethers, particularly the nearby hydroxymethyl one, precluded the endocyclic nitrogen atom to efficiently act as a nucleophile. Nevertheless, catalytic hydrogenolysis reaction to give DNJ 1, followed by nucleophilic displacement on 1-azido-5-iodopentane took place with acceptable yield (45% for the two steps). Reduction of the azido moiety, followed by coupling with readily-available aromatic isoselenocyanates [28], furnished title compounds 6aec in a 4.5e8.1% overall yield for the 9 steps (Scheme 1). The three targeted compounds chosen as representative examples of the hitherto unknown family of selenoureidoiminosugars bear either an electron-withdrawing group (Br, 6a), a strong electron-donating group (OMe, 6b), and a moderate electron-donating group (Me, 6c). 2.2. Biological assessment Compounds 6aec were tested as potential glycosidase inhibitors against a panel of seven glycosidases of medical relevance (a/b-glucosidases, a/b-galactosidases, a/b-mannosidases). Lineweaver-Burk plots (double reciprocal plot, 1/v vs. 1/[S]) allowed the determination of the inhibition type, together with the Ki values (e.g. Fig. 2). Compounds 6aec, in particular the p-bromo derivative 6a, were found to be strong and selective competitive b-glucosidase inhibitors, with Ki in the low micromolar range (Table 1, 1.6e5.5 mM). It is noteworthy mentioning that a remarkable increase in a/bglucosidase selectivity was found (up to 36-fold) when compared with parent DNJ (Ki, mM: 25 (a-glucosidase), 47 (b-glucosidase), 270 (a-mannosidase)) [29]. Surprisingly, a reversed selectivity was observed compared to DNJ, and particularly to analogues 2e3, which show preference for a-glucosidase. These results are presumably due to a combination of favourable p-stacking interactions involving the aromatic substituent of the selenoureido scaffold, and effective hydrogen bonding with polar residues of the b-glucosidase active site. Concerning the rest of glycosidases, negligible or

Fig. 2. Lineweaver-Burk plot for 6a against b-glucosidase.

weak activity was observed, a desirable effect for therapeutic applications. Derivatives 6aec were also tested against acetylcholinesterase (AChE from Electrophorus electricus); such enzyme catalyses [30] the breakdown of the cholinergic neurotransmitter acetylcholine. A well-known symptomatic feature of Alzheimer's disease is the profound decline in the cholinergic function. Therefore, one of the targets for treating this neurodegenerative disease in the early stages is the development of acetylcholinesterase inhibitors [31] which might increase both, the level and the duration of the neurotransmitter action. p-Bromophenyl selenourea 6a was found to be a strong mixed AChE inhibitor (Table 1, Kia ¼ 5.8 mM, Kib ¼ 11.2 mM); therefore, the p-bromophenyl moiety is again a crucial structural motif within the selenoureido-iminosugar to exert strong enzymatic inhibition. Until now, there is only one precedent [32] for the inhibition of AChE by imino- or azasugars. The capacity of compounds 6 to act as glutathione peroxidase

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Table 1 Inhibitory properties of compounds 6aec (Ki, mM). Enzyme

6a

6b

6c

a-Glucosidase (Bakers' yeast) b-Glucosidase (Almonds) a-Galactosidase (Green coffee beans) b-Galactosidase (Asp. Oryzae) b-Galactosidase (E. coli) a-Mannosidase (Jack bean)

30.7 ± 10.7 1.6 ± 0.2 7%a 42%a 12%a Kia ¼ 390b Kib ¼ 290c (Mixed) 18%a Kia ¼ 5.8 ± 1.3 Kib ¼ 11.2 ± 2.5 (Mixed)

36.0 ± 8 5.5 ± 0.8 20%a 29%a 15%a 510

35.0 ± 11 3.7 ± 0.8 2%a 20%a 31%a 46%a

7% Kia ¼ 83.1 ± 31.3 Kib ¼ 58.8 ± 22.1 (Mixed)

16% Kia ¼ 105.1 ± 40.0 Kib ¼ 16.9 ± 2.8 (Mixed)

b-Mannosidase (Helix pomatia) Acetylcholin-esterase (electric eel)

a b c

Percentage of inhibition at 0.5 mM concentration. Inhibition constant for the E-I binding. Inhibition constant for the ES-I binding.

