Rapid discovery of potent α-fucosidase inhibitors by in situ screening of a library of (pyrrolidin-2-yl)triazoles

Article abstract of DOI:10.1039/c4ob00931b

The synthesis of a small library of (pyrrolidin-2-yl)triazoles via copper catalysed cycloaddition of an alkynyl iminocyclopentitol and a set of commercial and synthetic azides has been achieved. The in situ screening for the activity towards α-fucosidase of the resulting triazoles has allowed the identification of one of the most potent and selective pyrrolidine derived inhibitors of this enzyme (K_i = 4 nM). This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00931b

Organic &
Biomolecular Chemistry
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Rapid discovery of potent α-fucosidase inhibitors
by in situ screening of a library of (pyrrolidin-2-yl)-
triazoles†
Cite this: Org. Biomol. Chem., 2014,
12, 5898
Pilar Elías-Rodríguez, Elena Moreno-Clavijo, Ana T. Carmona,
Antonio J. Moreno-Vargas* and Inmaculada Robina
Received 6th May 2014,
Accepted 12th June 2014
The synthesis of a small library of (pyrrolidin-2-yl)triazoles via copper catalysed cycloaddition of an
alkynyl iminocyclopentitol and a set of commercial and synthetic azides has been achieved. The in situ
screening for the activity towards α-fucosidase of the resulting triazoles has allowed the identification of
one of the most potent and selective pyrrolidine derived inhibitors of this enzyme (Ki = 4 nM).
DOI: 10.1039/c4ob00931b
www.rsc.org/obc
Introduction
α-Fucosidases are glycosidases involved in the biosynthesis of
cell surface O-fucosylated oligosaccharides which play an
important role in cell recognition, bacterial adhesion and viral
invasion. Thus, α-fucosidases are associated with certain
disorders such as inflammation,1 metastasis of certain cancer
cells2 and cystic fibrosis.3 Recently, it has been reported that
the activity of human α-fucosidase 2 is critical for the patho-
genesis of Helicobacter pylori including gastric cancer4 among
other diseases. Moreover, α-fucosidases have been found
in human seminal plasma and in the membrane of human
sperm cells and facilitate sperm transport and sperm–egg
interactions.5 For these reasons, α-fucosidases are clinically
important targets and the design of efficient routes for the syn-
thesis of potent and selective inhibitors remains an attractive
goal.
In recent years, we have been actively working on the
development of new iminocyclitols with inhibitory activity
towards α-fucosidases.6–9 We have shown that the presence of
an additional aromatic or heteroaromatic binding component
close to the five membered iminocyclitol increases notably
their inhibitory activity (1 10 vs. 2,8 Fig. 1). This fact has also
been shown by Behr in the case of pyrrolidine 311 and by
Wong in the case of six-membered iminocyclitols (4 vs. 5).12
Nevertheless, attempts to explore the chemical diversity on
five-membered iminocyclitols by the systematic variation of
the aromatic group remains a cumbersome task.
Fig. 1 Inhibitory activity of representative iminocyclitols towards
α-fucosidase from bovine kidney.
The Cu(I)-catalyzed alkyne–azide cycloaddition13 (CuAAC)
has become a widely used strategy for chemical space explora-
tion in drug design14 due to its quickness at room temperature
and compatibility with water-rich solvent systems, which
makes it very suitable for in situ screening. In the search for
potent glycosidase inhibitors, the strategy of in situ screening
of a library of compounds generated by a combinatorial
procedure has been very scarcely explored to date.12,15 Thus,
the search for α-mannosidase inhibitors has been performed
through imine condensation of the appropriate configured
pyrrolidine carbaldehyde and amines.15a In the case of α-fuco-
sidase inhibitors, L-fucopiperidine derivatives were sought
through amide condensations from 1-aminofuconojirimycin
as the starting material.12,15c For the synthesis of pyrrolidine
derivatives as hexosaminidase inhibitors, amide condensation
and non-catalyzed Huisgen cycloaddition were used starting
from convenient 1-amino- and 1-azidoiminocyclitols, respecti-
vely.15b Particularly, CuAAC has been only used in the in situ
screening of aminocyclitols as glucocerebrosidase (a type of
β-glucosidases) inhibitors,15d in spite of that CuAAC has been
Department of Organic Chemistry, Faculty of Chemistry, University of Seville,
C/Prof. García González, 1, 41012-Seville, Spain. E-mail: ajmoreno@us.es
†Electronic supplementary information (ESI) available: Experimental details for
the preparation of 6 and azides a–u, enzymatic test details, 1H- and 13C-NMR
spectra. See DOI: 10.1039/c4ob00931b
5898 | Org. Biomol. Chem., 2014, 12, 5898–5904
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Scheme 1 Proposed strategies for the preparation of libraries of
inhibitors.
