Exploring the effect of chirality on the therapeutic potential of N-alkyl-deoxyiminosugars: anti-inflammatory response to Pseudomonas aeruginosa infections for application in CF lung disease

Article abstract of DOI:10.1016/j.ejmech.2019.04.061

In the frame of a research program aimed to explore the relationship between chirality of iminosugars and their therapeutic potential, herein we report the synthesis of N-akyl L-deoxyiminosugars and the evaluation of the anti-inflammatory properties of selected candidates for the treatment of Pseudomonas aeruginosa infections in Cystic Fibrosis (CF)lung disease. Target glycomimetics were prepared by the shortest and most convenient approach reported to date, relying on the use of the well-known PS-TPP/I₂ reagent system to prepare reactive alkoxyalkyl iodides, acting as key intermediates. Iminosugars ent-1-3 demonstrated to efficiently reduce the inflammatory response induced by P. aeruginosa in CuFi cells, either alone or in synergistic combination with their D-enantiomers, by selectively inhibiting NLGase. Surprisingly, the evaluation in murine models of lung disease showed that the amount of ent-1 required to reduce the recruitment of neutrophils was 40-fold lower than that of the corresponding D-enantiomer. The remarkably low dosage of the L-iminosugar, combined with its inability to act as inhibitor for most glycosidases, is expected to limit the onset of undesired effects, which are typically associated with the administration of its D-counterpart. Biological results herein obtained place ent-1 and congeners among the earliest examples of L-iminosugars acting as anti-inflammatory agents for therapeutic applications in Cystic Fibrosis.

Full text of DOI:10.1016/j.ejmech.2019.04.061

Accepted Manuscript
Exploring the effect of chirality on the therapeutic potential of N-alkyl-
deoxyiminosugars: Anti-inflammatory response to Pseudomonas aeruginosa
infections for application in CF lung disease
Maria De Fenza, Daniele D'Alonzo, Anna Esposito, Silvia Munari, Nicoletta Loberto,
Alessandra Santangelo, Ilaria Lampronti, Anna Tamanini, Alice Rossi, Serena
Ranucci, Ida De Fino, Alessandra Bragonzi, Massimo Aureli, Rosaria Bassi, Matteo
Tironi, Giuseppe Lippi, Roberto Gambari, Giulio Cabrini, Giovanni Palumbo, Maria
Cristina Dechecchi, Annalisa Guaragna
PII:
S0223-5234(19)30379-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.061
EJMECH 11296
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2019
Revised Date: 4 April 2019
Accepted Date: 21 April 2019
Please cite this article as: M. De Fenza, D. D'Alonzo, A. Esposito, S. Munari, N. Loberto, A. Santangelo,
I. Lampronti, A. Tamanini, A. Rossi, S. Ranucci, I. De Fino, A. Bragonzi, M. Aureli, R. Bassi, M. Tironi,
G. Lippi, R. Gambari, G. Cabrini, G. Palumbo, M.C. Dechecchi, A. Guaragna, Exploring the effect
of chirality on the therapeutic potential of N-alkyl-deoxyiminosugars: Anti-inflammatory response to
Pseudomonas aeruginosa infections for application in CF lung disease, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.061.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Exploring the Effect of Chirality on the Therapeutic Potential of N-
Alkyl-Deoxyiminosugars: Anti-Inflammatory Response to Pseudomonas
aeruginosa Infections for Application in CF Lung Disease
Maria De Fenza,‡ Daniele D'Alonzo,*,‡ Anna Esposito,‡ Silvia Munari,≠ Nicoletta Loberto,§
Alessandra Santangelo,≠ Ilaria Lampronti,¶ Anna Tamanini,≠ Alice Rossi,₸ Serena Ranucci,₸ Ida
De Fino,₸ Alessandra Bragonzi,₸ Massimo Aureli,§ Rosaria Bassi,§ Matteo Tironi,§ Giuseppe
Lippi,≠ Roberto Gambari,¶ Giulio Cabrini,≠ Giovanni Palumbo,‡ Maria Cristina Dechecchi,*,≠
and Annalisa Guaragna‡
≠
‡Department of Chemical Sciences, University of Napoli Federico II, via Cintia, 80126 Napoli (Italy); Laboratory
of Molecular Pathology-Department of Pathology and Diagnostics, University Hospital of Verona, Verona (Italy);
§Department of Medical Biotechnology and Translational Medicine, University of Milano, Milano (Italy);
¶Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara (Italy); ₸CFaCore, Infection and CF
Unit, San Raffaele Scientific Institute, Milano (Italy).
* Corresponding authors: daniele.dalonzo@unina.it, cristina.dechecchi@aovr.veneto.it
KEYWORDS:
NLGase
L-Deoxyiminosugars, L-NBDNJ, cystic fibrosis, polymeric triphenylphosphine,
ABSTRACT. In the frame of a research program aimed to explore the relationship between chirality of iminosugars
and their therapeutic potential, herein we report the synthesis of N-akyl -deoxyiminosugars and the evaluation of
L
the anti-inflammatory properties of selected candidates for the treatment of Pseudomonas aeruginosa infections in
Cystic Fibrosis (CF) lung disease. Target glycomimetics were prepared by the shortest and most convenient
approach reported to date, relying on the use of the well-known PS-TPP/I2 reagent system to prepare reactive
alkoxyalkyl iodides, acting as key intermediates. Iminosugars ent-1-3 demonstrated to efficiently reduce the
inflammatory response induced by P. aeruginosa in CuFi cells, either alone or in synergistic combination with their
D
-enantiomers, by selectively inhibiting NLGase. Surprisingly, the evaluation in murine models of lung disease
showed that the amount of ent-1 required to reduce the recruitment of neutrophils was 40-fold lower than that of the
corresponding -enantiomer. The remarkably low dosage of the -iminosugar, combined with its inability to act as
inhibitor for most glycosidases, is expected to limit the onset of undesired effects, which are typically associated
with the administration of its -counterpart. Biological results herein obtained place ent-1 and congeners among the
D
L
D
earliest examples of
Fibrosis.
