Dual targeting of PTP1B and glucosidases with new bifunctional iminosugar inhibitors to address type 2 diabetes

Article abstract of DOI:10.1016/j.bioorg.2019.03.053

The diffusion of type 2 diabetes (T2D) throughout the world represents one of the most important health problems of this century. Patients suffering from this disease can currently be treated with numerous oral anti-hyperglycaemic drugs, but none is capab

Full text of DOI:10.1016/j.bioorg.2019.03.053

BioorganicChemistry87(2019)534–549
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Dual targeting of PTP1B and glucosidases with new bifunctional iminosugar
inhibitors to address type 2 diabetes
Xhenti Ferhatia,1, Camilla Matassinia,1, Maria Giulia Fabbrinia, Andrea Gotia,b, Amelia Morronec,
⁎
⁎
Francesca Cardonaa,b, , Antonio J. Moreno-Vargasd, Paolo Paolie,
a Department of Chemistry ‘Ugo Schiff’, University of Firenze, via della Lastruccia 3-13, Sesto Fiorentino, (FI), Italy
b Associated with Consorzio Interuniversitario Nazionale di ricerca in Metodologie e Processi Innovativi di Sintesi (CINMPIS), Italy
c Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children’s Hospital, and Department of Neurosciences, Pharmacology and Child Health.
University of Florence, Viale Pieraccini n. 24, 50139 Firenze, Italy
d Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, n/Prof. García González 1, E-41012 Sevilla, Spain
e Department of Experimental and Clinical Biomedical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
The diffusion of type 2 diabetes (T2D) throughout the world represents one of the most important health pro-
blems of this century. Patients suffering from this disease can currently be treated with numerous oral anti-
hyperglycaemic drugs, but none is capable of reproducing the physiological action of insulin and, in several
cases, they induce severe side effects. Developing new anti-diabetic drugs remains one of the most urgent
challenges of the pharmaceutical industry. Multi-target drugs could offer new therapeutic opportunities for the
treatment of T2D, and the reported data on type 2 diabetic mice models indicate that these drugs could be more
effective and have fewer side effects than mono-target drugs. α-Glucosidases and Protein Tyrosine Phosphatase
1B (PTP1B) are considered important targets for the treatment of T2D: the first digest oligo- and disaccharides in
the gut, while the latter regulates the insulin-signaling pathway. With the aim of generating new drugs able to
target both enzymes, we synthesized a series of bifunctional compounds bearing both a nitro aromatic group and
an iminosugar moiety. The results of tests carried out both in vitro and in a cell-based model, show that these
bifunctional compounds maintain activity on both target enzymes and, more importantly, show a good insulin-
mimetic activity, increasing phosphorylation levels of Akt in the absence of insulin stimulation. These com-
pounds could be used to develop a new generation of anti-hyperglycemic drugs useful for the treatment of
patients affected by T2D.
Type 2 diabetes
Bifunctional compounds
α-glucosidase inhibitors
PTP1B inhibitors
Iminosugars
Insulin-mimetic activity
1. Introduction
options aim to stabilise the glycaemia of patients under the physiolo-
gical range to avoid acute symptoms (hyperglycaemic peaks) and, in
Non-insulin dependent diabetes mellitus (NIDDM), or T2D, is cur-
rently the most prevalent form of diabetes and the number of people
affected by this pathology is continuously increasing worldwide [1].
Although non-genetic factors, such as obesity, a sedentary lifestyle and
hypercaloric diets are the main causes of T2D, the disease also affects
non-obese and physically active people, highlighting the importance of
further research focussed on the onset mechanism. T2D is typically
considered a pathology of the elderly, even though in the last century
the number of diagnoses in men and women of middle age, or even in
children began to rise [2]. In addition, it is a chronic pathology for
which no definitive treatment is available. All current therapeutic
the long term, the risk of pathological complications [3]. Therapeutic
options include oral anti-hyperglycaemic drugs such as metformin, gut
amylase and α-glucosidase inhibitors, sulfonylureas, thiazolidine-
diones, glucagon-like peptide-1 receptor agonists, inhibitors of the de-
grading enzyme dipeptidyl peptidase 4, or, as a last resort, the injection
of exogenous insulin [4]. No currently available drug is able to mimic
the action of physiological insulin, therefore the search for new anti-
diabetic drugs remains an important challenge.
Protein Tyrosine Phosphatase 1B (PTP1B) is one of the most pro-
mising targets for the treatment of T2D and metabolic syndrome.
Administration of PTP1B inhibitors to diabetic mice models showed
⁎ Corresponding authors at: Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale Morgagni 50, 50134
Florence, Italy (Paolo Paoli), Department of Chemistry ‘Ugo Schiff’, University of Firenze, via della Lastruccia 3-13, Sesto Fiorentino (FI), Italy (Francesca Cardona).
E-mail addresses: francesca.cardona@unifi.it (F. Cardona), paolo.paoli@unifi.it (P. Paoli).
1 These authors contributed equally to this work
https://doi.org/10.1016/j.bioorg.2019.03.053
Received 15 January 2019; Received in revised form 4 March 2019; Accepted 18 March 2019
Availableonline22March2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

X. Ferhati, et al.
BioorganicChemistry87(2019)534–549
normalization of glycaemia and loss of body weight in obese mice [5].
Since T2D can be managed by addressing different targets, an al-
ternative approach based on bifunctional inhibitors could be more ef-
fective and could have fewer side effects.
decreases postprandial hyperglycaemia [27].
Due to a long-standing interest in the total synthesis of natural
iminosugars and non-natural analogues with potential activity as gly-
cosidase inhibitors, we screened a collection of iminosugars with dif-
ferent structures against PTP1B. The monocylic pyrrolidines 5a-j
[28–31] and piperidines 6a-f [32,33], and bicyclic pyrrolizidines 7a-m
[34–38] and indolizidines 8a-e) [39,40] shown in Figs. 2 and 3 were
assayed.
Very recently, structurally complex polycyclic natural compounds
isolated from different sources able to target both α-glucosidase and
PTP1B were identified and characterized [6–12], suggesting that the
use of such bifunctional drugs may delay the absorption of carbohy-
drates and, at the same time, enhance insulin sensitivity by modulating
the insulin signalling pathway.
Taking into consideration the activity shown by compound 5c (see
Discussion), we selected the pyrrolidine as the most promising structure
for further development, because it is easier to synthesize and func-
tionalize. We synthesized a series of new compounds characterized by
the presence of the DAB-1 moiety (common to compound 5c and to
some of the bicyclic structures tested) linked to the nitroaromatic
moiety with spacers of variable length (2, 3 and 4 carbon atoms) via a
triazole (compounds 9–11) or an amide (compounds 12–14) moiety
(Fig. 4).
Several studies correlated insulin resistance with type 1 Gaucher
Disease (GD1), the most prevalent lysosomal storage disorder, caused
by an accumulation of glycosphingolipids in the lysosomes as an effect
of hydrolytic enzyme malfunctioning (e.g. acid β-glucocerebrosidase
(GCase), which is a β-glucosidase) [13]. GD1 patients show insulin
resistance as well as other abnormalities in whole body metabolism
[14–17], suggesting a connection between GD1 and T2D probably due
to the influence of glycosphingolipids on insulin receptor functioning
[18].
These new iminosugar hybrids were assayed in vitro against PTP1B,
and kinetic analyses were performed to evaluate their potency and
mechanism of action. The activity of these bifunctional compounds was
also evaluated towards commercial and human α- and β-glucosidases to
assess their potential as dual inhibitors for the treatment of T2D.
Finally, assays with HepG2 cells were carried out to evaluate their ex
vivo effectiveness. The results of this multidisciplinary work are re-
ported herein.
Since a β-glucosidase inhibitor has the potential to act as pharma-
cological chaperone rescuing the activity of the deficient enzyme in
GD1 [19,20], an inhibitor addressing both β-glucosidase and PTP1B
enzymes may act as a bifunctional drug to treat GD1 patients with
diabetic complications.
With the aim of identifying new compounds with a dual activity to
target T2D, we accessed new glycomimetics obtained by linking a
phosphotyrosine mimetic group (namely a p-nitrophenyl moiety) and
an iminosugar moiety. Among the many phosphotyrosine mimetic
groups developed to target PTP1B inhibition, the p-nitrophenyl moiety
proved to be effective due to its resemblance to the natural enzyme
substrate [21] and, at the same time, it lacks of a net negative charge
that hampers cell permeability and bioavailability [22]. The iminosugar
moiety was introduced in order to impart α-/β- glucosidase inhibition
and determine whether it could enhance PTP1B inhibition.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of bifunctional inhibitors
The synthesis of DAB-1 derivatives functionalized with an azido
(compounds 17, 22, 23) or an amino (compounds 24–26) terminal
moiety, suitable for further coupling with the nitro aromatic moiety, is
shown in Scheme 1. The most straightforward approach was adopted
for the 4-carbon atom linker, with the reaction of the benzylated DAB-1
15 [29] with 1-azido-4-bromobutane 16 [41] under microwave heating
in THF as a solvent, which produced compound 17 with an excellent
90% yield. For the 2- and 3-carbon atom linkers a different procedure
was followed to avoid problems due to volatility and/or instability of
low-molecular-weight azido derivatives. Compound 15 was reacted
with 2-bromoethanol or 3-bromopropanol in THF at room temperature,
to give the corresponding alcohols 18 and 19 with very good yields
(85% and 94%, respectively). To introduce the terminal azido moiety,
nucleophilic displacement with NaN3 on the corresponding mesyl de-
rivatives 20–21 was performed, leading to azides 22 and 23. Com-
pound 23 was produced with a 72% yield over two steps starting from
alcohol 19; the lower yield of compound 22 (6%) was ascribed to the
formation of bicyclic spiro aziridinium by-products, which were visible
in the crude reaction mixture. To overcome this problem, in the case of
the 2-carbon atom linker, a direct Mitsunobu reaction with diphenyl
phosphoryl azide (DPPA) on alcohol 19 was performed, giving a sa-
tisfactory 88% yield for compound 22. The azido-amino reduction step
was achieved either with catalytic hydrogenation under acidic condi-
tions (for compound 26), that also removed the O-benzyl protecting
groups, or via the Staudinger reaction (compounds 24–25), that al-
lowed us to work with O-benzyl-protected derivatives in the subsequent
conjugation step as shown in Scheme 2.
Iminosugars are carbohydrate analogues with a nitrogen atom re-
placing the endocylic oxygen of carbohydrates and are widely known as
glycosidase inhibitors [23]. In particular, the pyrrolidine 1,4-dideoxy-
1,4-imino-D-arabinitol (DAB-1, 1) [24], N-alkylated deoxynojirimycin
(DNJ) derivatives such as compound 2 (Fig. 1), [25] and the natural
pyrrolizidine compound casuarine (3, Fig. 1) [26] have significant
potential for treating T2D. The first oral drug for the treatment of
diabetes based on an iminosugar compound, Miglitol (4, Fig. 1), is now
commercially available. Miglitol inhibits the α-glucosidase enzymes in
the brush border of the small intestine, delays glucose absorption and
The O-benzyl protected azido derivatives 17, 22 and 23 were re-
acted with 1-ethynyl-4-nitrobenzene in the presence of Cu(I) salts [42].
Since the reaction in THF gave only moderate yields at room tem-
perature (53% for compound 27), microwave (MW) irradiation and in-
situ generated Cu(I) were performed for compounds 28 and 29, leading
to the much better yields of 87% and 92% respectively (Scheme 2).
Treatment of 27–29 with BCl3 in dichloromethane at room temperature
allowed us to remove the O-benzyl groups without reducing the nitro
Fig. 1. Examples of iminosugars with potential application (1–3) or on the
market (4) for T2D.
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BioorganicChemistry87(2019)534–549
Fig. 2. Monocyclic iminosugars (pyrrolidines 5a-j and piperidines 6a-f) tested in this work as PTP1B inhibitors.
Fig. 3. Bicyclic iminosugars (pyrrolizidines 7a-m and indolizidines 8a-e) tested in this work as PTP1B inhibitors.
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2.2. Biology
2.2.1. Preliminary screening on PTP1B of the available iminosugars
Based on the observation that polyhydroxylated compounds are
able to target both α-glucosidase and PTP1B [6–12], we envisaged that
our iminosugars 5–8, bearing several hydroxyl groups, could be simi-
larly effective. Therefore, the whole collection of iminosugars 5–8
(Figs. 2 and 3) available in our laboratories was initially screened
against PTP1B using a fixed inhibitor concentration (100 μM) as shown
in Table 1. This preliminary evaluation showed that iminosugars 5–8
behave as moderate-to-good PTP1B inhibitors. Of all the compounds,
5c, 6d, 7g, 7k-m were the most potent, reducing PTP1B activity by up
to 70%. Interestingly, by repeating the same test with a different
phosphatase, namely the low molecular weight protein tyrosine phos-
phatase (LMW-PTP), we observed that the above-mentioned com-
pounds were less active, suggesting a certain specificity for PTP1B over
LMW-PTP.
Fig. 4. Compounds 9–14 bearing the DAB-1 and the nitroaromatic moieties
synthesized and tested in this work as bifunctional inhibitors.
moiety, as previously reported with the NeO bond of nitrones [43],
producing the target compounds 9–11 with a yield of 63–85% after
basic treatment with the resin Ambersep 900 OH in MeOH as a solvent.