Scheme 3
broadly used in the last few years as click reaction in the prepa-
ration of multivalent glycosidase inhibitors.16
We report herein the application of the CuAAC followed by
in situ screening towards α-fucosidase for the preparation of
analogues of compound 2 with improved inhibitory properties.
As far as we are aware, no other optimization of the glycosidase
inhibitory properties of iminocyclitols using this strategy has
been reported. To achieve this goal and choose the appropriate
lead compound, we planned two possible strategies
(Scheme 1): (1) CuAAC between an unprotected alkynyl imino-
cyclitol with synthetic or commercial azides, and (2) CuAAC
between an azidomethyl iminocyclitol and terminal alkynes.
In both cases the biological analysis of the combinatorial
library should be carried out by in situ screening, allowing
determination of whether the resulting substituted
pyrrolidine–triazole derivatives are α-fucosidase inhibitors and
if they could improve the activity of the parent pyrrolidines.
Table 1 Inhibitory activities of compounds 14, 14a and 15 towards
glycosidases. Percentage of inhibition at 1 mM of inhibitor, IC50 and Ki.
Optimal pH for each enzyme, 37 °Ca,b
Compounds/enzyme
14
14a
15
α-L-Fucosidase
(from bovine kidney)
99%
IC50 = 0.5 μM
Ki = 44 nM
99%
IC50 = 0.3 μM
Ki = 24 nM
99%
IC50 = 2.1 μM
Ki = 500 nM
a No inhibition was detected towards β-galactosidases from Aspergillus
oryzae and from Escherichia coli, α-glucosidase from rice,
α-mannosidase from Jack beans, β-N-acetylglucosaminidase from Jack
beans, α-galactosidase from coffee beans, amyloglucosidase from
Aspergillus niger, α-glucosidase from Saccharomyces cerevisiae,
β-glucosidase from almonds and β-mannosidase from snail.
b Competitive inhibition was observed in all the cases, according to the
Lineweaver–Burk plots (see ESI).
Results and discussion
Synthesis of the lead compound for the generation of the
carried out from D-lyxose following the procedure reported by
Behr17 but using the tert-butyldiphenylsilyl ether as the pro-
tecting group.18 Treatment of 6 with TBAF followed by stereo-
selective hydrogenation of 7 over Pd/C in MeOH at 200 psi gave
8. N-Boc protection and oxidation furnished carbaldehyde
10 19 which was transformed into alkyne derivative 11 after
reaction with the Bestmann–Ohira reagent. On its side, N-Cbz
protection of 8 and introduction of the azido moiety through
tosylation and displacement, provided azido derivative 13.
In order to choose the best strategy (strategy 1 or 2,
Scheme 1) for the combinatorial preparation of a library of
potential inhibitors, alkyne 11 and azide 13 were transformed
into isomeric triazoles 14a and 15, respectively. Thus, CuAAC
of 11 with benzyl azide using CuI/DIPEA followed by acid
deprotection afforded triazole 14a (Scheme 3). Azide 13 was
transformed into triazole 15 by CuAAC with phenyl acetylene
followed by deprotection. The inhibitory activity of both tri-
azoles 14a and 15 was studied towards eleven commercial
glycosidases (Table 1). Triazole 14a was twenty times better
fucosidase inhibitor than the isomeric triazole 15. The pres-
ence of a methylene group between the pyrrolidine skeleton
and the triazole moiety proved to be detrimental for the inhi-
bition of α-fucosidase. Thus, strategy 1 was chosen for the
generation of the combinatorial library. For this reason,
unprotected alkyne 14 was also synthesized from 11 and bio-
logically analyzed.