L-iminosugars acting as anti-inflammatory agents for therapeutic applications in Cystic
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1. Introduction
Cystic Fibrosis (CF) is the most common life-threatening Mendelian disorder worldwide that
affects respiratory, digestive, and reproductive systems. It is caused by mutations in the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR) gene, encoding for a cAMP-activated
chloride channel, expressed at the apical membrane of most of the surface epithelial cells lining
the airways, gastrointestinal tract, exocrine pancreas, airway submucosal and sweat glands [1,2].
CFTR protein plays a key role in hydrating airway secretions [3,4]. In the lung, the defective
CFTR causes dehydration of the airway surface liquid and drives the cascade of pathological
events characterized by progressive chronic infections and inflammation of the airways. The last
ones then lead to irreversible lung damage and fibrosis, which represent the major cause of
mortality in CF patients [3]. Accordingly, increasing attention is currently focusing on anti-
inflammatory therapies, with the aim to ameliorate CF lung pathology [5]. High-dose ibuprofen
is the only anti-inflammatory drug recommended to date for use in CF, however, it is not widely
prescribed due to the onset of important side-effects as the gastrointestinal bleeding [6]. Hence,
the development of novel and more potent anti-inflammatory drugs as well as candidate targets
for precision therapies directed towards pathways regulating inflammatory response in CF
airway disease remains a compelling need.
Sphingolipids (SLs) represent a large group of lipids with emerging importance for the
pathophysiology of several human pathology including respiratory disorders [7-12]. They are
one of the active constituents of the mucus secreted by alveolar epithelium, which protects the
lung tissue from invading pathogens, thus representing effective novel specific drug targets for
controlling pulmonary infections [13]. An increasing number of studies indicate that SLs play an
important regulatory role in CF with respect to pulmonary infections and inflammation and
support the use of modulators of SL metabolism as therapeutic agents for CF lung disease [11].
As a matter of fact, novel drugs are being developed to selectively target different enzymes
involved in SL metabolism and, in this regard, iminosugars (sugar analogues having an amino
group in place of the endocyclic oxygen) open up exciting new opportunities for therapeutic
agent discovery, due to their good oral bioavailability and very specific immune modulatory and
chaperoning activity [14].
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As previously reported [15,16], the iminosugar N-butyl
D-deoxynojirimycin (D-NBDNJ, 1,
Miglustat) exhibits anti-inflammatory effect in vitro and in vivo and reduces the P. aeruginosa
induced immunoreactive ceramide expression by targeting non-lysosomal glucosidase 2 (GBA2-
encoded NLGase) [17]. NLGase is an important player in the regulation of SL homeostasis at the
plasma membrane level, due to its ability to modify the lipid organization at this site [11,18].
Therefore, by modulating plasma membrane ceramide levels, it is possible to determine the
susceptibility to inflammatory stimuli. It must be noted that the pharmacological use of
D-
NBDNJ and its congeners has typically been associated to the onset of numerous (even severe)
adverse effects [19], due to the side inhibition of a variety of carbohydrate-processing enzymes.
In search for more selective NLGase inhibitors, a new generation of iminosugars, obtained either
varying the N-alkyl chain length/structure [20] and/or changing the configuration of one or more
stereogenic centres within the iminosugar core [21], is currently under development. On one
hand, iminosugars bearing long alkyl chains including N-nonyl
adamantanemethoxypentyl -DNJ ( -AMP-DNM, 3), N-hexyloxypentyl
and N-nonyloxypentyl -DNJ ( -NPDNJ, 5) have demonstrated to be increasingly powerful
NLGase inhibitors [21]. On the other hand, novel therapeutic opportunities are recently being
offered by -iminosugars and their N-alkylated derivatives [22,23]. While typically acting as
weak [24] inhibitors of most glycosidases, in an increasing number of reports -iminosugars
have displayed more efficient pharmacological properties than their -isomers toward specific
enzymes, acting as either inhibitors [25] or activators [26-28]. As an example, N-alkyl
iminosugars with -ido [20,29] and -altro [29] configuration have drawn attention because of
their remarkably high selectivity and potency in the inhibition of various glycosidases and
glycosyltransferases, including NLGase, compared to the activity of the corresponding -gluco
congeners. Inspired by these observations, we have conceived to widen the repertoire of
therapeutic -iminosugars, exploring the biological properties of N-alkyl iminosugars with
gluco configuration ent-(1-5) (Figure 1). In preliminary studies, ent-1 (i.e. -NBDNJ) has already
D
-DNJ (
D
-NNDNJ, 2),
D
D
D
-DNJ (
D
-HPDNJ, 4)
D
D
L
L
D
L
L
D
L
L-
L
demonstrated to be able to increase the levels of exogenous GAA (acid α-glucosidase,
Myozyme) in fibroblasts from patients with Pompe disease (PD), while not working as a
glycosidase inhibitor [28]. Herein, the analysis of the pharmacological potential of ent-1, as well
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as that of other N-alkyl congeners, has been extended to NLGase inhibition, with the aim to
study the role of iminosugar chirality in the anti-inflammatory treatment of P. aeruginosa
infections.
Figure 1. N-alkylated D- and L-DNJ derivatives.
2. Results and Discussion
2.1 Chemistry
The synthesis of N-alkyl
L-deoxyiminosugars ent-(2-5) was devised as depicted in Scheme 1,
whereas the preparation of ent-1, as well as the total synthesis of enantiomerically pure
L-DNJ
(11), was reported previously [28,30]. Particularly, herein our attention was focused on the
preparation of alkyl iodides 7a-c and on a straightforward route, leading to alkoxyalkyl iodides
9a-c, to be used in the N-alkylation reaction of 11 by alternative procedures to the existing
methods [20,21,29,31]. Our synthetic approaches are based on the use of the well-known
polymer supported triphenylphosphine (PS-TPP) and iodine [32] as iodination system, which has
been already used by us for other synthetic purposes [33]. The PS-TPP/I2 reagent system, in this
case, can be employed in the absence of the commonly used imidazole, thus enabling to devise a
synthetic protocol with a sequence of steps not requiring extractive work-up and
chromatographic purifications. Indeed, the only reaction by-product, i.e. the resin-bound
phosphine oxide, can be easily filtered off and reduced to the original phosphine form by
treatment with trichlorosilane [34]. In early studies, the procedure was based on two iodination
reactions, the first one to convert alcohols 6a-c into desired alkyl iodides 7a-c, that were
subsequently coupled with 1,5-pentandiol (NaH) to afford alcohols 8a-c; the second one to
obtain 9a-c from 8a-c (Scheme 1a). In order to find the best iodination conditions, different
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solvents and temperatures were used: the use of refluxing ACN provided the best results for both
6a,b and 8a,b, whereas for the lower reactive adamantanemethanol (6c) and the corresponding
derivative 8c, refluxing toluene was the solvent of choice.