To prepare the amido derivatives 12–14, direct acylation of 24–26 with
4-nitrobenzoyl chloride in pyridine as a solvent at room temperature
produced compounds 13, 34 and 35 with a 49–62% yield. With pro-
tected derivatives 34–35, final treatment with BCl3 in dichloromethane
at room temperature followed by FCC (Flash Column Chromatography)
with a basic eluent gave compounds 12 and 14 with yields of 70% and
85% (Scheme 2).
It is worth noting that all the most active derivatives (30–40% re-
sidual activity) displayed the same absolute configuration at the pyr-
rolidine ring stereogenic centres. The results of this preliminary
screening prompted us to choose compound 5c for further functiona-
lization, as it is one of the most active compounds against both PTP1B
and glucosidases and much easier to synthesize and functionalize than
other bicyclic pyrrolizidine iminosugars such as 7g and 7k-m.
2.1.2. Synthesis of a multivalent bifunctional inhibitor
The concept of multivalency, defined as an increase in binding af-
finity when several bioactive units are simultaneously linked to a
common scaffold [44], has been exploited with lectins [45–48]. Re-
cently, research into the “multivalent effect” has also been carried out
on enzymes [49–52]. In particular, impressive enhancement of activity
was observed with multivalent iminosugars towards some glycosidases
and several applications with therapeutically relevant enzymes have
also been reported [53].
2.2.2. Analysis of inhibitory properties of functionalized iminosugars 9–14
and 39
Iminosugars 9–14 and 39 (Schemes 2 and 3) were analysed to
evaluate their inhibitory power on the PTP1B enzyme. Preliminary
screening carried out using both a fixed substrate and inhibitor
(100 μM) concentration showed that compounds 9 and 13 were the
most potent, whereas compounds 12 and 39 were weaker inhibitors.
Finally, compounds 10, 11 and 14 resulted completely inactive (Fig. 5).
These results suggest that the linker length plays a pivotal role in the
activity of these compounds. The highest activity is shown by com-
pound 13, with a five-atom linker (amide bond included) between the
DAB-1 moiety and the nitro aromatic ring. A shorter linker (four atoms,
as in compound 12) reduces inhibitory potency, while a longer one (six
or seven atoms, as in compounds 10 and 11) results in a complete loss
of activity. Interestingly, the triazole based compound 9 and the amide
based compound 13 show a similar inhibitory profile and both have a
five-atom spacer between the two bioactive moieties, demonstrating
that the triazole moiety is able to replace the amide bond without
detrimental effects on biological activity, as expected since the 1,2,3-
triazole moiety is well known as an amide bond isostere [55,56].
To confirm these results, we calculated the IC50 values of active
With the aim of investigating whether PTP1B could accommodate
multivalent ligands bearing more than one iminosugar unit, we syn-
thesized the new ligand 39 in which the nitro aromatic active moiety
was linked to a trimeric DAB-1 moiety. Compound 39 was synthesized
through a reaction of azido derivative 22 and propargylated TRIS 36
[54], as shown in Scheme 3. Compound 22 bearing a two-carbon atom
spacer was chosen since this linker length proved to impart the best
inhibitory activity towards PTP1B (vide infra). Copper catalyzed azido
alkyne cycloaddition (CuAAC) between 22 and 36 under MW irradia-
tion as described above gave compound 37 with a 68% yield. Sub-
sequent acylation with p-nitrobenzoyl chloride, followed by O-benzyl
deprotection with BCl3 produced the trivalent derivative 39 with a
good yield (Scheme 3).
Scheme 1. Synthesis of key azido and amino
DAB-1 based intermediates 24–26. Reaction
conditions: (a) NEt3, THF, MW 150 °C, 4 h, 90%;
(b) NEt3, THF, r.t., 3 days, 85% for 18, 94% for
19; (c) MsCl, NEt3, CH2Cl2, r.t., 2 h, 25% for 20,
77% for 21; (d) NaN3, 18-crown 6 ether, CH3CN,
reflux, 5 h, 25% for 22, 94% for 23; (e) DPPA,
PPh3, DIAD, THF, r.t., 88% for 22; (f) PPh3, H2O,
THF, reflux, 16 h, 84% for 24, 63% for 25; (g)
H2, Pd/C, HCl, MeOH, r.t., 2 days, 86%.
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BioorganicChemistry87(2019)534–549
Scheme 2. Synthesis of the target triazole-
derivatives 9–11 and amide derivatives
12–14. Reaction conditions: (a) CuI, DIPEA,
THF, r.t., 2 h, 53% for 27; (b) CuSO4, so-
dium ascorbate, THF, H2O, MW, 80 °C,
45 min, 87% for 28, 92% for 29; (c) BCl3,
CH2Cl2, r.t., 15–18 h, 63–85% for 9–11,
70% for 12 and 85% for 14; (d) Ambersep
900 OH, MeOH, r.t., 30 min, quantitative; e)
Py, r.t., 22–40 h, 49% for 13, 56% for 34,
62% for 35.
compounds, including compound 5c (data obtained are reported in
Table 2). Compound 13 was the most potent of the screened molecules,
with an IC50 value of 31 μM, a value equivalent to half of that calculated
for compound 5c. Compounds 9, 12 and 39 were weaker inhibitors,
with IC50 values of 103, 180 and 119 μM, respectively.
control systems (Fig. 7). Compared to compound 13, compound 40 [57]
lacks the iminosugar moiety DAB-1 and compound 41 lacks the two
hydroxy groups and the hydroxymethyl on the pyrrolidine ring.
We found that both compounds are inactive, demonstrating that
both the pyrrolidine group and the hydroxyl functionalities are essen-
tial in imparting inhibitory activity to the final compound towards
PTP1B (Fig. S50 of the Supporting Information).
To gain insight into the mechanism of action of the best nitrophenyl
substituted inhibitors, compounds 9 and 13 (see Fig. 4), the dependence
of the main kinetic parameters, Km and Vmax, on inhibitor concentration
was investigated. Data obtained were analysed using the Lineweaver-
Burk plot. It was found that compounds 9 and 13 behave as non-
competitive inhibitors (Fig. 6, see also Fig. S49 of the Supporting
Information), as demonstrated by the fact that the compounds have a
very limited effect on Km while causing the reduction of Vmax (see Fig.
S47 and S48 of the Supporting Information). By using an appropriate
equation (see Materials and Methods section), we calculated the Ki
2.2.4. Effect of compounds on α-glucosidases
The inhibition of amylase and intestinal α-glucosidase prevents the
digestion of carbohydrates, thus representing a second mechanism
through which our compounds could act as anti-diabetic drugs.
Synthetic drugs that inhibit α-glucosidase are widely used in the clinic
to treat T2D mellitus, causing a reduction in postprandial blood glucose
and insulin levels [58]. Therefore, commercial porcine pancreatic
amylase was chosen as a model and compounds 5c, 9–14 and 39 were
assayed towards this enzyme using p-nitrophenyl-α-D-glucopyranoside
as a synthetic substrate. We did not find these compounds to be capable
of inhibiting this enzyme. However, a good inhibitory profile was found
towards α-glucosidase from Saccharomyces cerevisiae and towards
amyloglucosidase from Aspergillus niger for compounds 5c and 13,
values for each compound, which resulted 153
32 3 μM for compounds 9 and 13, respectively.
41 μM and
2.2.3. Analysis of inhibitory properties of control compounds
To verify the role played by the iminosugar moiety in PTP1B in-
hibition, we also prepared and evaluated compounds 40 and 41 as
Scheme 3. Synthesis of trivalent derivative 39. Reaction conditions: (a) CuSO4, sodium ascorbate, THF, H2O, MW, 80 °C, 45 min, 68% of 37; (b) DIPEA, CH2Cl2, r.t.,
2 days, 85% of 38; (c) BCl3, CH2Cl2, r.t., 18 h, 95% of 39.
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Table 1
(IC50 = 4.1 μM and 4.0 μM, respectively) and good-to-moderate in-
hibitors of α-glucosidase from Saccharomyces cerevisiae (IC50 = 6.4 μM
and 25.0 μM, respectively, see Table 3).
Preliminary enzymatic assays. Tests were carried out on PTP1B and LMW-PTP
by using a fixed inhibitor concentration (100 μM) and a fixed substrate con-
centration (2.5 mM). Data reported in the Table represent the mean
S.E.M.
Selectivity towards different α-glucosidases is an important issue.
Compounds 5c, 9–14 and 39 were thus evaluated as lysosomal α-glu-
cosidase inhibitors in human lymphocytes homogenate [59,60]. Com-
pounds 9–12 and 14 inhibited lysosomal α-glucosidase (82–94%) at
1 mM and their IC50 values were subsequently determined by mea-
suring human enzyme activity at different iminosugar concentrations
(Table 4). To our delight, compounds 13 and 5c, the best PTP1B in-
hibitors, were only weak α-glucosidase inhibitors (percentage of in-
hibition around 40%).
(n = 3).
Compounds
Residual activity (%)
PTP1B
100
LMW-PTP
Pyrrolidiness
Ctr
5a
5b
5c
5d
5e
5f
100
116
82
141
84
1
15
9
3
2
1
2
34
89
71
8
81
In the development of novel candidates as potential antidiabetic
agents, the inhibition of lysosomal α-glucosidase is undesirable as it can
result in the accumulation of non-hydrolyzed substrate in the lysosome,
with clinical manifestations similar to those observed in Pompe Disease,
a lysosomal storage disorder caused by a mutation in the gene encoding
for lysosomal α-glucosidase (GAA) [61].
92
3
109
86
2
110
82
6
1
5g
5h
5i
4
147
154
80
2
73
1
14
62
2
2
5j
64
4
96
5
Piperidines
6a
6b
6c
6d
6e
6f
61
68
82
47
84
76
12
78
78
103
78
86
98
2
1
8
2.2.5. Effect of compounds on human lysosomal β-glucosidase (GCase)
Since a lysosomal β-glucosidase inhibitor has the potential to act as
pharmacological chaperone rescuing the activity of the deficient en-
zyme in Gaucher Disease, we tested the synthesized compounds to-
wards acid β-glucosidase (glucocerebrosidase, GCase) from human
leukocytes homogenates. We found that a residual enzymatic activity
no higher than 18% was observed for the whole set of compounds
(except for the trivalent derivative 39) when assayed at 1 mM con-
centration, as shown in Fig. 8. Since compounds 9 and 13 are also good
PTP1B inhibitors, further investigations of their GCase inhibitory pro-
file/kinetics would be of particular interest in view of the potential
application of these compounds as bifunctional PTP1B/GCase in-
hibitors/chaperones in the treatment of T2D related to Gaucher disease.
In this regard, both therapeutic actions (inhibitory and chaperone)
may be relevant. While a pharmacological chaperone for GCase rescues
the activity of the endogenous enzyme, which is deficient in the pa-
thology, it can also stabilize the exogenous GCase enzyme in patients
treated with enzyme replacement therapy (ERT). From the existing
literature, it is not clear if the onset of T2D is related to Gaucher Disease
(even untreated) or only to Gaucher Disease patients treated with
Enzyme Replacement Therapy (ERT) [18]. Metabolic abnormalities are
apparent in Gaucher Disease but the administration of the recombinant
enzyme to reduce glucosylceramide levels in patients seems to have its
own metabolic consequences, such as peripheral insulin resistance [15].
This may be due to reductions of glucosylceramide levels per se or
secondary glycosphingolipids alterations, such as transient increases in
ceramide as the excess glucosylceramide passes through the catabolic
pathways [15]. Inhibition of GCase (the enzyme responsible for the
conversion of glucosylceramide into glucose and ceramide) could avoid
accumulation of ceramide inside the cells, a metabolite that is able to
impair the translocation of Akt and promote Akt dephosphorylation by
PP2A, thereby resulting in Akt inactivation [62].
7
5
5
16
2
7
3
3
Pyrrolizidines
7a
7b
7c
7d
7e
7f
83
67
60
73
57
63
38
83
103
55
31
40
38
7
3
2
7
8
4
6
2
7
10
9
3
2
102
5
1
8
2
3
4
4
2
3
6
8
3
4
85
112
102
62
74
7g
7h
7i
68
103
116
118
88
7j
7k
7l
132
120
7m
Indolizidines
8a
8b
8c
8d
8e
134
2
135
1
68
4
85
5
131
82
6
132
102
109
2
12
5
5
113
3
2.2.6. Ex vivo assay
In order to evaluate whether compounds 13 and 9 possessed insulin-
mimetic activity, further tests using HepG2 cells were performed. Cells
were starved for 20 h and then treated with compound 9 or 13, or
stimulated with 10 nM insulin. The proteins of extracts were separated
by SDS-PAGE and transferred to PVDF membrane by western blot.
Membranes were probed with specific antibodies to evaluate levels of
the phosphorylate form of Akt (Fig. 9). As expected, after insulin sti-
mulation, a great increase in the phosphorylation level of Akt was ob-
served. An increase in Akt phosphorylation was already detectable after
15 min incubation with both compounds, suggesting that 9 and 13
possess insulin mimetic activity.
