Synthesis and Therapeutic Applications of Iminosugars in Cystic Fibrosis

Abstract

Iminosugars are sugar analogues endowed with a high pharmacological potential. The wide range of biological activities exhibited by these glycomimetics associated with their excellent drug profile make them attractive therapeutic candidates for several medical interventions. The ability of iminosugars to act as inhibitors or enhancers of carbohydrate-processing enzymes suggests their potential use as therapeutics for the treatment of cystic fibrosis (CF). Herein we review the most relevant advances in the field, paying attention to both the chemical synthesis of the iminosugars and their biological evaluations, resulting from in vitro and in vivo assays. Starting from the example of the marketed drug NBDNJ (N-butyl deoxynojirimycin), a variety of iminosugars have exhibited the capacity to rescue the trafficking of F508del-CFTR (deletion of F508 residue in the CF transmembrane conductance regulator), either alone or in combination with other correctors. Interesting results have also been obtained when iminosugars were considered as anti-inflammatory agents in CF lung disease. The data herein reported demonstrate that iminosugars hold considerable potential to be applied for both therapeutic purposes.

Keywords: CFTR correctors; anti-inflammatory agents; cystic fibrosis; cystic fibrosis transmembrane conductance regulator (CFTR); glycomimetics; glycosidase inhibitors; iminosugars; miglustat.

Figure 1

Iminosugar structural motifs of natural and synthetic origin.

Figure 2

Iminosugars: carbohydrate mimetics.

Figure 3

Marketed iminosugars.

Scheme 1

Chemo-enzymatic synthesis of NBDNJ (reproduced with permission from [1]).

Figure 4

Rescue of functional F508del-CFTR (deletion of F508 residue in the cystic fibrosis transmembrane conductance regulator) activity by NBDNJ, low temperature or 7 as resulting by iodide efflux experiments. This figure is adapted with permission from [75].

Scheme 2

Synthesis of fluorinated and unsaturated N-alkyl DNJ derivatives [88].

Figure 5

F508del-CFTR correction by saturated, unsaturated and fluorinated N-alkyl DNJ derivatives. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. This figure is republished with permission of the Royal Society of Chemistry, from [88]; permission conveyed through Copyright Clearance Center, Inc.

Figure 6

AMP-DNJ and the corresponding triazole-containing derivatives [98].

Scheme 3

Synthesis of DNJ-adamantane conjugates [98].

Figure 7

(A) Functional evaluation of F508del-CFTR by DiSBAC2(3) assay in CF-KM4 cells treated or not with iminosugar (100 μM, 2 h). (B) F508del-CFTR was assayed with the iodide efflux technique in the presence of forskolin/genistein. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. This figure is republished with permission of the Royal Society of Chemistry, from [98]; permission conveyed through Copyright Clearance Center, Inc.

Figure 8

Multivalent iminosugars as F508del-CFTR correctors.

Scheme 4

Synthesis of monovalent iminosugar-based analogues [121].

Scheme 5

Synthesis of (A) tri- and tetravalent iminosugars and (B) hepta- and tetra-decavalent DNJ-cyclodextrin iminosugars [106,116].

Figure 9

(A) Rescue of functional F508del-CFTR activity: iodide efflux curves on untreated or iminosugar-treated CF-KM4 cells in the presence of forskolin/genistein. (B) Summary of the functional evaluation of F508del-CFTR: histograms represent the mean (± SEM) of four experiments obtained by the iodide efflux technique in cystic fibrosis (CF) cells untreated or treated (100 μM, 2 h) in the presence of forskolin/genistein. *** p < 0.001; n.s., not significant. This figure is adapted with permission from [106]).

Figure 10

Carbon-branched pyrrolidine iminosugars.

Scheme 6

Synthesis of (A) isoDAB and (B) isoLAB [136].

Figure 11

Functional evaluation of F508del-CFTR activity by single-cell fluorescence imaging assay in CF-KM4 cells treated with isoLAB (100 μM, 2 h). (A) Time courses obtained with untreated cells or with cells treated with isoLAB in the presence of forskoline/genistein. (B) Histograms summarizing the results collected from separate experiments. *** p < 0.001; ns, not significant. This figure is reprinted from [136], with permission from Elsevier.

Scheme 7

Synthesis of l-isoDMDP [139].

Figure 12

Effect of l-isoDMDP, isoLAB and NBDNJ on the activity of F508del-CFTR in human airway epithelial CF-KM4 cell line, assessed by single-cell fluorescence imaging. (A) Cells were incubated with the iminosugars (100 μM, 4 h) in the presence of forskolin/genistein, followed by inhibition with CFTR_inh-172. (B) Histograms summarizing the results collected from separate experiments. *** p < 0.001. This figure is adapted with permission from [139]. Copyright (2013) American Chemical Society.