Scheme 1. Synthetic routes to N-alkylated L-DNJ derivatives ent-(2-5).
NMR analysis of the crude mixtures of 9a-c demonstrated that high yield conversions (70-88%
o.y.) could be obtained in all cases, with virtually no side products attributable to the limiting
reagents. The strategy reported in Scheme 1a represents a faster and more convenient alternative
to most procedures [20,21,29,31a-b] devised for the preparation of alkyl and alkoxypentyl chains
to be used in the N-alkylation reaction of DNJ. In search for an even more expeditious protocol,
in subsequent studies an alternative route was conceived, involving a PS-TTP mediated double
iodination of 1,5-pentandiol, to provide 1,5-diiodopentane (10), followed by two sequential
coupling reactions, with alcohols 6a-c first and with
L-DNJ (11) then (Scheme 1b). The
efficiency of the double iodination reaction was markedly affected by the reaction conditions:
over a variety of solvents tested (Table S1), the most convenient conversion (95%) was observed
using DCM for 1h at rt. The subsequent coupling reaction of 10 with alcohols 6a-c proceeded
smoothly with hexanol (6a) and nonanol (6b), providing after 48h at rt the corresponding
alkoxyalkyl iodides 9a,b in 75 and 74% yields, respectively. On the other hand, the reaction with
adamantanemethanol (6c) proceeded slower, and the corresponding iodide 9c was obtained with
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a 72% yield after 72h at rt (Scheme 1b) [35]. The eventual reaction of nonyl iodide 7b and
alkoxyalkyl iodides 9a-c with
L-DNJ (11) [28] was carried out under standard conditions
(K2CO3), smoothly providing ent-2 (80% o.y., Scheme 1a) and ent-(3-5) in satisfactory 75-80%
yields (Scheme 1b), respectively.
2.2 Biochemistry
The effect of iminosugar chirality on NLGase inhibition and thereby on the P. aeruginosa
triggered lung inflammation was then explored. In first studies, the inhibitory effect of the
synthesized D- and L-iminosugars [1-5, ent-(1-5)] on NLGase activity was examined by in vitro
assays in model SH-SY5Y cells, a human neuroblastoma cell line having high activity of both
GCase (encoding the lysosomal enzyme glucocerebrosidase) and NLGase (encoding the non-
lysosomal glucosylceramidase). As summarized in Table 1,
NLGase inhibitors (entries 2, 5, 8, 11 and 14). Nevertheless, the inhibition potency was always
lower (in the micromolar range) than those of -iminosugars (IC50 down to 3 nM, entries 1, 4, 7,
10 and 13). Ent-3 and ent-5 displayed the highest inhibitory activity among -iminosugars (IC50
65 nM, entry 8; IC50 137 nM, entry 14), in agreement with the role of the alkyl chain length
regarding NLGase inhibition [20,21]. In several cases, -iminosugars (entries 2, 5 and 12) when
L-iminosugars ent-(1-5) acted as
D
L
L
tested as GCase inhibitors, showed higher IC50 with respect to those observed against NLGase
demonstrating, as expected, high enzymatic selectivity.
The effect of the combined incubation of D- and L-iminosugars [26], leading to rac-(1-5), was
also considered. While no significant synergistic effect was found for rac-1 and rac-2,
conversely rac-3-5 displayed a similar activity to that exerted by 3-5 when taken alone (entries 9,
12 and 15) with a total inhibition of the NLGase activity.
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Table 1. Effect of iminosugar chirality on glycohydrolase activity.
GCase
NLGase
entry Compound
IC50 (µM) M.I. (%) IC50 (µM) M.I. (%)
118
62
57
0.014
7.7
100
100
100
100
100
100
100
10
1
2
1
ent-1
rac-1
2
24
N.D.
5.6
N.D.
100
0.260
0.003
4.6
3
4
ent-2
rac-2
3
136
51
5
N.D.
N.D.
N.D.
N.D.
5.4
N.D.
N.D.
N.D.
N.D.
100
0.035
0.003
0.065
0.004
0.0004
36.7
6
7
ent-3
rac-3
4
8
100
100
100
100
100
10
9
10
11
12
13
14
15
ent-4
rac-4
5
603
65
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.001
0.034
0.137
0.044
ent-5
rac-5
100
The assays were performed on lysates of SH-SY5Y cells, using different concentrations of each
compound (from 1nM to 1mM) in the presence of 5nM AMP-DNM or 750µM CBE. IC50: inhibitor
concentration that produces 50% inhibition. M.I.: maximal inhibition. N.D.: Not Determined.
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2.3 Biological evaluation
2.3.1 In vitro studies
A selection of N-alkylated iminosugar enantiomers were tested for their effect on P.
aeruginosa stimulated IL-8 mRNA expression in CF bronchial epithelial cells. CuFi-1 cells were
treated with either 1, 2, 3, ent-1, ent-2, ent-3, or the corresponding racemic mixtures (rac-1, rac-
2, rac-3) 1 h before infection with P. aeruginosa and for the following 4 h. We found that both
ent-1 and ent-3 (0.1 µM) reduced the IL-8 mRNA expression (up to a 20% and 29% reduction,
respectively), with an effect comparable to that of the corresponding enantiomers. Conversely,
ent-2 exhibited a smaller effect (16% reduction) under the same conditions, although it was
greater than that of 2 (8% reduction). Interestingly, the combination of iminosugar enantiomers
(0.05 + 0.05 µM) produced an even more pronounced effect. As an example, the reduction of IL-
8 mRNA expression by rac-1 and rac-3 was down to 30% and 42%, respectively. Analogously,
rac-2 inhibited the expression of IL-8 down to 22% (Figure 2). These results suggest that the
combination of
D and L enantiomers could reduce the concentration of D enantiomers, thus
minimizing the undesired side effects.