Fig. 5. Preliminary enzymatic assay on PTP1B. Tests were carried out in 3,3-
dimethylglutarate buffer pH 7.0 containing 0.1 mM DTT and 1 mM EDTA, in the
presence of a fixed substrate concentration (2.5 mM, corresponding to the Km
value of the enzyme) and a fixed inhibitor concentration (100 μM). Data re-
ported in the figure represent the mean value
S.E.M (n = 3).
which are the best PTP1B inhibitors we have analysed. Preliminary
screening carried out using a fixed inhibitor concentration (1 mM)
showed the inhibition percentage of both α-glucosidases to be higher
than 90%. Upon IC50 determination, we found that compounds 5c and
13 are both good inhibitors of amyloglucosidase from Aspergillus niger
Interestingly, no increase in the Akt phosphorylation level was ob-
served on HepG2 cells treated with 100 μM of compound 5c (data not
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Table 2
3. Conclusions
IC50 values for compounds 5c, 9, 12, 13 and 39 towards PTP1B. To determine
the IC50 values, we calculated residual activity of PTP1B in the presence of
The development of effective drugs for the treatment of T2D or
insulin resistance is one of the most important goals of pharmaceutical
companies. A growing body of evidence shows that multi-target drugs
could be good alternatives to traditional therapies, offering the possi-
bility of simultaneously controlling the activity of different targets,
without the risk of generating undesirable secondary effects.
Protein Tyrosine Phosphatase 1B (PTP1B) is one of the most pro-
mising targets for the treatment of T2D and metabolic syndrome. On
the other hand, α-glucosidase inhibitors have the potential to be de-
veloped as oral anti-hyperglycaemic drugs.
increasing inhibitor concentrations and
a fixed substrate concentration
(2.5 mM). Each assay was carried out in triplicate.
Compound
9
12
13
39
5c
IC50 (μM)
103
4
180
4
31
2
119
9
66
2
shown). This finding suggested that compound 5c does not possess an
evident insulin-mimetic activity, thereby confirming that the presence
of the nitro aromatic moiety is essential to confer an insulin-mimetic
activity to compounds 9 and 13.
In this work, we show that our new bifunctional inhibitors, obtained
by linking an iminosugar moiety with a phosphotyrosine mimetic
group, are able to target both PTP1B and α-glucosidases.
To date, we can only speculate on the mechanisms of the ex vivo
effect of compounds 9 and 13, which could be due to PTP1B inhibition,
or to β-glucosidase inhibition.
The privileged iminosugar moiety (a polyhdroxylated pyrrolidine)
was selected on the basis of a preliminary screening towards PTP1B of
more than 30 iminosugars with different structures. We synthesized a
Fig. 6. Double reciprocal plots for compounds 9 (A) and 13 (B). pNPP (p-nitrophenylphosphate) is employed as a synthetic substrate. (A) The concentration of
compound 9 are: ■, 0 μM; ○, 80 μM; ▴, 100 μM; ∇, 120 μM. Data reported in the figures represent the mean values S.E.M. (n = 3). (B) The concentration of
compound 13 are: ■, 0 μM; ○, 15 μM; ▴, 25 μM; ∇, 35 μM; ♦, 45 μM; data reported in the figures represent the mean values S.E.M. (n = 3).
Fig. 7. Chemical structure of compounds 13, 40,
and 41. Compounds 40 and 41 were used as
control compounds to verify the importance of
the iminosugar moiety in PTP1B inhibition.
Table 3
IC50 values determined by measuring the residual activity of α-glucosidases in the presence of increasing inhibitor concentration for iminosugars 5c and 13.
Compound
Amyloglucosidase from Aspergillus niger, IC50, μM
α-Glucosidase from Saccharomyces cerevisiae, IC50, μM
5c
13
4.1
4.0
6.4
25.0
Table 4
Inhibition of lysosomal α-glucosidase (in an extract from human lymphocytes) by compounds 5c, 9–14 and 39. IC50 values determined for percentages of inhibition
higher than 80% by measuring the residual activity of α-glucosidase in the presence of increasing inhibitor concentration. For each compound, we used at least 10
different inhibitor concentrations. Each assay was carried out in triplicate.
Compound
5c
9
10
11
12
13
14
39
IC50 (μM)
a
———a
240
10
210
10
170
5
250
10
———a
170
10
———a
Compounds 5c, 13 and 39 showed percentage of inhibition lower than 80% at 1 mM inhibitor concentration.
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BioorganicChemistry87(2019)534–549
4. Materials and methods
4.1. Chemistry
Commercial reagents were used as received. All reactions were
carried out under magnetic stirring and monitored by TLC on 0.25 mm
silica gel plates (Merck F254). Column chromatographies were carried
out on Silica Gel 60 (32–63 μm) or on silica gel (230–400 mesh, Merck).
Yields refer to spectroscopically and analytically pure compounds un-
less otherwise stated. 1H NMR spectra were recorded on a Varian
Mercury-400 or on a Varian INOVA 400 instrument at 25 °C. 13C NMR
spectra were recorded on a Varian Gemini-200 or on a Varian Gemini-
300. Chemical shifts are reported relative to TMS (1H: δ = 0.00 ppm)
and CDCl3
(
13C: δ = 77.0 ppm). Integrals are in accordance with as-
signments, coupling constants are given in Hz. For detailed peak as-
signments 2D spectra were measured (COSY, HSQC, NOESY, and NOE
as necessary). Small scale microwave assisted syntheses were carried
out in a CEM Discover microwave apparatus for synthesis with an open
reaction vessel and an external surface sensor. IR spectra were recorded
with a BX FT-IR Perkin-Elmer System spectrophotometer. ESI-MS
spectra were recorded with a Thermo Scientific™ LCQ Fleet Ion Trap
Mass Spectrometer. HRESI-MS spectra were recorded with a Thermo
Scientific™ Orbitrap Elite or with an ABSCIEX Triple TOF® 5600+ in-
strument. Optical rotation measurements were performed on a JASCO
DIP-370 polarimeter.
Fig. 8. Residual activity of β-glucosidase (GCase) in an extract from human
leukocytes isolated from healthy donors, incubated with 1 mM iminosugars 5c,
9–14 and 39. *The percentage of inhibition and the IC50 value (108
14 μM)
are reported in Ref. 28, where the same assay conditions were employed.
series of six novel compounds 9–14 in which the iminosugar moiety and
the nitro aromatic phosphotyrosine mimetic group were connected
through linkers that differ in nature (amide or a triazole ring) and
length (2, 3 or 4 carbon atoms), and the trivalent compound 39. Some
of the new compounds (namely 9, 12, 13, and 39), maintained the
ability to inhibit the PTP1B enzyme with IC50 values that ranged be-
tween 30 and 180 μM. Analysis of kinetic parameters showed that
compounds 9 and 13, the best PTP1B inhibitors bearing the nitrophenyl
moiety, behave as non-competitive inhibitors (Fig. 6). Moreover, the
presence of the iminosugar moiety was demonstrated to be essential in
imparting inhibitory activity towards PTP1B, since compounds 40 and
41, lacking the polyhydroxylated pyrrolidine moiety, did not show any
inhibitory activity. Our best PTP1B inhibitors 13 and 5c were also as-
sayed towards commercial α-glucosidases, namely α-glucosidase from
Saccharomyces cerevisiae and amyloglucosidase from Aspergillus niger,
and moderate to good inhibition was found for both compounds (IC50
values ranging from 4 to 25 μM). These results pave the way for a new
generation of bifunctional PTP1B/α-glucosidase inhibitors. Undesired
inhibition of lysosomal α-glucosidase was negligible for both com-
pounds.
Synthesis of (2R,3R,4R)-1-(4-azidobutyl)-3,4-bis(benzyloxy)-2-
[(benzyloxy)methyl]pyrrolidine (17): A solution of pyrrolidine 15
[29] (86 mg, 0.21 mmol) in THF (2 mL) was prepared in a MW vial, and
NEt3 (60 μL, 0.42 mmol) and 1-azido-4-bromobutane 16 [41] (86 mg,
0.48 mmol) were added. The reaction mixture was heated in MW re-
actor at 150 °C for 45 min until a TLC analysis showed the dis-
appearance of the starting material and the formation of a new product.
The solvent was removed under reduced pressure and the crude was
purified by FCC (CH2Cl2/Et2O 100:1) affording pure 17 (93 mg,
0.19 mmol) with a 90% yield. [α]D25 = −31.1 (c = 0.94, CHCl3). 1H
NMR (400 MHz, CDCl3) δ ppm = 7.35–7.25 (m, 15H, Ar), 4.55–4.42
(m, 6H, O-CH2-Ph), 3.92 (d, J = 5.4 Hz, 1H, H-4), 3.86 (d, J = 4.3 Hz,
1H, H-3), 3.54 (m, 2H, H-6), 3.26 (m, 2H, H-10), 3.20 (d, J = 10.5 Hz,
1H, Ha-5), 2.87 (m, 1H, Ha-7), 2.72 (q, J = 5.2 Hz, 1H, H-2), 2.55 (dd,
J = 10.5, 5.1 Hz, 1H, Hb-5), 2.36 (m, 1H, Hb-7), 1.66–1.52 (m, 4H, H-
8, H-9). 13C NMR (50 MHz, CDCl3) δ ppm = 138.5, 138.4 (s, 3C, Ar),
128.3, 127.6 (d, 15C, Ar), 85.6 (d, 1C, C-3), 81.8 (d, 1C, C-4), 73.2, 71.1
(t, 4C, O-CH2-Ph, C-6), 69.3 (d, 1C, C-2), 57.2 (t, 1C, C-5), 54.8 (t, 1C,
C-7), 51.4 (t, 1C, C-10), 26.8–25.4 (t, 2C, C-8, C-9). IR (CDCl3)
ν = 3032, 2934, 2865, 2808, 2099, 1496, 1453, 1365, 1350, 1282,
1259, 1098, 1076 cm−1. MS-ESI (m/z, %) = 501.28 (MH+, 100),
523.28 (MNa+, 62).
Furthermore, ex vivo tests carried out on HepG2 cells demonstrated
that the new nitrobenzene iminosugar hybrids 9 and 13 show a good
insulin-mimetic activity, increasing phosphorylation levels of Akt also
in the absence of insulin stimulation, while the iminosugar 5c was not
active in the ex vivo assay.
To the best of our knowledge, examples of iminosugar-based PTP1B
inhibitors are unprecedented, thus this study represents proof of con-
cept for the possible use of iminosugars as new bifunctional drugs in the
treatment of T2D. Work is underway to improve the potency of in-
hibitors towards the selected enzymatic targets and to elucidate the
mechanism underlying their ex vivo activity.
Synthesis of 2-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzyloxy)
methyl]pyrrolidin-1-yl}ethanol (18): [63,64] To a pyrrolidine 15
[29]; solution (82 mg, 0.20 mmol) in THF (3 mL), NEt3 (141 μL,
1.00 mmol) and 2-bromoethanol (85 μL, 1.22 mmol) were added. The
reaction mixture was stirred at room temperature for 3 days until a TLC
Fig. 9. Effect of compound 9 and 13 on insulin
signalling pathway. HepG2 cells were starved
and then stimulated with 10 nM insulin for
Compound 9
60’
Compound 13
60’
15’
30’
120’ 15’ 30’
120’
15 min, or treated with compound 9 and 13 for
increasing times. At the end of each interval time,
cells were lysed and analysed to evaluate phos-
phorylation levels of Akt using specific antibodies.
The final concentration of compounds 9 and 13 in
the test was 42 μM.
Ctr
I
Time (min)
pAkt
Actin
541

X. Ferhati, et al.
BioorganicChemistry87(2019)534–549
analysis (CH2Cl2/MeOH 30:1) showed the disappearance of the starting
material (Rf = 0.43) and the formation of a new product (Rf = 0.81).
After evaporation under reduced pressure, the crude was purified by
FCC (AcOEt/PE 1:1) affording pure 18 (Rf = 0.30, 75 mg, 0.17 mmol,
85% yield) as a yellow oil. [α]D25 = −20.3 (c = 0.92 in CHCl3). 1H
NMR (400 MHz, CDCl3): δ = 7.35–7.26 (m, 15H, H-Ar), 4.55–4.42 (m,
6H, O-CH2-Ph), 3.99–3.97 (m, 1H, H-4), 3.89 (dd, J = 3.6, 1.2 Hz, 1H,
H-3), 3.64–3.48 (m, 4H, H-7, H-8), 3.25 (d, J = 10.4 Hz, 1H, Ha-5),
3.06 (ddd, J = 12.9, 9.1, 4.7 Hz, 1H, Ha-6), 2.88 (dd, J = 9.6, 5.6 Hz,
1H, H-2), 2.67 (dd, J = 10.4, 5.2 Hz, 1H, Hb-5), 2.58 (dt, J = 12.6,
3.8 Hz, 1H, Hb-6).
8.1 Hz, 1H, Ha-7), 2.91 (s, 3H, Me), 2.74 (m, 1H, H-2), 2.56 (dd,
J = 10.2, 5.3 Hz, 1H, Hb-5), 2.46 (m, 1H, Hb-7), 1.91 (m, 2H, H-8). 13
C
NMR (50 MHz, CDCl3) δ ppm = 138.3–138.1 (s, 3C, Ar), 128.3–127.7
(d, 15C, Ar), 85.3 (d, 1C, C-3), 81.7 (d, 1C, C-4), 73.2, 71.4, 71.1 (t, 4C,
O-CH2-Ph, C-6), 69.3 (d, 1C, C-2), 68.5 (t, 1C, C-9), 57.2 (t, 1C, C-5),
51.1 (t, 1C, C-7), 37.1 (q, 1C, Ms), 27.9 (t, 1C, C-8). IR (CDCl3)
ν = 3088, 3031, 2900, 2864, 2815, 1603, 1496, 1454, 1358, 1206,
1176, 1097 cm−1. MS-ESI (m/z, %) = 561.00 (MNa+, 67).