library
Protected precursors of the above iminocyclitols were prepared
as described in Scheme 2. The synthesis of pyrroline 6 was
Scheme 2 Synthesis of protected alkynyl/azidomethyl pyrrolidines.
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Fig. 2 Inhibitory activities towards bovine kidney α-fucosidase (pH 6,
37 °C) measured for triazole derivatives 14a–u at 0.5 µM on the well.
were studied towards eleven commercial glycosidases (footnote
in Table 1). At 1 mM concentration, 14p only showed inhi-
Scheme 4 Reaction of (2-ethynyl)iminocyclitol 14 with azides a–u for
in situ screening in a microtiter plate.
bition towards α-fucosidase from bovine kidney, with IC50
=
17 nM and Ki = 4 nM (competitive inhibition), which confirms
the high potency of this inhibitor.
Generation of a library of (pyrrolidin-2-yl)triazoles and in situ
screening as α-fucosidase inhibitors
In order to obtain a library of triazoles from alkyne 14, alkyl
and aryl azide reactants (Scheme 4) were selected from com-
mercial sources or prepared from commercially available
materials following standard protocols (see ESI†). As we had
previously reported that appropriate configured furyl imino-
cyclitols showed good α-fucosidase inhibition,6,7 a batch of
synthetic azides containing the furan moiety (compounds
o–u)20 was also used.
Conclusions
In conclusion, we have demonstrated that the fucosidase
inhibitory activity of a library of (pyrrolidin-2-yl)triazoles
generated by CuAAC can be in situ analysed after the click reac-
tion, avoiding the tedious isolation/purification steps. By this
strategy, one of the best α-fucosidase inhibitor reported so far
belonging to the pyrrolidine-iminosugar family has been
identified. This is the first combinatorial method for the rapid
discovery of α-fucosidase inhibitors that employs the combi-
nation of a CuAAC click reaction and in situ screening.
Parallel CuAAC between (2-ethynyl)iminocyclitol 14 and
azides a–u were carried out under the same reaction con-
ditions using H2O–tBuOH as the solvent system (Scheme 4).21
The in situ screening of the resulting crude (pyrrolidin-2-yl)-
triazoles towards α-fucosidase was carried out in a 96-well
microtiter plate. Each inhibition assay was performed in a well
containing 0.5 µM of the potential inhibitor (see Experimental
section for details). Blank experiments with the CuAAC
reagents (CuSO4 and sodium ascorbate) and with each of the
azides a–u were also carried out: no inhibition was observed.
Interestingly, several potent inhibitors were found among the
21 library members studied, their % inhibition values at
0.5 µM are shown in Fig. 2. All the triazole derivatives resulted
in better inhibitors than the alkyne precursor. Compounds
containing the triazole linked to other aromatic moieties
showed better inhibition than when linked to non-aromatic
ones (% inhibition of 14b, d, e, o–t >85% vs. % inhibition of
14h–l <68%). It is worth noting the high inhibition presented
by the derivatives bearing the furan moiety (14o–14u), of
which 14p being the best inhibitor of the library (93%
inhibition at 0.5 µM). These results were used as criteria for
preliminary screening and compound selection.
Experimental
General methods
Optical rotations were measured in a 1.0 cm or 1.0 dm tube
with a Jasco P-2000 spectropolarimeter. Infrared spectra were
recorded with a Jasco FTIR-410 spectrophotometer. H and 13C
1
NMR spectra were recorded with a Bruker AMX300, AV300,
AV500 and AVIII500 for solutions in CDCl3, CD3OD and DMSO-
d6 at room temperature except when indicated. δ are given in
ppm and J in Hz. All the assignments were confirmed by COSY
and HSQC experiments. Mass spectra (CI and LSI) were
recorded on Micromass AutoSpeQ and QTRAP spectrometers.