Figure 2. Effect of sugar chirality of NBDNJ (1, ent-1 ), NNDNJ (2, ent-2 ) and AMPDNM (3, ent-3 ) 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. The inflammatory response was
evaluated by studying the expression of IL-8 mRNA, measured by Real-time qPCR and obtained by
comparing the ratio IL-8 and the housekeeping gene β-actin between not infected and infected cells. The
results, expressed as percentage of untreated cells, are mean ± standard error of the mean of four
independent experiments in duplicate. Comparisons between groups were made by using Anova.
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Subsequently, dose-response experiments were performed. IC50 values related to the inhibition of
IL-8 mRNA expression were calculated by adding increasing doses of rac-1, rac-2 and rac-3
(from 1 nM to 1 mM) to CF bronchial cells 1 h before infection (Table 2). We found that rac-1
inhibited at very low concentration (IC50 0.95 nM; CI 0.54-1.36), with a maximal inhibition of
36% (CI 29-38). The IC50 value of rac-2 was even lower (IC50 0.6 nM; CI 0-2), although
maximal inhibition was only 12% (CI 7-15). Similarly, the IC50 value of rac-3 was 1.84 nM (CI
0.3-2.1), while maximal inhibition was 29% (CI 27-33). In the same cell model, 1 alone
displayed an IC50 value of about 2 µM and a maximal inhibition of 50% [17]; conversely, 3 alone
displayed an IC50 value very similar to that obtained using the racemic mixture.
The effect of racemic mixtures reported in Figure 2 was also tested in CF bronchial primary
cells infected by P. aeruginosa (Figure 3). Primary airway epithelial cells, i.e., mainstem human
bronchi, derived from CF individuals [36], were treated with rac-1, rac-2 or rac-3 (10 nM)
before the infection with P. aeruginosa strain PAO1. As shown in Figure 3, rac-1 and rac-3
significantly reduced the inflammatory response to P. aeruginosa in CF primary cells.
Whether as single enantiomer or as racemic mixtures, N-alkylated iminosugars did not induce
significant apoptosis in CF bronchial cells when compared with vehicle after 48h treatment
(Figure S1). In addition, all tested iminosugars did not significantly decrease cell viability after
48h incubation in the same experimental model (Figure S2).
Table 2. IC50 and Maximal Inhibition of racemic mixtures on IL-8 mRNA expression
Compound IC50 (nM) CI (nM) M.I. (%) CI (%)
rac-1
rac-2
rac-3
0.95
0.6
0.54-1.36 36
29-38
7-15
0-2
12
29
1.84
0.3-2.1
27-33
CuFi-1 cells were treated as indicated in Figure 2. Data obtained in three independent experiments in
duplicate were used to calculate IC50 values by “Graph Pad Prism” 6.0.
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Figure 3. 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. The inflammatory response was evaluated as indicated in Figure 2. The
results, expressed as relative to not infected cells, are mean ± standard error of the mean of three
independent experiments in duplicate. Comparisons between groups were made by using Anova.
2.3.2 In vivo studies
The combined effect of iminosugar enantiomers was eventually studied in murine models of lung
inflammation. Since we were interested to evaluate the role of chirality on anti-inflammatory
activity of such compounds, we needed of a complete overview of the in vitro and in vivo
behavior, as well as of pharmacokinetics and safety profile data of a reference iminosugar.
Among all the synthesized compounds, N-butyl iminosugar derivative rac-1 was chosen for
preliminary studies since 1 was already widely studied [15,37-40]. Treatment with 100 mg/kg of
1 for 3 days reduced the recruitment of neutrophils into the bronchoalveolar space after
intranasal instillation of LPS [15]. Moreover, an oral dose of 400 mg/kg of 1 given to mice
before the intra-tracheal inoculum with P. aeruginosa was effective in reducing strongly the
inflammatory response associated with acute pneumonia in terms of leukocyte recruitment and
myeloperoxidase activity in the airways [16]. Therefore, we evaluated at first the effect of rac-1
on acute infection and inflammation in C57Bl/6NCr male mice. Considering the IC50 values
obtained in vitro with rac-1 (Table 2), lower doses than those previously described [15] were
used in these studies. Mice were treated by gavage 24 and 1 h before the infection with rac-1 at
10 mg/kg or 100 mg/kg (Figure 4). After infection (6h), mice were killed with an overdose of
carbon dioxide, and murine lungs tested for lung infection, while BAL was processed for
inflammation as previously described [41]. We found that treatment of mice with rac-1 at 100
mg/kg provided a slight reduction of the amount of neutrophils, although this decrease was not
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statistically significant (Figure 4B). Interestingly, we also found a marked increase of alveolar
macrophages in mice treated with the lower dose of rac-1 (Figure 4C). In line with this last
result, treatment with rac-1 at 10 mg/kg was accompanied by a reduction of CFU both in BAL
and in lungs (Figure S3).
Figure 4. Effect of rac-1 on cells recruited in BAL of C57Bl/6NCr mice infected by P. aeruginosa. A.
Total cells; B. Neutrophils; C. Alveolar macrophages.
Figure 5. Effect of 1 and ent-1 on cells recruited in BAL of C57Bl/6NCr mice infected by P. aeruginosa.
A. Total cells; B. Neutrophils; C. Alveolar macrophages.
Administration of rac-1 proved to be safe as demonstrated by the weight of mice after treatment.