Synthesis of (2R,3R,4R)-1-(2-azidoethyl)-3,4-bis(benzyloxy)-2-
[(benzyloxy)methyl]pyrrolidine (22), Strategy a): A solution of
mesylate 20 (40 mg, 0.076 mmol), 18-crown-6 ether (6.04 mg,
0.022 mmol) and sodium azide (9.89 mg, 0.15 mmol) in dry acetonitrile
(1.5 mL) was stirred under nitrogen atmosphere at reflux for 3.5 h. The
mixture was transferred to a separatory funnel and washed with H2O.
The aqueous layer was then extracted with CH2Cl2 (3 × 5 mL) and the
combined organic layers were dried over Na2SO4. The solvent was
evaporated under a vacuum and the crude was purified by FCC
(CH2Cl2/Et2O 100 : 1) affording pure 22 (9 mg, 0.019 mmol) with a
25% yield. [α]D24 = −12.9 (c = 0.34, CHCl3). 1H NMR
(400 MHz,CDCl3) δ ppm = 7.28–7.18 (m, 15H, Ar), 4.49–4.34 (m, 6H,
O-CH2-Ph), 3.88 (d, J = 4.4 Hz, 1H, H-4), 3.76 (d, J = 3.5 Hz, 1H, H-3),
3.48 (m, 2H, H-6), 3.31 (m, 1H, Ha-8), 3.21 (m, 2H, Hb-8, Ha-5) 3.09
(m, 1H, Ha-7), 2.77 (m, 1H, H-2), 2.59 (m, 2H, Hb-5, Hb-7). 13C NMR
(50 MHz, CDCl3) δ ppm = 138.4–138.2 (s, 3C, Ar), 128.6–127.6 (d,
15C, Ar), 85.2 (d, 1C, C-3), 81.8 (d, 1C, C-4), 73.3, 71.5, 71.1, (t, 4C, O-
CH2-Ph, C-6), 69.4 (d, 1C, C-2), 57.5 (t, 1C, C-5), 54.3 (t, 1C, C-7), 49.9
(t, 1C, C-8). IR (CDCl3) ν = 3066, 3032, 2926, 2857, 2103, 1602, 1496,
1454, 1365, 1271, 1206 cm−1. MS-ESI (m/z, %) = 473.32 (MH+, 100),
495.31 (MNa+, 81.6).
Synthesis of 2-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzyloxy)
methyl]pyrrolidin-1-yl}propan-1-ol (19): [64] To a solution of 15
[29] (132 mg, 0.33 mmol) in 6 mL of THF, NEt3 (230 μL, 1.65 mmol)
and 3-bromo-1-propanol (179 μL, 1.98 mmol) were added. The reaction
mixture was stirred at room temperature for 3 days until a TLC analysis
(CH2Cl2/MeOH 10:1) showed the disappearance of the starting material
(Rf = 0.51) and the formation of a new product (Rf = 0.85). After
evaporation under reduced pressure, the crude was purified by FCC
(AcOEt/PE 1:1) affording pure 19 (Rf = 0.30, 143 mg, 0.31 mmol, 94%
yield) as a yellow oil. [α]D24 = −39.1 (c = 0.47 in CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 7.34–7.24 (m, 15H, H-Ar), 4.56–4.48 (m, 6H, O-
CH2-Ph), 3.93 (d, J = 4.8 Hz, 1H, H-4), 3.82–3.77 (m, 3H, H-9 and H-
3), 3.63 (dd, J = 9.7, 5.8 Hz, 1H, Ha-6), 3.55 (dd, J = 9.8, 6.4 Hz, 1H,
Hb-6), 3.45 (d, J = 10.8 Hz, 1H, Ha-5), 3.14 (td, J = 11.7, 3.9 Hz, 1H,
Ha-7), 2.73 (q, J = 5.2 Hz, 1H, H-2), 2.64 (dt, J = 12.2, 3.9 Hz, 1H, Hb-
7), 2.50 (dd, J = 10.8, 4.7 Hz, 1H, Hb-5), 1.94–1.82 (m, 1H, Ha-8),
1.56–1.49 (m, 1H, Hb-8). 13C NMR (50 MHz, CDCl3): δ = 138.3, 138.2,
138.1 (s, 3C, C-Ar), 128.3–127.5 (d, 15C, C-Ar), 85.3 (d, 1C, C-3), 81.4
(d, 1C, C-4), 73.3, 71.4, 71.2, 70.9 (t, 4C, C-Bn and C-6), 69.7 (d, 1C, C-
2), 64.0 (t, 1C, C-9), 56.7 (t, 1C, C-5), 55.2 (t, 1C, C-7), 29.3 (t, 1C, C-8).
IR (CDCl3): ν = 3312, 3066, 3032, 2923, 2861, 1496, 1453, 1366,
1333, 1282, 1208, 1100, 1071, 1028 cm−1. MS-ESI (m/z, %) = 484.33
(MNa+, 100), 462.30 (MH+, 74).
Synthesis of (2R,3R,4R)-1-(2-azidoethyl)-3,4-bis(benzyloxy)-2-
[(benzyloxy)methyl]pyrrolidine (22), Strategy b): To a solution of
alcohol 18 (83 mg, 0.18 mmol) in dry THF (2 mL) at 0 °C under nitrogen
atmosphere, PPh3 recrystallized from Et2O (146 mg, 0.56 mmol) was
added, followed by DIAD (109 μL, 0.56 mmol) and DPPA (133 μL,
0.59 mmol). The mixture was left to reach room temperature and
maintained under nitrogen atmosphere for 2.5 h. The solvent was re-
moved under reduced pressure and the crude purified by FCC (Et2O:EtP
1:3), to yield compound 22 (77 mg, 0.16 mmol, 88%).
Synthesis of 2-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzyloxy)
methyl]pyrrolidin-1-yl}ethyl methanesulfonate (20): A solution of
alcohol 18 (130.0 mg, 0.29 mmol) in anhydrous CH2Cl2 (5 mL) was
stirred under nitrogen atmosphere and cooled at 0 °C. NEt3 (121 μL,
0.87 mmol) and MsCl (29 μL, 0.38 mmol) were added and the reaction
mixture was left to reach room temperature and stirred for 2 h. The
mixture was transferred to a separatory funnel and washed with H2O.
The aqueous layer was then extracted with CH2Cl2 (3 × 15 mL) and the
combined organic layers were dried over Na2SO4. The solvent was
evaporated under a vacuum and the crude was purified by FCC (AcOEt/
PE 1:1), leading to pure 20 (38.1 mg, 0.072 mmol) with a 25% yield.
Due to the instability of compound 20 (see main text), it was impossible
to obtain a complete characterization of this product. The 1H NMR
Synthesis of (2R,3R,4R)-1-(3-azidopropyl)-3,4-bis(benzyloxy)-
2-[(benzyloxy)methyl] pyrrolidine (23): A solution of mesylate 21
(83 mg, 0.15 mmol), 18-crown-6 ether (8 mg, 0.03 mmol) and sodium
azide (20 mg, 0.3 mmol) in dry acetonitrile (2.5 mL) was stirred under
nitrogen atmosphere at reflux for 5 h. TLC analysis (EtP:AcOEt 1:1)
showed the disappearance of the starting material (Rf = 0.49) and the
formation of a new product (Rf = 0.86). The mixture was transferred to
a separatory funnel and washed with H2O. The aqueous layer was then
extracted with CH2Cl2 (3 × 10 mL) and the combined organic layers
were dried over Na2SO4. The solvent was evaporated under a vacuum
and the crude was purified by FCC (CH2Cl2/Et2O 25:1) affording pure
23 (70 mg, 0.14 mmol) with a 94% yield. [α]D25 = −26.1 (c = 0.64,
CHCl3). 1H NMR (400 MHz,CDCl3) δ ppm = 7.36–7.25 (m, 15H, Ar),
4.56–4.43 (m, 6H, O-CH2-Ph), 3.94 (d, J = 4.9 Hz, 1H, H-4), 3.86 (d,
J = 3.9 Hz, 1H, H-3), 3.59–3.51 (m, 2H, H-6), 3.32 (td, J = 6.5, 3.1 Hz,
2H, H-9), 3.18 (d, J = 10.7 Hz, 1H, Ha-5), 2.97 (dt, J = 12.2, 8.0 Hz,
1H, Ha-7), 2.75 (q, J = 5.2 Hz, 1H, H-2), 2.56 (dd, J = 10.5, 5.1 Hz, 1H,
Hb-5), 2.42 (m, 1H, Hb-7), 1.45 (m, 2H, H-8). 13C NMR (50 MHz,
CDCl3) δ ppm = 138.4, 138.3 (s, 3C, Ar), 128.3, 127.5 (d, 15C, Ar),
85.5 (d, 1C, C-3), 81.9 (d, 1C, C-4), 73.3 (t, 1C, O-CH2-Ph), 71.4, 71.3
(t, 2C, O-CH2-Ph, C-6), 71.1 (t, 1C, O-CH2-Ph), 69.3 (d, 1C, C-2), 57.2 (t,
1C, C-5), 52.3 (t, 1C, C-7), 49.6 (t, 1C, C-9), 27.6 (t, 1C, C-8). IR (CDCl3)
ν = 3066, 3032, 2864, 2806, 2098, 1495, 1453, 1365, 1301, 1259,
1096, 1028 cm−1. MS-ESI (m/z, %) = 509.20 (MNa+, 100).
spectrum is reported below. 1H NMR (200 MHz, CDCl3)
δ
ppm = 7.38–7.26 (m, 15H), 4.53–4.35 (m, 7H), 4.26 (m, 1H), 3.94 (d,
J = 5.0 Hz, 1H), 3.80 (d, J = 4.2 Hz, 1H), 3.54 (d, J = 5.8 Hz, 2H), 3.29
(m, 2H), 2.96 (s, 3H), 2.73 (m, 3H).
Synthesis of 2-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzyloxy)
methyl]pyrrolidin-1-yl}propyl methanesulfonate (21): A solution
of alcohol 19 (34 mg, 0.073 mmol) in anhydrous CH2Cl2 (2 mL) was
stirred under nitrogen atmosphere and cooled at 0 °C. NEt3 (121 μL,
0.87 mmol) and MsCl (29 μL, 0.38 mmol) were added and the reaction
mixture was left to reach room temperature and stirred for 2 h. The
mixture was transferred to a separatory funnel and washed with H2O.
The aqueous layer was then extracted with CH2Cl2 (3 × 15 mL) and the
combined organic layers were dried over Na2SO4. The solvent was
evaporated under a vacuum and the crude was purified by FCC (AcOEt/
PE 1:2), affording pure 21 (30.2 mg, 0.056 mmol) with a 77% yield.
[α]D25 = −16.7 (c = 1.21, CHCl3). 1H NMR (400 MHz, CDCl3) δ
ppm = 7.38–7.23 (m, 15H, Ar), 4.61–4.39 (m, 6H, O-CH2-Ph), 4.27 (m,
2H, H-9), 3.94 (d, J = 5.3 Hz, 1H, H-4), 3.85 (d, J = 3.9 Hz, 1H, H-3),
3.54 (m, 2H, H-6), 3.18 (d, J = 10.2 Hz, 1H, Ha-5), 5.98 (dt, J = 12.5,
Synthesis of (4-{(2R,3R,4R)- 3,4-bis(benzyloxy)-2-[(benzyloxy)
methyl]pyrrolidin-1yl}butyl)amine (24): [For an alternative synth-
esis of 24 see: [65] To a solution of azide 17 (74 mg, 0.15 mmol) in dry
THF (2.5 mL) under nitrogen atmosphere PPh3 (47 mg, 0.18 mmol) and
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BioorganicChemistry87(2019)534–549
H20 (5.36 μL, 0.30 mmol) were added. The mixture was heated at reflux
overnight, then a TLC control (CH2Cl2:Et2O 100:1 showed the dis-
appearance of the starting material (Rf = 0.21). The mixture was di-
luted with H2O, transferred to a separatory funnel and washed with
H2O. The aqueous layer was then extracted with CH2Cl2 (3 × 15 mL)
and the combined organic layers were dried over Na2SO4. The solvent
was evaporated under a vacuum and the crude was purified by FCC
(AcOEt and then CH2Cl2: MeOH: NH4OH 6% 4: 1: 0.1) affording pure
0.18 mmol) were added: The solution was stirred at room temperature
for 2 h, when a TLC analysis (Et2O/PE 1:3) attested the disappearance
of the starting material 22 (Rf = 0.22) and the formation of a new
product (Rf = 0.03). The reaction mixture was concentrated under a
vacuum and the resulting crude was purified by FCC (PE/AcOEt 2:1),
affording 47 mg (0.08 mmol) of pure 27, with
a 53% yield.