The LSI was performed using thioglycerol as the matrix. NMR
and mass spectra were registered in CITIUS (University of
Seville). TLC was performed on silica gel HF254 (Merck), with
detection by UV light charring with H2SO4, p-anisaldehyde,
vanillin, ninhydrin or with Pancaldi reagent [(NH4)6MoO4,
Ce(SO4)2, H2SO4, H2O]. Silica gel 60 (Merck, 63–200 μm) was
used for preparative chromatography.
In order to perform a more accurate inhibition study,
selected triazole 14p was synthesized in a higher scale and
purified by column chromatography. Its inhibition properties
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(3R,4S,5S)-5-Hydroxymethyl-3,4-O-isopropylidene-2-methyl- (M + 2H − Boc)+]. HRCIMS m/z found 288.1806, calc. for
1-pyrroline-3,4-diol (7). To a solution of 6 (3.6 g, 8.5 mmol) C14H26O5N: 288.1811.
in THF (20 mL), TBAF (1 M in THF, 8.5 mL, 8.5 mmol)
(2R,3S,4R,5S)-N-tert-Butoxycarbonyl-2-formyl-3,4-O-isopropyl-
was added. After stirring at r.t. for 3 h, the solvent was idene-5-methylpyrrolidine-3,4-diol (10). To a solution of 9
evaporated and the residue purified by chromatography (50.7 mg, 0.176 mmol) in CH2Cl2 (3 mL), Dess–Martin reagent
column on silica gel (CH2Cl2–MeOH, 15 : 1) to give 7 (1.5 g, (112 mg, 0.26 mmol) was added. The mixture was stirred at r.t.
8.1 mmol, 95%) as a colourless oil. NMR and IR data are for 2 h. Then CH2Cl2 (18 mL), sat. aq. sol. of NaHCO3 (10 mL)
in accordance with those of its enantiomer.22 [α]D27 −78.2 and Na2S2O3·5H2O (250 mg, 1.0 mmol) were successively
(c 0.96, CH2Cl2). IR (ν cm−1) 3181 (OH), 2983, 2826, 1648, added and the resulting solution was stirred for 5 min. The
1207, 1069, 868. 1H-NMR (300 MHz, CDCl3, δ ppm, J Hz) organic phase was washed with sat. aq. sol. of NaHCO3 and
δ 4.88 (d, 1H, J4,3 = 5.6, H-3), 4.58 (d, 1H, H-4), 4.16 (br.s, 1H, brine, dried with Na2SO4, filtered and evaporated. The result-
2
H-5), 3.87 (dd, 1H, J1′a,1′b = 11.6, J1′a,5 = 3.3, H-1′a), 3.76 (dd, ing residue was purified by chromatography column on silica
1H, J1′b,5 = 3.5, H-1′b), 2.76 (br.s, 1H, OH), 2.09 (d, 3H, JH,H
=
gel (EtOAc–cyclohexane, 1 : 1) to give 10 (40.5 mg, 0.142 mmol,
1.1, Me), 1.35 (s, 3H, –C(CH3)2), 1.34 (s, 3H, –C(CH3)2). 81%) as a white solid. [α]2D5 +68.9 (c 0.95, CH2Cl2); IR (ν cm−1
)
13C-NMR (75.4 MHz, CDCl3, δ ppm) δ 176.1 (C-2), 111.9 2986, 1737 (CvO), 1684 (CvO), 1367, 1211, 1024, 865, 736. 1H
(–C(CH3)2), 87.4 (C-3), 80.8 (C-4), 78.1 (C-5), 62.6 (C-1′), 27.0 NMR (300 MHz, CDCl3, δ ppm, J Hz) δ 9.58 (br.s, 1H, CHO),
(–C(CH3)2), 25.8 (–C(CH3)2), 17.1 (Me). LSIMS m/z 186 [33%, 4.64–4.59 (m, 2H, H-4, H-3), 4.46–4.38 (m, 1H, H-2), 3.99–3.91
(M + H)+]. HRLSIMS m/z found 186.1134, calc. for C9H16NO3: (m, 1H, H-5), 1.52 (s, 3H, –C(CH3)2), 1.47–1.38 (m, 12H,
186.1130.