Furthermore, while infection typically induces a weight loss in untreated mice, this effect was
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not observed in mice treated with rac-1 at the higher dose (Figure S4). In order to investigate
whether this effect was due to the combined treatment of NBDNJ enantiomers or to singularly
administered iminosugars, in subsequent studies we separately treated mice with 1 or ent-1
before infection. Surprisingly, we found that ent-1 decreased the amount of neutrophils recruited
in BAL at a concentration as low as 10 mg/kg, i.e. 40-fold lower than that of 1 (Figure 5B). Very
importantly, the decreased recruitment of neutrophils did not increase the amount of bacteria
recovered in the airways (Figure S5). It was also noteworthy that in mice treated with ent-1 a
dose-dependent decrease of bacteria was detected in lungs, although it was not statistically
significant (Figure S5). Treatment with both 1 and ent-1 was safe as shown by the weights of
mice monitored during the experiment (Figure S6). Lastly, the NLGase activity in the lungs of
mice infected by P. aeruginosa in presence of 1, ent-1 and rac-1 was evaluated (Figure 6). If
rac-1 does not influence the enzymatic activity, ent-1 deeply inhibits NLGase. These results,
differently from the in vitro assays (Table 1) suggested, as previously observed for 1 [17], the
involvement of NLGase inhibition in the inflammatory response to P. aeruginosa infection.
Figure 6. NLGase activity in the lungs of mice infected by P. aeruginosa.
Mice were treated as indicated in Figure S3 (A) or S5 (B). Lung lysates were diluted in Mc Ilvaine buffer
(pH 6.0) and treated for 30 min at rt with 1mM CBE to inhibit GCase. Then lysates were incubated with
6mM methylumbelliferyl-β-D-glucopyranoside (MUB-Glc) at 37 °C under gently shaking. After 1h
incubation, the developed fluorescence was detected diluting an aliquot of the assay mixture with 20 vol
of 0.25 M glycine, pH 10.7. As background, we used boiled aliquots of each lung lysate. Data were
expressed as pmoles×mg tissue proteins-1× h-1. Data reported are Mean ± Standard Error Comparisons
between groups were made by Anova.
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3. Conclusion
The role of iminosugar chirality in the anti-inflammatory treatment of CF lung disease has
been herein explored through the analysis of the therapeutic potential of novel N-alkyl -DNJ
L
derivatives – from the workbench to in vivo studies. From the synthetic standpoint, the shortest
approach reported to date has been tuned, leading to the insertion of the bioactive alkoxypentyl
chains onto the iminosugar core through the efficient preparation of alkyl iodides 7a-c, 10 and
alkoxyalkyl iodides 9a-c by the PS-DPP/I2 reagent system.
L-Iminosugars ent-(1-3) have
demonstrated to act as selective NLGase inhibitors and they are able to reduce the inflammatory
response to P. aeruginosa in CF bronchial cells at very low concentration. In addition, a
synergistic effect deriving from the combined use of enantiomeric iminosugars has been
suggested both in NLGase inhibition and in the reduction of the IL-8 mRNA expression in CF
bronchial cells CuFi-1 cells. Importantly, the use of ent-1 in C57Bl/6NCr mice (which was
totally safe under our experimental conditions) has led to a decrease in the amount of neutrophils
recruited in BAL at a concentration 40-fold lower than 1, without increase of the amount of
bacteria recovered in the airways.
Taken together, these findings provide for the first time interesting indications on the
therapeutic potential of
activity (especially that of ent-1, which has been evaluated in greater detail) appears even
preferable to that of the corresponding -iminosugars. In particular, on the basis of these data,
L-iminosugars as anti-inflammatory agents in CF lung disease. Their
D
strong indications that ent-1 could be a promising molecule to reduce the inflammatory response
in CF patients with no side inhibition of glycosidases and without impairing host defences have
been given. Further studies aimed to more extensively define scope and limitation of the anti-
inflammatory properties of ent-1 and its congeners are currently ongoing and will be published in
due course.
4. Experimental Section
4.1 Chemical Synthesis
General information. All chemicals and solvents were purchased with the highest degree of
purity (Sigma-Aldrich, Alfa Aesar, VWR) and used without further purification. All moisture-
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sensitive reactions were performed under nitrogen atmosphere using oven-dried glassware. The
reactions were monitored by TLC (precoated silica gel plate F254, Merck) and the products were
detected by exposure to ultraviolet radiation, iodine vapor, chromic mixture and ninhydrin.
Column chromatography: Merck Kieselgel 60 (70-230 mesh); flash chromatography: Merck
Kieselgel 60 (230-400 mesh). The purity of the synthetic intermediates and the final compounds
was determined by CHNS analysis and was ≥ 95% in all cases. NMR spectra were recorded on
NMR spectrometers operating at 400 MHz (Bruker DRX, Bruker AVANCE) or 500 MHz
(Varian Inova), using CDCl3 solutions unless otherwise specified. Combustion analyses were
performed using a CHNS analyzer.
General procedure for 9a-c: PS-TPP/I2 as the iodination reagent. Step 1: formation of iodides
7a-c. Alcohol 6 (1.0 mmol) was dissolved in the appropriate anhyd solvent (6a and 6b: ACN; 6c:
toluene), then polymer supported triphenylphosphine (PS-TPP; 100-200 mesh, extent of labeling:
~3 mmol/g triphenylphosphine loading) (2.0 equiv) was added. The mixture was warmed to 40
°C and I2 (2.0 equiv) was added. After warming the reaction to reflux temperature and leaving
the suspension for the appropriate time (6a: 0.5h; 6b: 1h; 6c: 4h), the mixture was cooled at rt,
filtered and the solvent removed under reduced pressure at rt, affording iodide 7. 1-Iodohexane
1
(7a) and 1-iodononane (7b): H and 13C NMR spectra were fully in agreement with those
reported in the literature [42]. Adamantanemethyl iodide (7c): 1H NMR (500 MHz): δ 1.51 (d,
J = 2.5 Hz, 6H), 1.64-1.67 (m, 3H), 1.72-1.75 (m, 3H), 1.99 (s, 3H), 3.20 (s, 2H). 13C NMR (125
MHz): δ 27.1, 28.8, 36.7, 42.2. Anal. calcd for C11H17I: C, 47.84; H, 6.20. Found: C, 47.77; H,
6.22.