[α]D25 = +20.5 (c = 0.185, CHCl3). 1H NMR (400 MHz, CDCl3) δ
ppm = 8.04 (m, 2H, Ar o-NO2), 8.03 (s, 1H, triazole), 7.67 (m, 2H, Ar
m-NO2), 7.27–7.09 (m, 15H, Ar), 4.52–4.30 (m, 8H, O-CH2-Ph, H-8),
3.91 (d, J = 4.9 Hz, 1H, H-4), 3.77 (d, J = 3.9 Hz, 1H, H-3), 3.41 (m,
1H, Ha-6), 3.34–3.24 (m, 2H, Hb-6, Ha-7), 3.12 (d, J = 10.2 Hz, 1H,
Ha-5), 2.89–2.79 (m, 2H, H-2, Hb-7), 2.68 (dd, J = 10.2, 4.9 Hz, 1H,
Hb-5). 13C NMR (50 MHz, CDCl3) δ ppm = 147.1 (s, 1C, C-NO2), 145.1,
137.9, 137.2 (s, 5C, Ar p-NO2, Ar, triazole), 128.4–127.6 (d, 15C, Ar),
125.8 (d, 2C, Ar m-NO2), 124.1 (d, 2C, Ar o-NO2), 122.7 (d, 1C, tria-
zole), 85.0 (d, 1C, C-3), 81.9 (d, 1C, C-4), 73.3 (t, 1C, O-CH2-Ph), 71.6
(t, 1C, C-6), 71.3 (t, 2C, O-CH2-Ph), 69.2 (d, 1C, C-2), 57.3 (t, 1C, C-5),
53.7 (t, 1C, C-7), 49.1 (t, 1C, C-8). IR (CDCl3) ν = 3066, 3030, 2860,
2602, 1518, 1453, 1347, 1234, 1109 cm−1. MS-ESI (m/z, %) = 619.95
(MH+, 31), 642.05 (MNa+, 100).
23
24 (59 mg, 0.12 mmol) with a 84% yield. [α]D =−25.5 (c = 0.595,
CHCl3). 1H NMR (400 MHz, CDCl3) δ ppm = 7.34–7.25 (m, 15H, Ar),
4.56–4.42 (m, 6H, O-CH2-Ph), 3.91 (d, J = 4.9 Hz, 1H, H-4), 3.87 (d,
J = 4.4 Hz, 1H, H-3), 3.55 (m, 2H, H-6), 3.21 (d, J = 10.2 Hz, 1H, Ha-
5), 2.84 (m, 1H, Ha-7), 2.70 (m, 3H, H-2, H-10), 2.55 (dd, J = 10.2,
5.1 Hz, 1H, Hb-5), 2.34 (m, 1H, Hb-7), 1.55–1.41 (m, 4H, H-8, H-10).
13C NMR (50 MHz, CDCl3)
δ ppm = 138.5–138.4 (s, 3C, Ar),
128.3–127.5 (d, 15C, Ar), 85.6 (d, 1C, C-3), 81.8 (d, 1C, C-4), 73.2,
71.3, 71.1 (t, 4C, O-CH2-Ph, C-6), 69.3 (d, 1C, C-2), 57.2 (t, 1C, C-5),
55.3 (t, 1C, C-7), 42.0 (t, 1C, C-10), 31.6 (t, 1C, C-9), 25.6 (t, 1C, C-8).
IR (CDCl3) ν = 3320, 3306, 3090, 3066, 2931, 2863, 1601, 1495, 1454,
1366, 1095 cm−1. MS-ESI (m/z, %) = 474.96 (MH+, 100).
Synthesis of (2-{(2R,3R,4R)- 3,4-bis(benzyloxy)-2-[(benzyloxy)
methyl]pyrrolidin-1yl}ethyl)amine (25): To a solution of azide 22
(77 mg, 0.16 mmol) in dry THF (2.5 mL) under nitrogen atmosphere
PPh3 (50 mg, 0.19 mmol) and H20 (5.76 μL, 0.32 mmol) were added.
The mixture was heated at reflux overnight, then a TLC control
(CH2Cl2:Et2O 100:1 showed the disappearance of the starting material
(Rf = 0.36). The mixture was diluted with H2O, transferred to a se-
paratory funnel and washed with H2O. The aqueous layer was then
extracted with CH2Cl2 (3 × 15 mL) and the combined organic layers
were dried over Na2SO4. The solvent was evaporated under a vacuum
and the crude was purified by FCC (AcOEt and then CH2Cl2: MeOH 10:
Synthesis of 1-(3-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzy-
loxy)methyl]pyrrolidin-1-yl}propyl)-4-(4-nitrophenyl)-1H-1,2,3-
triazole (28): Azide 23 (32 mg, 0.066 mmol) was dissolved in a 2:1
THF/H2O mixture (2.7 mL total volume) in a MW vial reactor and
CuSO4 (3.1 mg, 0.021 mmol), sodium ascorbate (8.3 mg, 0.042 mmol)
and 1-ethynyl-4-nitrobenzene (11.6 mg, 0.083 mmol) were added. The
reaction mixture was heated in the MW at 80 °C for 45 min, when a TLC
analysis (eluent CH2Cl2/Et2O 30: 1) attested the disappearance of the
starting material 23 (Rf = 0.46) and the formation of a new product
(Rf = 0.17). The reaction mixture was filtered over Celite and con-
centrated under reduced pressure. The resulting crude product was
purified by FCC (PE/AcOet 1:1) affording 36 mg (0.057 mmol) of
1) affording pure 25 (45 mg, 0.10 mmol) with
a 63% yield.
[α]D23 = −20.3 (c = 0.785, CHCl3). 1H NMR (400 MHz, CDCl3) δ
ppm = 7.34–7.20 (m, 15H, Ar), 4.50–4.39 (m, 6H, O-CH2-Ph), 3.94 (d,
J = 4.8 Hz, 1H, H-4), 3.84 (d, J = 2.9 Hz, 1H, H-3), 3.52 (m, 2H, H-6),
3.19 (d, J = 10.3 Hz, 1H, Ha-5), 3.10 (dd, J = 10.5, 5.1 Hz, 1H, Hb-5),
2.99 (m, 1H, Ha-7), 2.79 (m, 3H, H-8, H-2), 2.53 (m, 1H, Hb-7), 1.96
(bs, 2H, NH2). 13C NMR (50 MHz, CDCl3) δ ppm = 138.1–138.0 (s, 3C,
Ar), 128.4–127.6 (d, 15C, Ar), 85.0 (d, 1C, C-3), 81.8 (d, 1C, C-4),
73.2–71.5–71.2 (t, 3C, O-CH2-Ph), 70.3 (t, 1C, C-6), 69.1 (d, 1C, C-2),
57.3 (t, 1C, C-5), 54.6 (t, 1C, C-7), 38.9 (t, 1C, C-8). IR (CDCl3)
ν = 3378, 3032, 2925, 2863, 1595, 1495, 1454, 1366, 1207, 1094,
1076, 1028 cm−1. MS-ESI (m/z, %) = 446.94 (MH+, 100).
compound 28 with a 87% yield. [α]D25 = −14.2 (c = 0.9, CHCl3). 1
H
NMR (400 MHz, CDCl3) δ ppm = 8.12 (m, 2H, Ar o-NO2), 7.89 (s, 1H,
triazole), 7.81 (m, 2H, Ar p-NO2), 7.27–7.15 (m, 15H, Ar), 4.46–4.33
(m, 8H, O-CH2-Ph, H-9), 3.88 (d, J = 5.8 Hz, 1H, H-4), 3.79 (d,
J = 4.3 Hz, 1H, H-3), 3.51–3.44 (m, 2H, H-6), 3.05 (d, J = 10.3 Hz, 1H,
Ha-5), 2.88–2.81 (m, 1H, Ha-7), 2.65 (q, J = 5.0 Hz, 1H, H-2), 2.48 (dd,
J = 10.3, 5.4 Hz, 1H, Hb-5), 2.38–2.32 (ddd, J = 4.9, 6.3, 11.2 Hz, 1H,
Hb-7), 2.08–2.00 (m, 2H, H-8). 13C NMR (50 MHz, CDCl3)
δ
ppm = 147.2 (s, 1C, C-NO2), 145.2, 138.1, 137.1 (s, 5C, Ar p-NO2, Ar,
triazole), 128.3–127.6 (d, 15C, Ar), 126.0 (d, 2C, Ar m-NO2), 124.1 (d,
2C, Ar o-NO2), 122.1 (d, 1C, triazole), 85.2 (d, 1C, C-3), 81.8 (d, 1C, C-
4), 73.3–71.2 (t, 3C, O-CH2-Ph), 70.9 (t, 1C, C-6), 69.3 (t, 1C, C-2), 57.3
(t, 1C, C-5), 51.8 (t, 1C, C-7), 48.5 (t, 1C, C-9), 28.6 (t, 1C, C-8). IR
(CDCl3) ν = 3066, 3031, 2926, 2858, 1607, 1521, 1454, 1344, 1232,
1205, 1110, 1075 cm−1. MS-ESI (m/z, %) = 656.31 (MNa+, 100).
Synthesis of 1-(4-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzy-
loxy)methyl]pyrrolidin-1-yl}butyl)-4-(4-nitrophenyl)-1H-1,2,3-
triazole (29): Azide 17 (21 mg, 0.042 mmol) was dissolved in a 2:1
THF/H2O mixture (1.50 mL total volume) in a MW vial reactor and
CuSO4 (2.0 mg, 0.012 mmol), sodium ascorbate (5.0 mg, 0.025 mmol)
and 1-ethynyl-4-nitrobenzene (7.4 mg, 0.05 mmol) were added. The
reaction mixture was heated in the MW at 80 °C for 45 min, when a TLC
analysis (eluent CH2Cl2/Et2O 50: 1) attested the disappearance of the
starting material 23 ((Rf = 0.51) and the formation of a new product
(Rf = 0.28). The reaction mixture was filtered over Celite and con-
centrated under reduced pressure. The resulting crude product was
purified by FCC (PE/AcOet 1:1) affording 25 mg (0.039 mmol) of
compound 29 with a 92% yield. [α]D25 = −13.9 (c = 0.15, CHCl3).
1H NMR (400 MHz, CDCl3) δ ppm = 8.17 (m, 2H, Ar o-NO2), 7.86 (m,
2H, Ar m-NO2), 7.75 (s, 1H, triazole), 7.26–7.16 (m, 15H, Ar),
4.43–4.30 (m, 8H, O-CH2-Ph, H-10), 3.86 (d, J = 5.3 Hz, 1H, H-4), 3.78
(d, J = 3.9 Hz, 1H, H-3), 3.49 (m, 2H, H-6), 3.09 (d, J = 10.7 Hz, 1H,
Ha-5), 2.86 (m, 1H, Ha-7), 2.65 (q, J = 5.0 Hz, 1H, H-2), 2.45 (dd,
J = 10.5, 5.1 Hz, 1H, Hb-5), 2.33 (m, 1H, Hb-7), 1.92 (m, 2H, H-9),
Synthesis of (3-{(2R,3R,4R)- 3,4-bis(benzyloxy)-2-[(benzyloxy)
methyl]pyrrolidin-1yl}propyl)amine (26): To a solution of azide 22
(34 mg, 0.07 mmol) in MeOH (2 mL) under nitrogen atmosphere Pd/C
(17 mg) was added, together with 3 drops of 37% aqueous HCl. The
mixture was stirred under hydrogen atmosphere (balloon) for 18 h. The
catalyst was filtered through Celite and the solvent evaporated under
reduced pressure. The crude was purified on DOWEX-50WX8 resin,
eluting sequentially with MeOH (10 mL), H2O (10 mL) and 6% NH4OH.
The free amine 26 (11 mg, 0.058 mmol) was recovered with a 86%
21
yield. [α]D
= −40.3 (c = 0.845, CH3OH). 1H NMR (400 MHz,
CD3OD) δ ppm = 3.94 (d, J = 5.1 Hz, 1H, H-4), 3.87 (dd, J = 4.3,
1.9 Hz, 1H, H-3), 3.68 (m, 2H, H-6), 3.05 (d, J = 10.4 Hz, 1H, Ha-5),
2.94 (dt, J = 12.0, 8.0 Hz, 1H, Ha-7), 2.81 (t, J = 6.5 Hz, 1H, Ha-9),
2.79 (t, J = 6.5 Hz, 1H, Hb-9), 2.61 (dd, J = 10.4, 5.1 Hz, 1H, Hb-5),
2.41 (m, 2H, Hb-7, H-2), 1.69 (m, 2H, H-8). 13C NMR (50 MHz, CD3OD)
δ ppm = 79.3 (d, 1C, C-3), 75.9 (d, 1C, C-4), 72.7 (d, 1C, C-2), 60.6 (t,
1C, C-6), 58.9 (t, 1C, C-5), 52.6 (t, 1C, C-7), 39.7 (t, 1C, C-9), 27.8 (t,
1C, C-8). MS-ESI (m/z, %) = 190.85 (MH+, 100).