(2S,3S,4R,5S)-2-Hydroxymethyl-3,4-O-isopropylidene-5-methyl- CDCl3,
pyrrolidine-3,4-diol (8). A solution of pyrroline (2.0 g, (–C(CH3)3), 80.6, 78.2 (C-3, C-4), 71.6 (C-2), 57.6 (C-5), 28.3
–C(CH3)3, Me), 1.33 (s, 3H, –C(CH3)2). 13C NMR (75.4 MHz,
ppm) 198.0 (CHO), 112.7 (–C(CH3)2), 81.1
δ
δ
7
10.8 mmol) in MeOH (40 mL) was stirred under H2 (200 psi) in (–C(CH3)3), 26.3, 25.2 (–C(CH3)2), 14.6 (Me). CIMS m/z 286
the presence of 10% Pd/C. After 24 h, the catalyst was filtered [10%, (M + H)+], 256 [49%, (M − CHO)+], 186 [100%, (M + 2H
through celite and washed with MeOH. The solvent was evapo- − Boc)+]. HRCIMS m/z found 286.1652, calc. for C14H24O5N:
rated and the residue purified by chromatography column on 286.1654.
silica gel (CH2Cl2–MeOH, 15 : 1→10 : 1, 1% Et3N) to give 8
(2S,3S,4R,5S)-N-tert-Butoxycarbonyl-2-ethynyl-3,4-O-iso-
(1.6 g, 8.6 mmol, 80%) as a white solid. NMR and IR data are propylidene-5-methylpyrrolidine-3,4-diol (11). To a solution of
in accordance with those of its enantiomer.22 [α]D27 −13.3 10 (31.7 mg, 0.111 mmol) in anhydrous MeOH (1.5 mL) at
(c 0.89, CH2Cl2). IR (ν cm−1) 3239 (OH, NH), 2977, 2874, 1369, 0 °C, K2CO3 (31 mg, 0.22 mmol) and Bestmann–Ohira reagent
1
841. H NMR (300 MHz, CDCl3, δ ppm, J Hz) δ 4.48 (dd, 1H, (25 μL, 0.166 mmol) were successively and slowly added. The
J4,3 = 5.4, J4,5 = 3.9, H-4), 4.40 (d, 1H, H-3), 3.50 (dd, 1H, 2J1′a,1′b mixture was stirred at r.t. for 7 h. Diethyl ether (5 mL) and sat.
= 10.2, J1′a,2 = 5.3, H-1′a), 3.32–3.24 (m, 2H, H-1′b, H-2), 3.09 aq. sol. of NaHCO3 were successively added and the aqueous
(qd, 1H, J5,Me = 6.6, H-5), 2.93 (s, 2H, NH, OH), 1.47, 1.29 (2 s, phase was extracted twice with CH2Cl2. The organic layers were
3H each, –C(CH3)2), 1.21 (d, 3H, Me). 13C NMR (75.4 MHz, washed with brine, dried with Na2SO4, filtered and evaporated.
CDCl3, δ ppm) δ 111.2 (–C(CH3)2), 84.1 (C-3), 83.3 (C-4), 66.0 The resulting residue was purified by chromatography column
(C-2), 60.1 (C-1′), 56.0 (C-5), 26.3, 24.1 (–C(CH3)2), 13.4 (Me). on silica gel (EtOAc–cyclohexane, 1 : 5) to give 11 (23.4 mg,
LSIMS m/z 188 [13%, (M + H)+]. HRLSIMS m/z found 188.1285, 0.083 mmol, 75%) as a yellow oil. [α]2D5 +145.6 (c 0.41, CH2Cl2);
calc. for C9H18NO3: 188.1287.