(GPx) mimics has also been evaluated as a model for the detoxification of deleterious peroxides. GPx is a selenoenzyme that catalyses the reduction of H2O2 and alkyl peroxides (examples of intracellular ROS) into water and alcohols, respectively, by using the redox properties of a selenocysteine residue [33], and the tripeptide glutathione as a cofactor. When the concentration of ROS species rise above a threshold level, oxidative stress is achieved, leading to the oxidative degradation of biomolecules, what has an active role in the etiology of numerous pathologies, like neurodegenerative diseases and cancer [24]. Therefore, it has been claimed [24] that the use of ROS scavengers can be a useful therapeutic approach to target some of the above-mentioned multifactorial diseases. In order to evaluate the GPx-like activity [34] the model reported by Iwaoka and Tomoda [35] was followed, PhSH being used as a substitute for glutathione. Compounds 6a,b were tested in a substoichiometric ratio (1% molar concentration) compared to PhSH (10.0 mM); reaction was started by using an excess of H2O2 (41.6 mM), and the subsequent formation of (PhS)2 was monitored at 305 nm. The activity was also compared with the background reaction, and also with (PhSe)2 and ebselen [36], two well-known organoselenium compounds with relevant GPx-like activity. Table 2 depicts the reaction rates for such experiment, together with the calculated half-life times. Remarkably, selenourea 6b, bearing a strong electron-donating group (OMe) behaved as an excellent bio-inspired GPx-mimic, with a 46 and 18-fold increase compared to the background reaction, and the reaction catalysed by (PhSe)2, respectively. The same situation was reported [37] for the GPx-like activity of aromatic seleninates, thus, strongly suggesting that the transition state of the rate determining step in the

catalytic cycle is stabilized by electron-donating substituents. The kinetic parameters (Km, Vmax) of the GPx-like activity were calculated by using different PhSH concentrations (Table 3), while keeping constant the concentrations of the catalyst and H2O2. Hanes-Woolf plot of tested compounds demonstrated that these organoselenium derivatives fulfill Michaelis-type kinetics. Remarkably, for p-methoxy derivative 6b the Kcat/Kuncat ratio was found to be 640, clearly indicating excellent H2O2 scavenging properties via a catalytic cycle; these results constitute a significant improvement compared to stoichiometric antioxidants. Finally, we have also explored the potential antiproliferative properties of compounds 6aec against a panel of six human solid tumor cell lines: A549 (non-small cell lung), HBL-100, (breast), HeLa (cervix), SW1573 (non-small cell lung), as drug sensitive lines, T-47D (breast) and WiDr (colon) as drug resistant lines. Selenourea 6a was found to exert good antiproliferative activity against A549, HBL-100 and T-47D cell lines (GI50 7.0e12.0 mM) and moderate properties against the other three ones (21e27 mM). 3. Conclusions In conclusion, N-alkylated deoxynojirimycin derivatives bearing a selenoureido motif have proved to be privileged structures that could be useful in the therapeutic treatment of Gaucher disease and its neurological complications. Thus, this prototype affords strong b-glucosidase and AChE inhibition, together with excellent catalytic scavenging properties against deleterious peroxides. 4. Experimental section 4.1. N-(50 -Azidopentyl)-1-deoxynojirimycin (13)

Table 2 GPx-like activity of compounds 6a,b.a

Compound

V (mM/min)b

t1/2 (min)

Relative rate to control

Control (MeOH) Control (DMSO) 9a (MeOH) 9b (MeOH) (PhSe)2 (MeOH) Ebselen (DMSO)

12.3 ± 2.3 18.9 ± 4.4 290.1 ± 3.2 567.4 ± 25.7 31.3 ± 1.5 10.6 ± 4.4

351.9 214.0 14.7 8.3 102.0 148

1.0 1.0 23.6 46.1 2.5 0.6

To a solution of per-O-benzyl-1-deoxynojirimycin 9 (1.1 g, 2.1 mmol) in dry MeOH (45 mL) was added 20% Pd(OH)2/C (0.15 g), and the suspension was acidified with 2 M aq. HCl, and then stirred under a H2 atmosphere for 14 h. The mixture was then filtered through a Celite® pad and the filtrate was concentrated in vacuo to give crude DNJ 1 (0.54 g). Compound 1 was dissolved in dry DMF

Table 3 Kinetic parameters for the GPx-like activity. Compound

Km (mM)

Vmax (mM/min)

Kcat (min1)

Kcat/Kuncat

6a 6b (PhSe)2

0.33 0.41 1.11

96.4 211.4 27.3

0.96 2.1 0.27

292 640 82

a

Experimental conditions: [PhSH]o ¼ 10.0 mM; [H2O2]o ¼ 41.6 mM; [Cat] ¼ 0.1 mM, solvent: MeOH for 6a, 6b, (PhSe)2, and 99:1 MeOHDMSO for ebselen. b The rate was corrected with the background reaction.