Synthesis of 1-(2-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzy-
loxy)methyl]pyrrolidin-1-yl}ethyl)-4-(4-nitrophenyl)-1-H-1,2,3-
triazole (27): To a solution of azide 22 (72 mg, 0.15 mmol) in THF
(1.5 mL), a catalytic amount (2.86 mg, 0.015 mmol) of CuI, 1-ethynyl-4-
nitrobenzene(26.5 mg, 0.18 mmol) and DIPEA (DIPEA, 31 μL,
543

X. Ferhati, et al.
BioorganicChemistry87(2019)534–549
1.45 (m, 2H, H-8). 13C NMR (50 MHz, CDCl3) δ ppm = 147.3 (s, 1C, C-
NO2), 145.4, 138.3, 138.1, 138.2, 137.1 (s, 5C, Ar p-NO2, Ar, triazole),
128.3, 127.7 (d, 15C, Ar), 126.1 (d, 2C, Ar m-NO2), 124.2 (d, 2C, Ar o-
NO2), 121.1 (d, 1C, triazole), 85.3 (d, 1C, C-3), 81.8 (d, 1C, C-4), 73.2
(t, 1C, O-CH2-Ph), 71.5–71.3 (d, 2C, C-6, O-CH2-Ph), 71.1 (t, 1C, O-CH2-
Ph), 69.3 (d, 1C, C-2), 57.3 (t, 1C, C-5), 54.3 (t, 1C, C-7), 50.1 (t, 1C, C-
10), 28.2 (t, 1C, C-9), 24.7 (t, 1C, C-8). IR (CDCl3) ν = 3087, 3032,
2928, 2862, 1606, 1497, 1454, 1230, 1207, 1109 cm−1. MS-ESI (m/z,
%) = 670.36 (MNa+, 100), 648.35 (MH+, 14).
starting material 29 (Rf = 0.65). EtOH was added until a homogeneous
mixture was obtained, then the solvents were evaporated under a va-
cuum. The resulting solid was dissolved in the minimum amount of H2O
and the basic resin Ambersep 900-OH was added. The suspension was
stirred for 30 min and filtered on cotton, extensively washing with
MeOH. The mixture was concentrated under reduced pressure and the
crude was purified by FCC (CH2Cl2/MeOH 7:1) to obtain pure 11
(10.3 mg, 0.027 mmol) with a 80% yield. [α]D25 = −25.2 (c = 0.44,
CH3OH). 1H NMR (400 MHz, CD3OD) δ ppm = 8.58 (s, 1H, triazole),
8.31 (m, 2H, Ar o-NO2), 8.07 (m, 2H, Ar m-NO2), 4.52 (t, J = 7.0 Hz,
2H, H-10), 3.94 (d, J = 5.2 Hz, 1H, H-4), 3.89 (m, 1H, H-3), 3.67 (m,
2H, H-6), 3.02 (d, J = 10.3 Hz, 1H, Ha-5), 2.94 (m, 1H, Ha-7), 2.66 (dd,
J = 10.3, 5.2 Hz, 1H, Hb-5), 2.45 (m, 2H, Hb-7, H-2), 2.01 (m, 2H, H-
9), 1.56 (q, J = 7.4 Hz, 2H, H-8). 13C NMR (50 MHz, CD3OD) δ
ppm = 147.3 (s, 1C, C-NO2), 145.3, 136.9 (s, 2C, Ar p-NO2, triazole),
125.9 (d, 2C, Ar m-NO2), 123.8 (d, 2C, o-NO2), 122.6 (d, 1C, triazole),
79.3 (d, 1C, C-3), 75.8 (d, 1C, C-4), 73.3 (d, 1C, C-2), 61.1 (t, 1C, C-6),
59.0 (t, 1C, C-5), 54.2 (t, 1C, C-7), 49.9 (t, 1C, C-10), 27.6 (t, 1C, C-9),
24.4 (t, 1C, C-8). MS-ESI (m/z, %) = 400.22 (MNa+, 100). HRESI-MS
Synthesis
of
(2R,3R,4R)-2-(hydroxymethyl)-1-{2-[4-(4-ni-
trophenyl)-1H-1,2,3-triazol-1-yl]ethyl}pyrrolidine-3,4-diol (9): A
solution of compound 27 (35 mg, 0.056 mmol) in dry CH2Cl2 (5.6 mL)
was cooled at 0 °C (ice bath) under nitrogen atmosphere and a 1 M
solution of BCl3 in hexane (0.51 mL) was added. The reaction mixture
was left to reach room temperature and stirred for 18 h, when a TLC
analysis (eluent PE/AcOEt 1:1) attested the disappearance of the
starting material 27 (Rf = 0.5). EtOH was added until a homogeneous
mixture was obtained, then the solvents were evaporated under a va-
cuum. The resulting solid was solved in the minimum amount of H2O
and the basic resin Ambersep 900-OH was added. The suspension was
stirred for 40 min and filtered on cotton, extensively washing with
MeOH. The mixture was concentrated under reduced pressure and the
crude was purified by FCC (CH2Cl2/MeOH 5:1) to obtain pure 9
(12.3 mg, 0.035 mmol) with a 63% yield. [α]D22 = −6.2 (c = 0.21,
CH3OH). 1H NMR (400 MHz, D2O) δ ppm = 8.32 (s, 1H, triazole), 8.05
(m, 2H, Ar o-NO2), 7.70 (m, 2H, Ar m-NO2), 4.45 (t, J = 6.1 Hz, 2H, H-
8), 3.98 (m, 1H, H-4), 3.77 (m, 1H, H-3), 3.48 (m, 2H, H-6), 3.25 (m,
1H, Ha-7), 2.85 (m, 2H, Hb-7, Ha-5), 2.67 (m, 1H, Hb-5), 2.48 (q,
J = 4.9 Hz, 1H, H-2). 13C NMR (50 MHz, D2O) δ ppm = 147.2, 145.6,
136.2 (s, 3C, C-NO2, Ar p-NO2, triazole), 126.3 (d, 2C, Ar m-NO2), 124.5
(d, 2C, Ar o-NO2), 124.1 (d, 1C, triazole), 78.9 (d, 1C, C-3), 75.7 (d, 1C,
C-4), 71.7 (d, 1C, C-2), 61.0 (t, 1C, C-6), 58.4 (t, 1C, C-5), 53.8 (t, 1C, C-
7), 49.2 (t, 1C, C-8). MS-ESI (m/z, %) = 372.12 (MNa+, 100).
m/z found 378.1767, calc. for C17H24N5O5 [MH+]: 378.1772.
+
Synthesis of N-{3-[(2R,3R,4R)-3,4-dihydroxy-2-(hydroxymethyl)
pyrrolidin-1-yl]propyl}-4-nitrobenzamide (13): Amine 26 (32 mg,
0.17 mmol) was solved in dry pyridine (2 mL) and 4-nitrobenzoyl
chloride (31 mg, 0.17 mmol) was added. The reaction mixture was
stirred at room temperature for 22 h. Then, the pyridine was evaporated
under vacuum and the resulting crude was purified by FCC (CH2Cl2/
MeOH/6% NH4OH 5:1:0.1), leading to compound 13 (28 mg,
0.083 mmol) with a 49% yield. [α]D25 = −13.2 (c = 0.82, CH3OH). 1H
NMR (400 MHz, CD3OD) δ ppm = 8.30 (m, 2H, Ar o-NO2), 8.05 (m, 2H,
Ar m-NO2), 3.98 (m, 1H, H-4), 3.92 (m, 1H, H-3), 3.69 (m, 2H, H-6),
3.62–3.37 (m, 2H, H-9), 3.09 (m, 2H, Ha-7, Ha-5), 2.70 (dd, J = 10.3,
4.1 Hz, 1H, Hb-5), 2.52 (m, 2H, Hb-7, H-2), 1.83 (m, 2H, H-8). 13C NMR
(50 MHz, CD3OD) δ ppm = 166.1 (s, 1C, C]O), 149.6, 140.0 (s, 2C, C-
NO2, Ar p-NO2), 128.3 (d, 2C, Ar m-NO2), 123.1 (d, 2C, Ar o-NO2), 79.1
(d, 1C, C-3), 75.8 (d, 1C, C-4), 73.5 (d, 1C, C-2), 60.8 (t, 1C, C-6), 59.1 (t,
1C, C-7), 53.2 (t, 1C, C-5), 38.6 (t, 1C, C-9), 26.7 (t, 1C, C-8). MS-ESI (m/
Synthesis
of
(2R,3R,4R)-2-(hydroxymethyl)-1-{3-[4-(4-ni-
trophenyl)-1H-1,2,3-triazol-1-yl]propyl} pyrrolidine-3,4-diol (10):
A solution of compound 28 (50 mg, 0.079 mmol) in dry CH2Cl2
(7.9 mL) was cooled at 0 °C (ice bath) under nitrogen atmosphere and a
1 M solution of BCl3 in hexane (0.71 mL) was added. The reaction
mixture was left to reach room temperature and stirred for 18 h, when a
TLC analysis (eluent PE/AcOEt 1:2) attested the disappearance of the
starting material 28 (Rf = 0.65). EtOH was added until a homogeneous
mixture was obtained, then the solvents were evaporated under a va-
cuum. The resulting solid was dissolved in the minimum amount of H2O
and the basic resin Ambersep 900-OH was added. The suspension was
stirred for 30 min and filtered on cotton, extensively washing with
MeOH. The mixture was concentrated under reduced pressure and the
crude was purified by FCC (CH2Cl2/MeOH 9:1) to obtain pure 10
(21.2 mg, 0.058 mmol) with a 74% yield. [α]D24 = −34.7 (c = 0.80,
CH3OH). 1H NMR (400 MHz, CD3OD) δ ppm = 8.61 (s, 1H, triazole),
8.29 (m, 2H, Ar o-NO2), 8.06 (m, 2H, Ar m-NO2), 4.58 (m, 2H, H-9),
3.94 (m, 2H, H-3, H-4), 3.64 (m, 2H, H-6), 3.06 (d, J = 10.3 Hz, 1H, Ha-
5), 2.83 (dt, J = 12.2, 8.1 Hz, 1H, Ha-7), 2.63 (dd, J = 10.2, 5.1 Hz, 1H,
Hb-5), 2.39 (m, 2H, Hb-7, H-2), 2.13 (m, 2H, H-8). 13C NMR (50 MHz,
CD3OD) δ ppm = 147.3, 145.2, 136.9 (s, 3C, C-NO2, Ar p-NO2, tria-
zole), 125.8 (d, 2C, Ar m-NO2), 123.8 (d, 2C, Ar o-NO2), 123.0 (d, 1C,
triazole), 79.4 (d, 1C, C-3), 75.9 (d, 1C, C-4), 73.0 (d, 1C, C-2), 60.9 (t,
1C, C-6), 58.7 (t, 1C, C-5), 51.0 (t, 1C, C-7), 47.9 (t, 1C, C-9), 28.3 (t,
1C, C-8). MS-ESI (m/z, %) = 394.21 (MH+,100). HRESI-MS m/z found
z, %) = 340.12 (MH+, 100). HRESI-MS m/z found 340.1495, calc. for
+
C
15H22N3O6 [MH+]: 340.1503.
Synthesis of N-(2-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzy-
loxy)methyl]pyrrolidin-1-yl}ethyl)-4-nitrobenzamide (34): Amine
25 (28 mg, 0.063 mmol) was dissolved in dry pyridine (1.5 mL) and 4-
nitrobenzoyl chloride (17 mg, 0.092 mmol) was added. The reaction
mixture was stirred at room temperature for 28 h, when a TLC analysis
attested the disappearance of the starting material 25. The pyridine was
evaporated under a vacuum and the resulting crude was purified by
FCC (CH2Cl2/MeOH 50:1), leading to compound 34 (21 mg,
23
0.035 mmol) with a 56% yield. [α]D
−15.8 (c = 1.175, CHCl3).
=
1H NMR (400 MHz, CDCl3) δ ppm = 7.87 (m, 2H, Ar o-NO2), 7.67 (m,
2H, Ar m-NO2), 7.20 (m, 15H, Ar), 4.43 (m, 6H, O-CH2-Ph), 3.67 (d,
J = 5.4 Hz, 1H, H-4), 3.86 (d, J = 3.9 Hz, 1H, H-3), 3.56 (m, 3H, H-6,
Ha-8), 3.29 (m, 1H, Hb-8), 3.18 (d, J = 10.2 Hz, 1H, Ha-5), 3.02 (m,
1H, Ha-7), 2.79 (m, 1H, H-2), 2.63 (m, 2H, Hb-5, Hb-7). 13C NMR
(50 MHz, CDCl3) δ ppm = 165.1 (s, 1C, C]O), 149.3 (s, 1C, C-NO2),
140.0 (s, 1C, Ar p-NO2), 137.9, 137.8 (s, 3C, Ar), 128.4, 127.5 (d, 17C,
Ar, Ar m-NO2), 123.5 (d, 2C, Ar o-NO2), 84.8 (d, 1C, C-3), 81.6 (d, 1C,
C-4), 73.4, 71.7, 71.3 (t, 3C, O-CH2-Ph), 70.2 (t, 1C, C-6), 68.6 (d, 1C,
C-2), 56.8 (t, 1C, C-5), 51.9 (t, 1C, C-7), 38.0 (t, 1C, C-8). IR (CDCl3)
ν = 3357, 3088, 3067, 3032, 2960, 2863, 1659, 1601, 1526, 1496,
364.1616, calc. for C16H22N5O5 [MH+]: 364.1612.