IR (ν cm−1) 2986, 2933, 1706 (CvO), 1366, 1163, 1024, 859.
(2S,3S,4R,5S)-N-tert-Butoxycarbonyl-2-hydroxymethyl-3,4-O- 1H NMR (300 MHz, CDCl3, δ ppm, J Hz) δ 4.66–4.64 (m, 2H,
isopropylidene-5-methylpyrrolidine-3,4-diol (9). To a solution H-4, H-3), 4.58 (br.s, 1H, H-2), 3.79–3.71 (m, 1H, H-5), 2.28 (d,
of 8 (198 mg, 1.06 mmol) in MeOH (8 mL), (Boc)2O (302 mg, 1H, J2′,2 = 2.4, H-2′), 1.47 (s, 12H, –C(CH3)2, –C(CH3)3), 1.42 (d,
1.4 mmol) was added. The mixture was left at r.t. for 6 h. The 3H, JMe,5 = 6.3, Me), 1.31 (s, 3H, –C(CH3)2). 13C NMR
solvent was evaporated and the obtained residue was purified (75.4 MHz, CDCl3, δ ppm) δ 155.7 (CvO), 111.8 (–C(CH3)2),
by column chromatography on silica gel (CH2Cl2–MeOH, 82.9, 81.6 (C-4, C-3), 80.8 (–C(CH3)3), 80.6 (C-1′), 72.5 (C-2′),
40 : 1→5 : 1) to give 9 (281.2 mg, 0.98 mmol, 92%) as a colour- 56.5 (C-2), 55.6 (C-5), 28.5 (–C(CH3)3), 26.2, 25.2 (–C(CH3)2),
less oil. [α]2D3 +59.1 (c 1.01, CH2Cl2); IR (ν cm−1) 3433 (OH), 14.8 (Me). CIMS m/z 282 [8%, (M + H)+], 226 [100%,
1
2986, 2938, 1665 (CvO), 1367, 1165, 1024, 857, 774. H NMR (M − C(CH3)3 + 2H)+]. HRCIMS m/z found 282.1707, calc. for
(500 MHz, DMSO-d6, 363 K, δ ppm, J Hz) δ 4.64 (dd, 1H, J3,4
=
C15H24O4N: 282.1705.
6.2, J3,2 = 0.75, H-3), 4.59 (t, 1H, J4,5 = 6.2, H-4), 3.82–3.77 (m,
(2S,3S,4R,5S)-N-Benzyloxycarbonyl-2-hydroxymethyl-3,4-O-
2H, H-2, H-5), 3.57–3.51 (m, 2H, H-1′a, H-1′b), 1.42 (s, 9H, isopropyliden-5-methylpyrrolidine-3,4-diol (12). To a solution
–C(CH3)3), 1.41 (s, 3H, –C(CH3)2), 1.30 (s, 3H, –C(CH3)2), 1.25 of compound 8 (265.2 mg, 1.42 mmol) in EtOH–H2O (1 : 1,
(d, 3H, J5,Me = 6.5, Me). 13C NMR (125.7 MHz, DMSO-d6, 363 K, 20 mL), NaHCO3 (120 mg, 1.43 mmol) and CbzCl (224 μL,
δ ppm) δ 153.4 (CvO), 109.7 (–C(CH3)2), 80.3 (C-3), 79.7 (C-4), 1.56 mmol) were added. After stirring for 2 h at r.t., sat. aq.
78.1 (–C(CH3)3), 63.9, 56.4 (C-2, C-5), 59.9 (C-1′), 27.7 sol. of NaHCO3 was added and the mixture was extracted with
(–C(CH3)3), 25.4, 24.5 (–C(CH3)2), 14.9 (Me). CIMS m/z 288 ethyl acetate. The organic phase was dried over Na2SO4,
[63%, (M + H)+], 256 [56%, (M − CH2OH)+], 188 [100%, filtered and concentrated. The resulting residue was purified
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by chromatography column on silica gel (EtOAc–cyclohexane, (C-3), 74.5 (C-4), 73.2 (C-2′), 56.6 (C-5), 54.3 (C-2), 14.6 (Me).