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(5.5 mL), and to the corresponding solution, 1-azido-5-iodopentane (0.66 g, 2.76 mmol) and K2CO3 (0.88 g, 6.4 mmol) were added, and stirred at 80  C for 4.5 h. After that, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography (4:1 / 7:3 EtOAcMeOH with 1% NH4OH) to 1 afford 13 (0.26 g, 45% over two steps). [a]26 D 7 (c 0.84, MeOH); H NMR (500 MHz, CD3OD) d 3.95 (dd, 1H, J5,6a ¼ 2.3 Hz, J6a,6b ¼ 12.2 Hz, H-6a), 3.89 (dd, 1H, J5,6b ¼ 2.8 Hz, H-6b), 3.59e3.54 (m, 1H, H-2), 3.45 (t, 1H, J3,4 ¼ J4,5 ¼ 9.5 Hz, H-4), 3.34e3.31 (m, 2H, CH2), 3.24 (t, 1H, H-3), 3.21e3.17 (m, 1H, H-1a), 3.09e3.02 (m, 1H, CH2 pentyl), 2.88e2.82 (m, 1H, CH2 pentyl), 2.56e2.49 (m, 2H, H1b, H-5), 1.69e1.59 (m, 4H, 2CH2), 1.48e1.37 (m, 2H, CH2); 13C NMR (125.7 MHz, CD3OD) d 79.5 (C-3), 70.7 (C-4), 69.5 (C-2), 67.5 (C-5), 57.7 (C-6), 56.5 (C-1), 53.7 (CH2), 52.3 (CH2), 29.6 (CH2), 25.4 (CH2), 24.5 (CH2); HRESI-MS m/z calcd for C11H23N4O4 ([MþH]þ): 275.1714, found: 275.1708.

4.2.3. N-{5-[N0 -(p-Methylphenyl)-selenoureido]pentyl}-1deoxynojirimycin (6c) p-Methylyphenyl isoselenocyanate 16c (90 mg, 0.46 mmol) was used. Column chromatography (1:3 MeOHEtOAc) furnished 6c 1 (37 mg, 37%). [a]26 D 3 (c 1.06, CD3OD); H NMR (500 MHz, CD3OD) d 7.30e7.21 (m, 2H, AreH), 7.14e7.03 (m, 2H, AreH), 3.90e3.86 (m, 2H, H-1a, H-1b), 3.67e3.61 (m, 2H, CH2), 3.50 (ddd, 1H, J4,5 ¼ 9.2 Hz, J5,6a ¼ 4.9 Hz, J5,6b ¼ 10.6 Hz, H-5), 3.38 (t, 1H, J2,3 ¼ J3,4 ¼ 9.4 Hz, H3), 3.16 (dd, 1H, H-4), 3.06 (dd, 1H, J6a,6b ¼ 11.3 Hz, H-6a), 2.93e2.84 (m, 1H, CH2a pentyl), 2.72e2.61 (m, 1H, CH2b pentyl), 2.34 (s, 3H, Me), 2.32e2.22 (m, 2H, H-2, H-6b), 1.68e1.53 (m, 4H, 2CH2), 1.42e1.30 (m, 2H, CH2); 13C NMR (125.7 MHz, CD3OD) d 138.1 (AreC), 131.3 (AreC), 130.2 (AreC), 126.5 (AreC), 80.3 (C-4), 71.6 (C3), 70.4 (C-5), 67.4 (C-2), 58.9 (C-1), 57.3 (C-6), 53.7 (CH2), 48.8 (CH2), 30.0 (CH2), 25.5 (CH2), 24.8 (CH2), 21.1 (CH3); HRESI-MS: m/z calcd for C19H32N3O4Se ([MþH]þ): 446.1553, found: 446.1543.