1485, 1454, 1348, 1261 cm−1. MS-ESI (m/z, %) = 618.37 (MNa+
,
+
Synthesis
of
(2R,3R,4R)-2-(hydroxymethyl)-1-{4-[4-(4-ni-
100).
trophenyl)-1H-1,2,3-triazol-1-yl]butyl}pyrrolidine-3,4-diol (11): A
solution of compound 29 (22 mg, 0.034 mmol) in dry CH2Cl2 (3.4 mL)
was cooled at 0 °C (ice bath) under nitrogen atmosphere and a 1 M
solution of BCl3 in hexane (0.71 mL) was added. The reaction mixture
was left to reach room temperature and stirred for 18 h, when a TLC
analysis (eluent PE/AcOEt 1:2) attested the disappearance of the
Synthesis of N-(4-{(2R,3R,4R)-3,4-bis(benzyloxy)-2-[(benzy-
loxy)methyl]pyrrolidin-1-yl}butyl)-4-nitrobenzamide (35): Amine
24 (37 mg, 0.078 mmol) was dissolved in dry pyridine (2 mL) and 4-
nitrobenzoyl chloride (19 mg, 0.10 mmol) was added. The reaction
mixture was stirred at room temperature for 40 h, when a TLC analysis
(eluent CH2Cl2/MeOH/6% NH4OH 4:1:0.1) attested the disappearance
544

X. Ferhati, et al.
BioorganicChemistry87(2019)534–549
of the starting material 24 (Rf = 0.54). The pyridine was evaporated
dissolved in a 2:1 THF/H2O mixture (7.5 mL total volume) in a MW vial
reactor and CuSO4 (9 mg, 0.06 mmol), sodium ascorbate (24 mg,
0.12 mmol) and 1-ethynyl-4-nitrobenzene (11.6 mg, 0.083 mmol) were
added. The reaction mixture was heated in the MW at 80 °C for 45 min,
when a TLC analysis (eluent AcOEt) attested the disappearance of the
scaffold 36 (Rf = 0.47). The reaction mixture was filtered over Celite
and concentrated under reduced pressure. The resulting crude product
was purified by FCC (CH2Cl2/MeOH 20:1) and subsequent size exclu-
sion chromatography with Sephadex, affording 67 mg (0.04 mmol) of
compound 37 with a 68% yield. [α]D24 = −14.6 (c = 1.34, CHCl3).
1H NMR (400 MHz, CDCl3) δ ppm = 7.58 (s, 3H, triazole), 7.34–7.24
(m, 45H, Ar), 4.49–4.33 (m, 30H, H-8, H-9, O-CH2-Ph), 3.94 (m, 3H, H-
4), 3.80 (m, 3H, H-3), 3.47 (m, 6H, H-6), 3.34 (m, 9H, H-10, Ha-7), 3.19
(d, J = 10.4 Hz, 3H, Ha-5), 2.86 (m, 6H, H-2, Hb-7), 2.68 (dd, J = 10.2,
5.1 Hz, 3H, Hb-5). 13C NMR (50 MHz, CDCl3) δ ppm = 144.6 (s, 3C,
triazole), 138.1, 138.0 (s, 9C, Ar), 128.4, 128.3, 127.7 (d, 45C, Ar),
123.4 (d, 3C, triazole), 85.1 (d, 3C, C-3), 81.6 (d, 3C, C-4), 73.2 (t, 3C,
O-CH2-Ph), 72.1 (s, 1C, scaffold), 71.5, 71.3, 71.1 (t, 12C, O-CH2-Ph, C-
6, C-10), 69.1 (d, 3C, C-2), 64.8 (t, 3C, C-9), 57.3 (t, 3C, C-5), 54.4 (t,
3C, C-7), 48.9 (t, 3C, C-8). IR (CDCl3) ν = 3377, 3089, 3066, 3032,
under a vacuum and the resulting crude was purified by FCC (CH2Cl2/
MeOH 30:1), leading to compound 35 (30 mg, 0.048 mmol) with a 62%
yield. [α]D
−2.3 (c = 1.05, CHCl3).1H NMR (400 MHz, CDCl3) δ
23
=
ppm = 8.00 (m, 2H, Ar o-NO2), 7.78 (m, 2H, Ar m-NO2), 7.29–7.11 (m,
15H, Ar), 4.47–4.26 (m, 6H, O-CH2-Ph), 3.86 (d, J = 5.3 Hz, 1H, H-4),
3.80 (d, J = 4.9 Hz, 1H, H-3), 3.50–3.32 (m, 4H, H-6, H-10), 5.13 (d,
J = 10.7 Hz, 1H, Ha-5), 2.85–2.79 (m, 1H, Ha-7), 2.54 (q, J = 5.0 Hz,
1H, H-2), 2.42 (dd, J = 10.7, 5.3 Hz, 1H, Hb-5), 2.26 (m, 1H, Hb-7),
1.68 (m, 1H, Ha-9), 1.55 (m, 3H, H-8, Hb-9). 13C NMR (50 MHz, CDCl3)
δ ppm = 165.5 (s, 1C, C]O), 149.3 (s, 1C, C-NO2), 140.4 (s, 1C, Ar p-
NO2), 137.9–137.7 (s, 3C, Ar), 128.4–127.7 (d, 17C, Ar, Ar m-NO2),
123.4 (d, 2C, Ar o-NO2), 84.9 (d, 1C, C-3), 81.4 (d, 1C, C-4), 73.2, 71.6,
71.3 (t, 3C, O-CH2-Ph), 70.1 (d, 1C, C-2), 69.7 (t, 1C, C-6), 57.5 (t, 1C,
C-5), 54.8 (t, 1C, C-7), 39.6 (t, 1C, C-10), 27.3 (t, 1C, C-9), 25.3 (t, 1C,
C-8). IR (CDCl3) ν = 3449, 3282, 3088, 2930, 2863, 2811, 1662, 1601,
1526, 1454, 1348, 1318, 1298, 1262, 1098 cm−1. MS-ESI (m/z,
%) = 624.28 (MH+, 63), 646.30 (MNa+, 100).
Synthesis
of
N-{2-[(2R,3R,4R)-3,4-dihydroxy-2-(hydro-
xymethyl)pyrrolidin-1-yl]ethyl}-4-nitrobenzamide (12): A solution
of compound 34 (21 mg, 0.035 mmol) in dry CH2Cl2 (3.5 mL) was
cooled to 0 °C (ice bath) under nitrogen atmosphere and a 1 M solution
of BCl3 in hexane (0.32 mL) was added. The reaction mixture was left to
reach room temperature and stirred for 15 h, when a 1H NMR control
attested the disappearance of the starting material 34 (disappearance of
multiplet at δ = 7.26 ppm, relative to benzylic protons). EtOH was
added until a homogeneous mixture was obtained, then the solvents
were evaporated under a vacuum. The resulting crude was passed
through an ion exchange resin DOWEX-50WX8, eluting with MeOH,
H2O and 6% NH4OH, and subsequently purified via FCC (CH2Cl2/
MeOH 5:1) affording pure 12 (8.0 mg, 0.025 mmol) with a 70% yield.
[α]D23 = −18.9 (c = 0.55, CH3OH). 1H NMR (400 MHz, CD3OD) δ
ppm = 8.31 (m, 2H, Ar o-NO2), 8.04 (m, 2H, Ar m-NO2), 4.00 (m, 1H,
H-4), 3.91 (m, 1H, H-3), 3.74–3.61 (m, 3H, H-6, Ha-8), 3.46 (m, 1H,
Hb-8), 3.16 (m, 2H, Ha-5, Ha-7), 2.83 (m, 1H, Hb-5), 2.71 (m, 1H, Hb-
7), 2.61 (m, 1H, H-2). 13C NMR (100 MHz, CD3OD) δ ppm = 166.9 (s,
1C, C]O), 149.6 (s, 1C, C-NO2), 139.9 (s, 1C, Ar p-NO2), 128.3 (d, 2C,
Ar m-NO2), 123.2 (d, 2C, Ar o-NO2), 78.9 (d, 1C, C-3), 75.8 (d, 1C, C-4),
73.4 (d, 1C, C-2), 60.8 (t, 1C, C-6), 60.0 (t, 1C, C-5), 53.7 (t, 1C, C-7),
38.3 (t, 1C, C-8). MS-ESI (m/z, %) = 348.16 (MNa+, 100). HRESI-MS
2924, 2864, 1722, 1496, 1454, 1363, 1263, 1206, 1097, 1054 cm−1
.
MS-ESI (m/z, %) = 1674.58 (MNa+, 92).
Synthesis of trivalent iminosugar bearing the 4-nitrobenzene
moiety 38: Trivalent iminosugar 37 (67 mg, 0.04 mmol) was dissolved
in dry CH2Cl2 (4 mL) and DIPEA (20 μL, 0.12 mmol) was added, under
nitrogen atmosphere. The reaction mixture was cooled at 0 °C (ice bath)
and 4-nitrobenzoyl chloride (9 mg, 0.049 mmol) was added. The reac-
tion mixture was stirred at room temperature for 2 days, when a TLC
analysis (eluent CH2Cl2/MeOH 30:1) attested the disappearance of the
starting material 37 (Rf = 0.22) and the formation of a new product
(Rf = 0.34). The reaction was transferred to a separatory funnel and
washed with 0.5 M HCl (2 × 5 mL) and H2O (3 × 10 mL). The com-
bined organic layers were dried over Na2SO4 and the solvent was re-
moved under a vacuum. The resulting crude was purified by FCC
(CH2Cl2/MeOH 30:1), leading to compound 38 (61 mg, 0.034 mmol)
with a 85% yield. [α]D25 = −18.7 (c = 0.92, CHCl3). 1H NMR
(400 MHz, CDCl3) δ ppm = 8.12 (m, 2H, Ar o-NO2), 7.95 (m, 2H, Ar m-
NO2), 7.50 (s, 1H, NH), 7.45 (s, 3H, triazole), 7.27–7.14 (m, 45H, Ar),
4.46–4.24 (m, 30H, O-CH2-Ph, H-8, H-9), 3.84 (m, 9H, H-10, H-4), 3.71
(d, J = 3.9 Hz, 3H, H-3), 3.38 (m, 6H, H-6), 3.24 (m, 3H, Ha-7), 3.09 (d,
J = 10.2 Hz, 3H, Ha-5), 2.77 (m, 6H, Hb-7, H-2), 2.60 (dd, J = 10.2,
5.1 Hz, 3H, Hb-5). 13C NMR (50 MHz, CDCl3) δ ppm = 165.4 (s, 1C,
C]O), 149.3 (s, 1C, C-NO2), 144.3 (s, 3C, triazole), 140.6 (s, 1C, Ar o-
NO2), 138.0–137.9 (s, 9C, Ar), 128.6–127.7 (d, 47C, Ar, Ar m-NO2),
123.5 (d, 2C, Ar o-NO2), 123.3 (t, 3C, triazole), 84.9 (d, 3C, C-3), 81.4
(d, 3C, C-4), 73.2, 71.5, 71.2 (t, 9C, O-CH2-Ph), 71.1 (t, 3C, C-6), 69.0,
68.7 (6C, C-2, C-10), 64.5 (t, 3C, C-9), 60.6 (s, 1C, scaffold), 57.3 (t, 3C,
C-5), 54.3 (t, 3C, C-7), 48.9 (t, 3C, C-9). IR (CDCl3) ν = 3415, 3088,
3066, 3032, 2925, 2863, 1724, 1667, 1603, 1526, 1496, 1454, 1347,
1279, 1054 cm−1. MS-ESI (m/z, %) = 1823.6 (MNa+, 97).
m/z found 326.1345, calc. for C14H20N3O6 [MH+]: 326.1347.
+
Synthesis
of
N-{4-[(2R,3R,4R)-3,4-dihydroxy-2-(hydro-
xymethyl)pyrrolidin-1-yl]butyl}-4-nitrobenzamide (14): A solution
of compound 35 (25 mg, 0.040 mmol) in dry CH2Cl2 (4 mL) was cooled
to 0 °C (ice bath) under nitrogen atmosphere and a 1 M solution of BCl3
in hexane (0.36 mL) was added. The reaction mixture was left to reach
room temperature and stirred for 15 h, when a 1H NMR control attested
the disappearance of the starting material 34 (disappearance of multi-
plet at δ = 7.26 ppm, relative to benzylic protons). EtOH was added
until a homogeneous mixture was obtained, then the solvents were
evaporated under a vacuum. The resulting crude was purified via FCC
(CH2Cl2/MeOH/ 6% NH4OH 5: 1:0.1) affording pure 14 (12.0 mg,
Synthesis of deprotected trivalent iminosugar bearing the 4-
nitrobenzene moiety 39:
A solution of compound 38 (38 mg,
0.034 mmol) with a 85% yield. [α]D = −21.3 (c = 0.53, CH3OH). 1H
0.021 mmol) in dry CH2Cl2 (2.1 mL) was cooled to 0 °C (ice bath) under
nitrogen atmosphere and a 1 M solution of BCl3 in hexane (0.57 mL)
was added. The reaction mixture was left to reach room temperature
and stirred for 15 h, when a TLC analysis (eluent AcOEt) attested the
disappearance of the starting material 38 (Rf = 0.45). EtOH was added
until a homogeneous mixture was obtained, then the solvents were
evaporated under a vacuum. The resulting crude was purified via FCC
(CH2Cl2/MeOH/ 6% NH4OH 6: 2:0.5) affording pure 39 (20 mg,
0.020 mmol) with a 95% yield. [α]D25 = −3.4 (c = 0.705, CH3OH).