1 : 2) to give 12 (385.7 mg, 1.20 mmol, 85%) as a colourless oil. CIMS m/z 142 [86%, (M + H)+]. HRCIMS m/z found 142.0866,
[α]2D4 +93.1 (c 0.53, CH2Cl2). IR (ν cm−1) 3447 (OH), 2986, 2938, calc. for C7H12O2N: 142.0868.
1681 (CvO), 1410, 1210, 1026, 697. 1H NMR (300 MHz, DMSO-
(2S,3S,4R,5S)-2-(1-Benzyl-1H-1,2,3-triazol-4-yl)-5-methyl-pyrrol-
d6, 363 K, δ ppm, J Hz) δ 7.37–7.31 (m, 5H, H-aromat.), 5.10 (d, idine-3,4-diol (14a). DIPEA (115 μL, 0.658 mmol) and CuI
2
1H, JH,H = 12.6, CH2 of Cbz), 5.05 (d, 1H, CH2 of Cbz), (10 mg, 0.05 mmol) were added to a solution of 11 (47.1 mg,
4.68–4.60 (m, 3H, H-3, H-4, OH), 3.92–3.84 (m, 2H, H-2, H-5), 0.167 mmol) and benzyl azide (29.7 mg, 0.223 mmol) in
3.56–3.53 (m, 2H, H-1′a, H-1′b), 1.39, 1.29 (2 s, 3H each, toluene (1.5 mL). The mixture was stirred at r.t. for 6 h and
–C(CH3)2), 1.27 (d, 3H, JMe,5 = 6.6, Me). 13C NMR (75.4 MHz, then, a sat. aq. sol. of NaHCO3 was added and extracted with
DMSO-d6 363 K, δ ppm) δ 154.1 (CvO of Cbz), 136.6, 127.9, EtOAc. The organic phases were dried, filtered and concen-
127.4, 127.2 (C-aromat.), 109.9 (–C(CH3)2), 80.5, 79.8 (C-3, C-4), trated under reduced pressure. The resulting residue was
65.5 (CH2 of Cbz), 64.3, 56.9 (C-2, C-5), 60.0 (C-1′), 25.5, 24.6 purified by chromatography column on silica gel (EtOAc–cyclo-
(–C(CH3)2), 14.9 (Me). CIMS m/z 322 [2%, (M + H)+], 290 [22%, hexane, 1 : 3) to give the corresponding protected triazole
(M
−
CH2OH)+]. HRCIMS m/z obsd 322.1647, calc. for derivative (47.9 mg, 0.116 mmol, 70%) as a white solid. Depro-
tection of this compound (47.9 mg, 0.116 mmol) was carried
C17H24NO5: 322.1654.
(2S,3S,4R,5S)-N-Benzyloxycarbonyl-2-azidomethyl-3,4-O-iso- out in HCl (4 M)–THF 1 : 1 (3 mL) at r.t. for 20 h. The solvent
propyliden-5-methylpyrrolidine-3,4-diol (13). To a solution of was then evaporated and the residue was purified by chromato-
the alcohol 12 at 0 °C (602 mg, 1.87 mmol) in dry pyridine graphy column on silica gel (CH2Cl2–MeOH–NH4OH, 9 : 1 : 0.1)
(16 mL), TsCl (717 mg, 3.76 mmol) was slowly added. After stir- to give 14a (29.9 mg, 0.11 mmol, 95%) as a yellow solid.