Scheme 8

Synthesis of carbon-branched pyrrolizidines [143].

Figure 13

Transepithelial short-circuit currents (Isc) recorded in F508del-CFTR-expressing CFBE cells. (A) Protocols used and model trace indicating ΔIsc. (B–D) Tracings of Isc recorded with CFBE cells incubated with: (B) DMSO or VX-809 (10 μM) (24 h); (C) water or 63 (100 μM) (4 h); (D) VX-809 (10 μM, 24 h) or VX-809+63 (100 μM, 4 h). F508del-CFTR was activated by application of CTR activators and inhibited by CFTRinh172. (E) Summary of the ratio ΔIsc/ΔIsc VX-809 for each compound; ** p < 0.01. A ratio above 1 means a potentiation. This figure is adapted with permission from [143].

Scheme 9

Synthesis of conformationally constrained iminosugars 68–70 [147,150].

Figure 14

(A) Rescue of functional F508del-CFTR activity. Iodide efflux curves on untreated or iminosugar-treated F508del-CFTR stably transfected HeLa cells in the presence of forskolin/genistein. (B) Summary of the functional evaluation of F508del-CFTR. Histograms represent the mean (± SEM) of four experiments obtained by the iodide efflux technique in CF cells untreated or treated with the iminosugar (100 μM, 4 h). ns, not significant. This figure is adapted from [146], published by the Royal Society of Chemistry.

Figure 15

Tetrahydroxylated azepanes evaluated by Désiré and co-workers [156].

Scheme 10

Synthesis of N-alkyl and C-alkyl azepane iminosugars [156].

Figure 16

Azepane iminosugars as F508del-CFTR correctors. ** p < 0.01; *** p < 0.001; ns, not significant. This figure is republished with permission of the Royal Society of Chemistry, from [156]; permission conveyed through Copyright Clearance Center, Inc.

Figure 17

Effect on the F508del-CFTR rescue after application of several correctors individually or in combinations. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. This figure is adapted with permission from [159].

Figure 18

Effect of NBDNJ on the P. aeruginosa stimulated IL-8 and ICAM-1 mRNA expression in (A,B) IB3-1 cells and (C,D) CuFi-1 cells. Cells were incubated for 24 h in the presence of NBDNJ (200 μM) and infected with PAO1 (50 CFU/cell) for 4 h. * p < 0.05; *** p < 0.001. This figure was reprinted from [78], with permission from Elsevier.

Figure 19

Iminosugars with anti-inflammatory potential.

Scheme 11

Synthetic route to AMP-DNM [69,70].

Figure 20

Effect of NBDNJ and AMP-DNM on NLGase inhibition in IB3-1 and CuFi-1 cells infected by P. aeruginosa. * p < 0.05; *** p < 0.001; ns, not significant. This figure is adapted from [173] published by PLOS.

Figure 21

Effect of AMP-DNM derivatives on β-glucosidase activity in CF bronchial cells. CuFi-1 cells were treated with (A–C) 0.5 μM or (D–F) 5.0 μM of compound for 1 h before the measurement (adapted from [176]).

Figure 22

N-Alkylated d- and l-DNJ derivatives. This figure is reprinted from [179], with permission from Elsevier.

Scheme 12

Synthesis of N-alkylated l-DNJ derivatives ent-(4,14,16,91,92) [179,186].

Figure 23

(A) Effect of sugar chirality of iminosugars 4,14,16 and ent-(4,14,16) on the inflammatory response to P. aeruginosa in CF bronchial epithelial cells. CuFi-1 cells were treated with the compounds (0.1 μM) for 1 h before P. aeruginosa infection. (B) Effect of racemic mixtures on the inflammatory response to P. aeruginosa in CF primary cells. CF bronchial primary cells were treated with the racemic mixtures (0.01 μM) for 1 h before P. aeruginosa infection. * p < 0.05; ** p < 0.01. This figure was reprinted from [179], with permission of Elsevier.

Figure 24

(A) Effect of 4 and ent-4 on cells recruited in bronchoalveolar lavage (BAL) of C57Bl/6NCr mice infected by P. aeruginosa (i, total cells; ii. neutrophils; iii. alveolar macrophages). (B) NLGase activity in the lungs of mice infected by P. aeruginosa. * p < 0.05; ** p < 0.01. This figure was reprinted from [179], with permission from Elsevier.