Step 2: formation of alkoxyalcohols 8a-c. An appropriate amount of 1,5-pentandiol (7a and 7b:
1.0 equiv; 7c: 5.0 equiv) was dissolved in dry DMF (7a and 7b: 1.0 mL; 7c: 5.0 mL) and NaH
(60% dispersion in mineral oil; 7a and 7b: 1.2 equiv; 7c: 5.0 equiv) was added. The mixture was
stirred for 30 min at room temperature. Then a solution of the crude iodide 7 in dry DMF (0.5
mL) was added dropwise. After 16h at 100 °C, the mixture was concentrated under reduced
pressure to furnish 8. For 8c, the crude was washed repetitively with hexane in order to remove
the excess of 1,5-pentandiol. NMR analysis of a small aliquot of the crude reaction mixture
1
confirmed the structure of desired alkoxyalcohols 8. 5-Hexyloxyl-1-pentanol (8a): H NMR
14

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(400 MHz): δ 0.88 (t, J = 6.4 Hz, 3H), 1.25-1.35 (m, 6H), 1.41-1.47 (m, 2H), 1.52-1.64 (m, 6H),
3.40 (q, J = 6.7 Hz, 4H), 3.65 (t, J = 6.5 Hz, 2H). 13C NMR (100 MHz): δ 14.2, 22.7, 22.8, 26.0,
29.6, 29.9, 31.9, 32.7, 63.0, 70.9, 71.2. Anal. calcd for C11H24O2: C, 70.16; H, 12.85; O, 16.99.
1
Found: C, 70.06; H, 12.89. 5-Nonyloxyl-1-pentanol (8b): H NMR (400 MHz): δ 0.87 (t, J =
6.6, 3H), 1.21–1.36 (m, 12H), 1.38–1.46 (m, 2H), 1.51–1.65 (m, 6H), 3.39 (q, J = 6.6, 4H), 3.65
13
(t, J = 6.5 Hz, 2H). C NMR (125 MHz): δ 14.2, 22.5, 22.7, 26.3, 29.3, 29.5, 29.6, 29.6, 29.8,
32.0, 32.6, 63.0, 70.8, 71.1. Anal. calcd for C14H30O2: C, 72.99; H, 13.12; O, 13.89. Found: C,
1
73.10; H, 13.06. 5-Adamantanemethoxypentanol (8c): H NMR (500 MHz): δ 1.39-1.45 (m,
2H), 1.52-1.53 (m, 6H), 1.56-1.61 (m, 4H), 1.63-1.72 (m, 6H), 1.95 (bs, 3H), 2.96 (s, 2H), 3.39
(t, J = 6.5 Hz, 2H), 3.66 (t, J = 6.6 Hz, 2H). 13C NMR (125 MHz): δ 22.6, 28.5, 29.4, 32.7, 34.3,
37.4, 39.9, 63.1, 71.7, 82.1. Anal. calcd for C16H28O2: C, 76.14; H, 11.18; O, 12.68. Found: C,
76.05; H, 11.22.
Step 3: formation of iodides 9. Alcohol 8 was dissolved in the appropriate anhyd solvent (8a and
8b: ACN, 2.5 mL; 8c: toluene, 5 mL), then PS-TPP (2.0 equiv) was added. The mixture was
warmed to 40 °C and I2 was added (2.0 equiv). After warming the reaction to reflux temp and
leaving the suspension under stirring at the same temp for the appropriate time (8a and 8b: 16h;
8c: 6h), the mixture was filtered and the filtrate evaporated under reduced pressure. Column
chromatography of the crude residue (hexane/EtOAc) gave the pure alkoxyalkyl iodide 9 (9a: 70
1
% o.y.; 9b: 88% o.y.; 9c: 80% o.y.). 1-Iodo-5-hexyloxyl-pentane (9a): H NMR (400 MHz): δ
0.89 (t, J = 6.7 Hz, 3H), 1.25-1.37 (m, 5H), 1.42-1.50 (m, 2H), 1.52-1.63 (m, 5H), 1.82-1.89 (m,
13
2H), 3.19 (t, J = 7.0 Hz, 2H), 3.39 (t, J = 5.0 Hz, 2H), 3.41 (t, J = 4.4 Hz, 2H). C NMR (100
MHz): δ 7.1, 14.2, 22.7, 26.0, 27.4, 28.8, 29.9, 31.9, 33.5, 70.6, 71.2. Anal. calcd for C11H23IO:
C, 44.30; H, 7.77; I, 42.56; O, 5.37. Found: C, 44.22; H, 7.44. 1-Iodo-5-nonyloxyl-pentane
1
(9b): H NMR (500 MHz): δ 0.88 (t, J = 6.8 Hz, 3H),1.25-1.34 (m, 12H), 1.43-1.49 (m, 2H),
1.53-.63 (m, 4H),1.82-1.88 (m, J = 7.1 Hz, 2H), 3.19 (t, J = 7.0 Hz, 2H), 3.4 (q, J = 6.4 Hz, 4H).
13C NMR (125 MHz): δ 7.3, 14.5, 23.1, 26.6, 27.7, 29.1, 29.7, 29.9, 30.0, 30.2, 32.3, 33.8, 70.9,
71.5. Anal. calcd for C14H29IO: C, 49.41; H, 8.59; I, 37.29; O, 4.70. Found: C, 49.50; H, 8.56. 1-
1
Iodo-5-adamantanemethoxyl-pentane (9c): H NMR (500 MHz): δ 1.41-1.50 (m, 2H), 1.50-
1.54 (m, 6H), 1.54-1.60 (m, 2H), 1.60-1.74 (m, 6H), 1.80-1.89 (m, 2H), 1.92-1.99 (m, 3H), 2.94
15

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(s, 2H), 3.19 (t, J = 7.1 Hz, 2H), 3.37 (t, J = 6.3 Hz, 2H). 13C NMR (125 MHz): δ 7.2, 27.4, 28.4,
28.5, 28.6, 28.7, 29.8, 31.4, 32.2, 33.5, 34.2, 37.4, 37.5, 39.9, 71.4, 82.1. Anal. calcd for
C16H27IO: C, 53.04; H, 7.51; I, 35.03; O, 4.42. Found: C, 53.16; H, 7.54
General synthetic procedure for 9a-c via bis-iodination. Step 1: 1,5-diiodopentane (10). To a
solution of PS-TPP (100-200 mesh, extent of labeling: ~3 mmol/g triphenylphosphine loading)
(4.0 equiv) and iodine (4.0 equiv) in anhyd DCM, 1,5-pentandiol (1.0 equiv) was added
dropwise. The reaction was stirred at room temperature for 1 h, then filtered, to remove
triphenylphosphine oxide, and washed with saturated aq Na2S2O3, brine and extracted with
DCM. Organic layers were dried (Na2SO4) and evaporated under diminished pressure at room
1
temperature to give the pure 1,5-diiodopentane (10) as a pale yellow oil (95% yield). H NMR
(500 MHz): δ 1.49-1.55 (m, 2H), 1.82-1.88 (m, 4H), 3.19 (t, J = 7.0 Hz, 4H). 13C NMR (100
MHz), δ: 6.3(2C), 31.1, 32.5(2C). Anal. calcd for C5H10I2: C, 18.54; H, 3.11; I, 78.35. Found: C,
18.62; H, 3.12. Step 2: NaH (60% dispersion in mineral oil; 1.0 equiv) was added to a
magnetically stirring solution of alcohol 6 (1.5 equiv) in dry THF (1.5 mL) at 0 °C. The reaction
mixture was stirred at the same temperature for 2 h. Bis-iodide 10 (0.70 mmol) was then added
and the mixture warmed to rt. After the appropriate time (6a and 6b: 48h; 6c: 72h) the mixture
was diluted with DCM and washed with aq NH4Cl and brine. The organic layer was dried with
Na2SO4 and the solvent evaporated under reduced pressure. Chromatography of the crude residue
over silica gel provided the pure iodide 9 (9a: 75% yield; 9b: 74% yield; 9c: 72% yield).