1H NMR (400 MHz, CD3OD) δ ppm = 8.29 (m, 2H, Ar o-NO2), 8.13 (s,
3H, triazole), 7.95 (m, 2H, Ar m-NO2), 4.75 (m, 6H, H-8), 4.60 (s, 6H,
H-9), 4.10 (m, 3H, H-4), 3.91 (m, 3H, H-3), 3.85 (s, 6H, H-10), 3.74 (m,
26
NMR (400 MHz, CD3OD) δ ppm = 8.31 (m, 2H, Ar o-NO2), 8.01 (m,
2H, Ar m-NO2), 3.98 (m, 1H, H-4), 3.90 (m, 1H, H-3), 3.71 (m, 2H, H-
6), 3.41 (t, J = 6.6 Hz, 2H, H-10), 3.12 (d, J = 10.5 Hz, 1H, Ha-5), 3.02
(m, 1H, Ha-7), 2.79 (dd, J = 10.5 Hz, 4.9 Hz, 1H, Hb-5), 2.60 (m, 2H,
Hb-7, H-2), 1.66 (m, 4H, H-8, H-9). 13C NMR (50 MHz, CD3OD) δ
ppm = 166.8 (s, 1C, C]O), 149.6 (s, 1C, C-NO2), 140.2 (s, 1C, Ar p-
NO2), 128.2 (d, 2C, Ar m-NO2), 123.2 (d, 2C, Ar o-NO2), 79.0 (d, 1C, C-
3), 75.7 (d, 1C, C-4), 73.7 (d, 1C, C-2), 60.8 (t, 1C, C-6), 59.1 (t, 1C, C-
5), 54.9 (t, 1C, C-7), 39.4 (t, 1C, C-10), 26.7, 24.6 (t, 2C, C-8, C-9). MS-
ESI (m/z, %) = 354.19 (MH+, 53), 376.24 (MNa+, 100). HRESI-MS m/
z found 354.1664, calc. for C16H24N3O6 [MH+]: 354.1660.
+
Synthesis of the trivalent iminosugar 37: Azide 22 (106 mg,
0.22 mmol) and trivalent scaffold 36 (14 mg, 0.059 mmol)were
9H, H-6, Ha-7), 3.31 (m, 6H, Hb-7, Ha-5), 3.13 (m, 6H Hb-5, H-2). 13
C
NMR (50 MHz, CD3OD) δ ppm = 167.2 (s, 1C, C]O), 149.5 (s, 1C, C-
545

X. Ferhati, et al.
BioorganicChemistry87(2019)534–549
NO2), 144.6 (s, 3C, triazole), 142.5 (s, 1C, Ar p-NO2), 128.5 (d, 2C, Ar
m-NO2), 124.7 (d, 2C, Ar o-NO2), 123.2 (d, 3C, triazole), 77.3 (d, 3C, C-
3), 76.0 (d, 3C, C-4), 75.0 (d, 3C, C-2), 67.8 (t, 3C, C-10), 63.7 (t, 3C, C-
9), 60.8 (s, 1C, scaffold), 60.0 (t, 3C, C-6), 59.6 (t, 3C, C-5), 55.0 (t, 3C,
C-7), 44.2 (t, 3C, C-8). MS-ESI (m/z, %) = 1013.34 (MNa+, 100).
Synthesis of 4-nitro-N-propylbenzamide (40): To a solution of 4-
nitrobenzoyl chloride (52 mg, 0.28 mmol) in dry pyridine, under ni-
trogen atmosphere, propan-1-amine (35 μL, 0.42 mmol) was added. The
reaction mixture was stirred at room temperature for 18 h, when a TLC
analysis (eluent PE/AcOEt 10:1) attested the disappearance of 4-ni-
trobenzoyl chloride (Rf = 0.58) and the formation of a new product
(Rf = 0.78). The pyridine was evaporated under a vacuum and the re-
sulting crude was purified by FCC (PE/AcOEt 3:2), leading to com-
pound 40 (44 mg, 0.021 mmol) with a 76% yield. 1H NMR (400 MHz,
CD3Cl3) [53] δ ppm =: 8.22 (d, J = 8.8 Hz, 2H, Ar o-NO2), 7.91 (d,
J = 8.8 Hz, 2H, Ar m-NO2), 6.56 (br s, 1H, NH), 3.41 (dd, J = 7.3,
6.9 Hz, 2H), 1.63 (sest, J = 7.4 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H).
Synthesis of 4-nitro-N-(3-pyrrolidin-1yl-propyl)benzamide
sample and fitted using the following equation:
Vi
V 0
Max Min
=
+ Min
X
slope
1 + (IC50
)
where Vi/V0, represents the ratio between the activity measured in the
presence of the inhibitor (Vi) and the activity of the enzyme measured
in the absence of inhibitor (V0), while the parameter “x” represents the
concentration of inhibitor. All assays were carried out in triplicate.
The action mechanism of compounds was determined by analysing
the dependence of main kinetic parameters, Km and Vmax, from the
inhibitor concentrations. The kinetic parameters, Km and Vmax, were
determined measuring the initial hydrolysis rates in the presence of
increasing substrate concentrations. Data obtained were fitted using the
Michaelis-Menten equation. Inhibition constant (Ki) were determined
using the following equation:
1/Vmaxapp = 1/(Vmax Ki)[I] + 1/Vmax
(1)
(41): To
a solution of N-(3-bromopropyl)-4-nitrobenzamide [66]
4.2.3. Enzymatic assays with porcine pancreatic α-glucosidase
(15 mg, 0.052 mmol) in CH3CN (1 mL), pyrrolidine (0.77 μL,
0.936 mmol) was added and the reaction mixture was stirred at reflux
for 18 h, until a \TLC analysis (eluent Hexane/AcOEt 1:1) attested the
disappearance of N-(3-bromopropyl)-4-nitrobenzamide (Rf = 0.57).
The mixture was concentrated under a vacuum and the resulting crude
was purified via FCC (Hexane/AcOEt 1:3), affording pure 41 (13.0 mg,
The ability of compounds to inhibit α-glucosidase was assayed by
using a spectrophotometric method. We prepared vials containing
300 μL of reaction mixture. Each vial contained 170 μL of 100 mM
phosphate buffer (pH 6.8), 100 μL of 2.5 mM pNPG, and 10 μL of the
compounds dissolved in DMSO. The final concentration of compounds
in each vial was 0.66 mM. Samples were incubated for 10 min at 37 °C
and then diluted with 20 μL of α-glucosidase [2 U/mL in 10 mM
phosphate buffer (pH 6.8)]. The vials were incubated at 37 °C for a
further 30 min. The reactions were stopped by the addition of 700 μL of
0.2 M sodium carbonate solution. The absorbance of samples was re-
corded at 405 nm using an Ultrospec 2000 UV/visible spectro-
photometer (Pharmacia Biotech). The control test was carried out using
the same reaction mixture containing 10 μL of DMSO, the solvent used
for dilute compounds.
0.047 mmol) with
a
90% yield. 1H NMR (400 MHz, CDCl3)
δ
ppm = 9.01 (br s, 1H, O = CNH), 8.26 (d, J = 8.6 Hz, 2H, Ar), 8.15 (d,
J = 8.6 Hz, 2H, Ar), 3.65 (dd, J = 10.8, 5.2 Hz, 2H, CH2NHC = O),
3.10–3.05 (m, 6H), 2.13–2.02 (m, 6H). 13C NMR (100 MHz, CDCl3) δ
ppm = 165.3 (s, 1C, C]O), 149.6 (s, 1C, C-NO2), 139.4 (s, 1C, Ar p-
NO2), 128.5 (d, 2C, Ar m-NO2), 123.6 (d, 2C, Ar o-NO2), 53.7, 53.4 (t,
3C), 37.8 (t, 1C,CH2NHC = O), 25.0, 23.3 (t, 3C). MS-ESI (m/z,
%) = 278.21 (MH+, 100).
4.2. Biology
4.2.4. Enzymatic assays with α-glucosidase from Saccharomyces cerevisiae
and towards amyloglucosidase from Aspergillus niger
4.2.1. Expression and purification of recombinant human PTP-1B
The PTP-1B enzyme was purified as a fusion protein from TB1
bacterial cell lines. pGEX-2 T expression vectors containing the full se-
quence of PTP-1B cloned downstream of the GST sequence were used to
transform the TB1 E. coli strain. The recombinant fusion protein was
purified from bacterial lysate using single step affinity chromatography
with a glutathione-agarose resin. Fractions containing the fusion pro-
tein were collected and concentrated up to a volume of 5 mL. The
cleavage of the fusion protein was carried out by incubating the solu-
tion with 1.25 U of human thrombin for 3 h at 37 °C. Then the active
phosphatase was purified from GST and thrombin by gel filtration on a
Superdex G75 column. The enzyme purity was determined by SDS-
PAGE.
The % of inhibition towards the corresponding glycosidase was
determined in the presence of 1 mM of the inhibitor on the well. Each
enzymatic assay (final volume 0.12 mL) contains 0.01 to 0.5 units/mL
of the enzyme and 10 mM aqueous solution of the p-nitrophenyl α-
glucopyranoside (substrate) buffered to the optimal pH of the enzyme
(pH = 7.0 for α-glucosidase from Saccharomyces cerevisiae and
pH = 5.0 for amyloglucosidase from Aspergillus niger). Enzyme and in-
hibitor were preincubated for 5 min at rt, and the reaction started by
the addition of the substrate. After 20 min of incubation at 37 °C, the
reaction was stopped by the addition of 0.1 mL of sodium borate so-
lution (pH 9.8). The p-nitrophenolate formed was measured by visible
absorption spectroscopy at 405 nm. Under these conditions, the p-ni-
trophenolate released led to optical densities linear with both reaction
time and concentration of the enzyme; thus, the measured absorbance
can be transformed into % of inhibition. The IC50 value (concentration
of inhibitor required for 50% inhibition of enzyme activity) was de-
termined from plots of % inhibition versus inhibitor concentration
(eight different concentrations were used). For the IC50 plot, each
percentage of inhibition was obtained in duplicate and the average
value was given (see IC50 graphs in Supplementary data).
4.2.2. Enzymatic assays with PTP1B
The assays were carried out at 37 °C using aliquots of purified re-
combinant human PTP1B and p-nitrophenylphosphate (pNPP) as a
synthetic substrate (2.5 mM, a value corresponding to the Km of the
enzyme). All assays were carried out in 0.075 M of β,β-dimethyl glu-
tarate pH 7.0 buffer, containing 1 mM EDTA and 1 mM dithiothreitol.
The final volume of each test was 1 mL. Assay solutions were incubated
at 37 °C for 10 min. Then, the reactions were started by adding aliquots
of the enzyme. The reactions were stopped by adding 4 mL of 0.1 M
KOH after 30 min. The released p-nitrophenolate ion was determined
by reading the absorbance at 400 nm (ε = 18,000 M−1 cm−1).
The IC50 values were determined by measuring the residual enzyme
activity in the presence of increasing inhibitor concentration and a fixed
substrate concentration corresponding to that of Km of the enzyme.
Fifteen different inhibitor concentrations were used for every com-
pound. Data obtained were normalized with respect to the control
4.2.5. Inhibition assay towards lysosomal enzyme acid α-glucosidase (EC
3.2.1.20) [59,60]
Our laboratories’ whole collection of iminosugars was tested to-
wards α-glucosidase from lymphocytes isolated from healthy donors’
flesh blood (controls), using the protocol with Lymphoprep™ [67].
Isolated lymphocytes were disrupted by sonication and the micro BCA
protein assay kit (Sigma-Aldrich) was used to set up the protein amount
for the enzymatic assay, according to the manufacturer’s instructions.
546

X. Ferhati, et al.
BioorganicChemistry87(2019)534–549
α-Glucosidase activity was measured in a flat–bottomed 96 well plate:
iminosugar solution (3 µL), 4.29 µg/µL lymphocytes homogenate (7 µL)
and 20 µL of substrate solution of 4-methylumbelliferyl-α-D-glucopyr-
anoside (Sigma-Aldrich) in Na acetate buffer (0.2 M, pH 4.0) were in-
cubated for 1 h at 37 °C. The reaction was stopped by the addition of a
solution of sodium carbonate (0.5 M, 0.0025% triton X100, pH 10.7,
200 µL) and fluorescence was measured in a SpectraMax M2 microplate
reader (Molecular-Devices) using an excitation wavelength of 365 nm
and an emission wavelength of 435 nm. Percentages of α-glucosidase
inhibition were given with respect to the control (without iminosugar).
Experiments were performed in triplicate.
University of Florence – Italy for financial support (Fondi di Ateneo, ex-
60%). We also thank Dr. Alessandro Pratesi for technical assistance.
Appendix A. Supplementary material
Supplementary data contains 1H and 13C NMR spectra of all new
compounds, kinetic analysis toward PTP1B of compounds 9 and 13,
evaluation of the inhibitory power of compound 40 and 41 on PTP1B,
and IC50 graphs towards glucosidases. Supplementary data to this ar-
ticle can be found online at https://doi.org/10.1016/j.bioorg.2019.03.
053.
The IC50 values against the human acid α-glucosidase inhibitors
were determined by measuring the initial hydrolysis rate under fixed 4-
methylumbelliferyl-α-D-glucoside concentration (1.47 mM). Data were
obtained using the previously reported method (see the Supporting
Information file for further details and graphs) [68].
References
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Isolated leukocytes were disrupted by sonication and the micro BCA
protein assay kit (Sigma-Aldrich) was used to set up the protein amount
for the enzymatic assay, according to the manufacturer instructions.
GCase activity was measured in a flat–bottomed 96 well plate: imino-
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200 µL) and fluorescence was measured in a SpectraMax M2 microplate
reader (Molecular-Devices) using an excitation wavelength of 365 nm
and an emission wavelength of 435 nm. Percentages of GCase inhibition
were given with respect to the control (without iminosugar).
Experiments were performed in triplicate.
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Declaration of interest
None.
Acknowledgements
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de Economía y Competitividad of Spain (CTQ2016-77270-R) is also
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Firenze for financial support and for a fellowship to CM (grant NO.
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