4.2. General procedure for the synthesis of selenoureas 6aec

Acknowledgements

To a solution of compound 13 (66 mg, 0.23 mmol) in MeOH (10 mL) was added a catalytic amount of Raney-Ni. The mixture was then stirred for 1 h under H2 atmosphere. After that it was filtered through a Celite® pad and concentrated in vacuo to afford amine 14. To a solution of crude 14 (56 mg, 0.23 mmol) in dry MeOH (10 mL) was added the corresponding isoselenocyanate 16aec (0.46 mmol), and the reaction was stirred at rt and in the dark for 24 h. Concentration to dryness and purification by column chromatography using the eluant indicated in each case afforded selenoureas 6aec. 4.2.1. N-{5-[N0 -(p-Bromophenyl)selenoureido]pentyl}-1deoxynojirimycin (6a) p-Bromophenyl isoselenocyanate 16a (120 mg, 0.46 mmol) was used. Column chromatography (1:3 MeOHEtOAc) furnished 6a: 1 75 mg, 66%. [a]25 D 8 (c 0.51, CH3OH); H NMR (500 MHz, CD3OD) d 7.54 (m, 2H AreH), 7.22 (m, 2H, AreH), 3.89 (m, 2H, H-1a, H-1b), 3.70e3.63 (m, 2H, CH2 pentyl), 3.55e3.47 (m, 1H, H-5), 3.42e3.37 (t, 1H, J2,3 ¼ J3,4 ¼ 9.2 Hz, H-3), 3.17 (t, 1H, J4,5 ¼ 9.1 Hz, H-4), 3.11e3.05 (m, 1H, H-6a), 2.97e2.87 (m, 1H, CH2a pentyl), 2.74e2.65 (m, 1H, CH2b pentyl), 2.39e2.23 (m, 2H, H-2, H-6b), 1.72e1.52 (m, 4H, 2CH2), 1.43e1.31 (m, 2H, CH2); 13C NMR (125.7 MHz, CD3OD) d 180.1 (CSe), 138.5 (AreC), 133.6 (AreC), 128.0 (AreC), 120.4 (AreC), 80.2 (C-4), 71.5 (C-3), 70.3 (C-5), 67.4 (C-2), 58.8 (C-1), 57.3 (C-6), 53.7 (CH2), 48.5 (CH2), 29.9 (CH2), 25.5 (CH2), 24.8 (CH2); HRESI-MS: m/z calcd for C18H29BrN3O4Se ([MþH]þ): 510.0501, found: 510.0485. 4.2.2. N-{5-[N0 -(p-Methoxyphenyl)-selenoureido]pentyl}-1deoxynojirimycin (6b) p-Methoxyphenyl isoselenocyanate 16b (98 mg, 0.46 mmol) was used. Column chromatography (1:3 / 3:7 MeOHEtOAc) 1 furnished 6b: 54 mg, 52%. [a]26 D 7 (c 0.46, CH3OH); H NMR (500 MHz, CD3OD) d 7.16e7.13 (m, 2H, AreH), 7.00e6.94 (m, 2H, AreH), 3.90e3.86 (m, 2H, H-1a, H-1b), 3.81 (s, 3H, OMe) 3.65e3.61 (m, 2H, CH2), 3.50 (ddd, 1H, J4,5 ¼ 9.2 Hz, J5,6a ¼ 4.9 Hz, J5,6b ¼ 10.6 Hz, H-5), 3.38 (t, 2H, J2,3 ¼ J3,4 ¼ 9.4 Hz, H-3), 3.16 (dd, 1H, H-4), 3.06 (dd, 1H, J6a,6b ¼ 11.3 Hz, H-6a), 2.92e2.83 (m, 1H, CH2a pentyl), 2.71e2.62 (m, 1H, CH2b pentyl), 2.32e2.22 (m, 2H, H2, H-6b), 1.66e1.54 (m, 4H, 2CH2), 1.36e1.27 (m, 2H, CH2); 13C NMR (125.7 MHz, CD3OD) d 179.2 (CSe), 160.2 (AreC), 131.2 (AreC), 128.5 (AreC), 116.0 (AreC), 80.3 (C-4), 71.7 (C-3), 70.4 (C-5), 67.4 (C-2), 59.0 (C-1), 57.4 (C-6), 56.0 (OMe), 53.7 (CH2), 48.5 (CH2), 30.1 (CH2), 25.5 (CH2), 24.8 (CH2); HRESI-MS: m/z calcd for C19H32N3O5Se ([MþH]þ): 462.1502, found: 462.1489.


n General de Investigacio
n of Spain We thank the Direccio (CTQ2011-28417-C02-01, CEI10/00018), the Junta de Andalucía (P11-CVI-7427, FQM134), the European Regional Development Fund (FEDER), and the EU Research Potential (FP7-REGPOT-2012CT2012-31637-IMBRAIN), for financial support. J.I.O. and G.B.P. also thank the Danish National Research Council (FNU) and the
n Caja Canarias, respectively, for the Obra Social La Caixa-Fundacio award of predoctoral grants. We would also like to thank the Ser
tica Nuclear, CITIUS (University of Sevvicio de Resonancia Magne ille) for the performance of NMR experiments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2016.07.021. References [1] a) A.E. Stütz, T.M. Wrodnigg, Imino sugars and glycosyl hydrolases: historical context, current aspects, emerging trends, Adv. Carbohydr. Chem. Biochem. 66 (2011) 187e298; b) T.M. Gloster, G.J. Davies, Glycosidase inhibition: assessing mimicry of the transition state, Org. Biomol. Chem. 8 (2010) 305e320;
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