General synthetic procedure for ent-2-5: iodide 7b, 9a-c (1.2 equiv) was dissolved in anhyd
DMF (3.5 mL) and added dropwise to a solution of L-DNJ (1.0 equiv) and K2CO3 (3.0 equiv) in
DMF (3.5 mL). The reaction was warmed to the appropriate temperature (7b: 70 °C; 9a-c: 80
°C) and stirred for 16 h. Afterwards, the solvent was removed under diminished pressure.
Chromatography of the crude residue over silica gel (ent-2-4: acetone/MeOH = 8:2; ent-5:
acetone/MeOH = 9:1) afforded the DNJ derivative ent-2-5 as an oil. The compound, dissolved in
water, was further purified with Dowex® 1X8, 50-100 mesh, ion-exchange resin.
L
-N-nonyl DNJ (ent-2): 80% yield; 1H NMR (400 MHz, CD3OD): δ 0.90 (t, J = 7.0 Hz, 3H),
1.22-1.38 (m, 12H), 1.45-1.57 (m, 2H), 2.18-2.20 (m, 1H), 2.24 (t, J = 10.9 Hz, 1H), 2.60-2.66
(m, 1H), 2.81-2.87 (m, 1H), 3.02 (dd, J = 4.8, 11.3 Hz, 1H), 3.15 (t, J = 9.1 Hz, 1H), 3.37 (t, J =
16

ACCEPTED MANUSCRIPT
9.3 Hz, 1H), 3.49 (dt, J = 4.9, 5.3 Hz, 1H), 3.86 (bs, 2H). 13C NMR (100 MHz, CD3OD), δ: 14.4,
23.7, 24.5, 27.9, 30.3(2C), 30.6, 33.0, 54.1, 55.5, 56.4, 67.4, 68.6, 69.7, 78.7. Anal. calcd for
C15H31NO4: C, 62.25; H, 10.80; N, 4.84; O, 22.11. Found: C, 62.40; H, 10.76; N, 4.81. LC-TOF
MS: m/z [M + H]+, calcd: 290.23; found: 290.23.
L
-N-Adamantanemethoxypentyl-DNJ (L
-AMP-DNM) (ent-3): 80% yield; 1H NMR (500
MHz, CD3OD): δ 1.31-1.40 (m, 2H), 1.50-1.64 (m, 10H), 1.69 (bd, J = 11.9 Hz, 3H), 1.76 (bd, J
= 12.0 Hz, 3H), 1.95 (bs, 3H), 2.09-2.15 (m, 1H), 2.16-2.22 (m, 1H), 2.55-2.63 (m, 1H), 2.77-
2.83 (m, 1H), 2.97 (s, 2H), 2.99 (dd, J = 4.8, 11.3 Hz, 1H), 3.13 (t, J = 9.1 Hz, 1H), 3.35 (t, J =
8.9 Hz, 1H), 3.39 (t, J = 6.2 Hz, 2H), 3.45-3.50 (m, 1H), 3.84 (dd, J = 1.7, 12.0 Hz, 1H), 3.87
13
(dd, J = 2.3, 12.2 Hz, 1H). C NMR (100 MHz, CD3OD), δ: 24.2, 24.7, 29.7, 30.2, 33.4, 38.3,
40.8, 55.1, 55.7, 67.4, 68.1, 69.2, 72.2, 72.6, 78.4, 83.1. Anal. calcd for C22H39NO5: C, 66.47; H,
9.89; N, 3.52; O, 20.12. Found: C, 66.37; H, 9.92; N, 3.51. LC-TOF MS: m/z [M + H]+, calcd:
398.29; found: 398.29.
L
-N-[5-(Hexoxy)pentyl]-DNJ (ent-4): 78% yield; 1H NMR 500 MHz, CD3OD): δ 0.93 (t, J =
6.7 Hz, 3H), 1.31-1.39 (m, 8H), 1.52-1.65 (m, 6H), 2.15-2.24 (m, 2H), 2.59-2.65 (m, 1H), 2.81-
2.87 (m, 1H), 3.02 (dd, J = 4.9, 11.2 Hz, 1H), 3.15 (t, J = 9.1 Hz, 1H), 3.37 (dd, J = 9.4, 12.0 Hz,
1H), 3.45 (q, J = 6.2 Hz, 4H), 3.49 (dt, J = 4.9, 10.3 Hz, 1H), 3.86 (dd, J = 2.7, 12.0 Hz, 1H),
3.89 (dd, J =2.6, 12.0 Hz, 1H). 13C NMR (500 MHz, CD3OD): δ 14.4, 23.7, 23.9, 24.5, 26.9,
30.1, 30.7, 32.8, 54.3, 54.9, 54.9, 67.4, 67.8, 68.8, 71.4, 72.1, 78.1. Anal. calcd for C17H35NO5:
C, 61.23; H, 10.58; N, 4.20; O, 23.99. Found: C, 61.13; H, 10.62; N, 4,21. LC-TOF MS: m/z [M
+ H]+, calcd: 334.26; found: 334.25.
1
L
-N-[5-(Nonyloxy)pentyl]DNJ (ent-5): 75% yield;. H NMR (500 MHz, CD3OD): δ: 0.92 (t,
J = 6.6 Hz, 3H), 1.29-1.41 (m, 14H), 1.51-1.64 (m, 6H), 2.13 (bd, J = 9.5 Hz, 1H), 2.19 (dd, J =
6.7, 10.8 Hz, 1H), 2.57-2.63 (m, 1H), 2.79-2.85 (m, 1H), 3.01 (dd, J = 4.9, 11.2 Hz, 1H), 3.14 (t,
J = 9.1 Hz, 1H), 3.37 (t, J = 9.0 Hz, 1H), 3.44 (q, J = 6.3 Hz, 4H), 3.49 (dt, J = 4.8, Hz, 1H), 3.85
(dd, J = 2.2, 11.9 Hz, 1H), 3.89 (dd, J = 1.9, 11.9 Hz, 1H). 13C NMR (100 MHz, CD3OD), δ:
14.4. 23.7, 24.0, 24.5, 27.3, 30.2, 30.4, 30.6, 30.7, 30.8, 33.0, 54.3, 54.8, 54.9, 67.4, 67.8, 68.8,
71.4, 72.1, 78.2. Anal. calcd for C20H41NO5: C, 63.96; H, 11.00; N, 3.73; O, 21.30. Found: C,
64.07; H, 10.96; N, 3,72. LC-TOF MS: m/z [M + H]+, calcd: 376.31; found: 376.30.
17

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4.2
Biochemical Studies.
General. SH-SY5Y cell line was cultured in DMEM High Glucose containing 10% inactivated
FBS (fetal bovine serum), 2 mM -glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.
L
At 80% of confluence, cells were scraped and pelleted and lysed in water containing protease
inhibitors. An aliquot of the cell homogenate was used to evaluate the protein concentration by
DC Protein Assay (Biorad). The enzymatic activity of GCase and NLGase associated with total
cell lysate were determined by an in vitro assay based on the use of the florigenic substrate 4-
methylumbelliferyl-β-D-glucopyranoside (MUB-Gluc) as previously described with some
modifications [18,43,44]. Aliquots corresponding to the same amount of cell lysate were
transferred in a 96-well plate and incubated or not (control), for 30 minutes at rt, in McIlvaine
buffer pH 5.8 containing the compounds at the concentrations of 1nM, 10nM, 5µM, 100µM, and
1mM respectively. For the detection of NLGase, the reaction mixtures were then incubated with
5nM AMP-DNM; whereas, for GCase were incubated with 750µM conduritol B epoxide, in both
cases for 30 minutes at the rt. The assay was started by adding MUB-Glc at the final
concentration of 6mM. After 2 h at 37°C of incubation, 10 µl of the reaction mixtures were
transferred to other 96-well plates and 190 µl of 0.25 M glycine at pH 10.7 was added to each
reaction. The plates were fluorometrically analyzed using a microplate reader (Victor, Perkin
Elmer). By the use of a free MUB standard the pmoles of the formed product were calculated
and the specific enzymatic activity was expressed as pmoles of product formed/mg
proteins/hour. The percentages of inhibition were calculated as ratio between the enzymatic
activity measured in presence of the different compounds with respect to that measured in their
absence. IC50 and maximal inhibition were determined by interpolating the data, expressed as %
of inhibition with respect to the inhibitor concentration, by one phase non-linear exponential
regression using the software Graphpad Prism 5.0.
In vitro and in vivo assays. Cells- CF bronchial epithelial CuFi-1 cells were grown as previously
described [17]. Primary airway epithelial cells, i.e., mainstem human bronchi, derived from CF
individuals, obtained from “Servizio Colture Primarie” of the Italian Cystic Fibrosis Research
Foundation were cultured as previously described [17].
18

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Inflammatory response in vitro- Cells were infected with P. aeruginosa laboratory strain
PAO1 as detailed [16]. As readout of the inflammatory response, IL-8 mRNA expression was
measured by Real-time qPCR, as described [17].
Toxicity and apoptosis- Annexin V and Dead Cell assays were performed with the Muse cell
analyzer (Millipore, Billerica, MA, USA) method, as described [15]. Data were acquired and
recorded utilizing the Annexin V and Dead Cell Software Module (Millipore, Billerica, MA,
USA). Cell counting and viability assays were carried out with the automated Muse cell analyzer
method. Data from samples were acquired and recorded utilizing the Count & Viability Software
Module.
Inflammatory response in vivo- C57Bl/6NCr male mice were treated by gavage 24 and 1 hour
before infection with 10 mg/kg or 100mg/kg 1 or ent-1 or rac-1. 6 hours after infection, mice
were killed with an overdose of carbon dioxide, and murine lungs tested for lung infection, and
BAL processed for inflammation as described [41].
Statistics- Statistical analysis was carried out withone-way ANOVA, using Prism 6.0 Software
(GraphPad Software, San Diego, CA). *P<0.05, **P <0.01, ***P <0.001and ****P <0.0001).
Conflicts of interest
The authors have no competing interests to declare and have no conflicts of interest.
Acknowledgment
This work was supported by Italian Cystic Fibrosis Research Foundation, grant #22/2015 to
MCD. and #24/2014 to SS and MA.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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Graphical abstract
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Highlights
•
•
•
•
•
Alkoxyalkyl iodides were conveniently prepared by PS-TPP/I2 reagent system.
N-alkyl-
N-alkyl-
L
-deoxyiminosugars were synthetized.
L-deoxyiminosugars selectively inhibit NLGase activity.
L
-NBDNJ reduces the inflammatory response to P.aeurginosa in CF bronchial cells.
-NBDNJ reduces the recruitment of neutrophils in mice at low dosage (10 mg/kg).
L