ring at r.t. overnight, the mixture was cooled to 0 °C, water was [α]2D6 −34.0 (c 0.85, MeOH); IR (ν cm−1) 3259 (OH, NH), 2919,
slowly added, and the mixture was allowed to warm to r.t. The 2360, 1453, 1051, 727, 611. 1H NMR (500 MHz, DMSO-d6,
solvent was then removed, and the residue was diluted with 363 K, δ ppm, J Hz) δ 8.00 (s, 1H, H-5′), 7.38–7.32 (m, 5H,
EtOAc, washed with HCl (1 N), sat. aq. sol. of NaHCO3 and H-aromat.), 5.57 (s, 2H, –CH2Ph), 4.24–4.20 (m, 2H, H-2, H-3),
brine, dried, filtered, and concentrated. To a solution of this 3.89 (t, 1H, J4,3 = J4,5 = 3.5, H-4), 3.47–3.42 (m, 1H, H-5), 1.13
compound in DMF (16 mL), NaN3 (305 mg, 4.69 mmol) was (d, 3H, JMe,5 = 6.5, Me). 13C NMR (125.7 MHz, DMSO-d6, 363 K,
added. After heating at 70 °C for 2 h, the solvent was evapor- δ ppm) 147.2 (C-4′), 135.5, 128.2, 127.6, 127.5 (C-aromat.),
ated and the residue diluted with CH2Cl2 and washed with 122.1 (C-5′), 77.5 (C-3), 72.3 (C-4), 57.1 (C-2), 55.1 (C-5), 52.5
water and brine. The organic phase was dried, filtered, and (–CH2Ph), 13.8 (Me). HRCIMS m/z found 275.1506, calc. for
concentrated. Purification by chromatography column (EtOAc– C14H19O2N4: 275.1508.
cyclohexane 1 : 6) afforded 13 (464 mg, 1.34 mmol, 72%) as a
(2S,3R,4S,5S)-2-Methyl-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-
colourless oil. [α]D28 +60.4 (c 0.55, CH2Cl2). IR (ν cm−1) 2986, pyrrolidine-3,4-diol (15). To a solution of azide 13 (50.1 mg,
1
2938, 2103 (N3), 1693 (CvO), 1403, 1210, 1026, 697. H NMR 0.145 mmol) in toluene (3 mL), phenylacetylene (19 μL,
(300 MHz, DMSO-d6, 363 K, δ ppm) δ 7.38–7.30 (m, 5H, 0.17 mmol), DIPEA (97 μL, 0.56 mmol) and CuI (7 mg,
2
H-aromat.), 5.14 (d, 1H, JH,H = 12.5, CH2 of Cbz), 5.08 (d, 1H, 0.037 mmol) were added. The mixture was stirred for 18 h at
CH2 of Cbz), 4.68 (t, 1H, J4,3 = J4,5 = 6.2, H-4), 4.56 (dd, 1H, r.t. Then, the mixture was diluted with EtOAc and washed with
J3,2 = 1.0, H-3), 4.01–3.98 (m, 1H, H-2), 3.90 (q, 1H, J5,Me = 6.4, sat. aq. sol. of NaHCO3. The organic layer was dried over
2
H-5), 3.66 (dd, 1H, J1′a,1′b = 12.8, J1′a,2 = 6.0, H-1′a), 3.53 (dd, Na2SO4, filtered and evaporated. The resulting residue was
1H, J1′b,2 = 3.5, H-1′b), 1.40, 1.30 (2 s, 3H each, –C(CH3)2,) 1.28 purified by column chromatography on silica gel (toluene–
(d, 3H, Me). 13C NMR (75.4 MHz, DMSO-d6, 363 K, δ ppm) δ acetone, 10 : 1), affording the corresponding protected triazole
153.6 (CvO of Cbz), 136.2, 127.8, 127.4, 127.2 (C-aromat.), derivative (47.8 mg, 0.107 mmol, 74%). A solution of this com-
110.4 (–C(CH3)2), 80.5 (C-3), 79.4 (C-4), 65.8 (CH2 of Cbz), 61.8 pound (40.9 mg, 0.091 mmol) in HCl (1 M)–THF (1 : 1, 3.5 mL)
(C-2), 56.6 (C-5), 50.5 (C-1′), 25.4, 24.4 (–C(CH3)2), 14.6 (Me). was stirred for 24 h, then the mixture was evaporated. The
CIMS m/z 347 [2%, (M + H)+], 290 [26%, (M − CH2N3)+]. resulting crude was purified by chromatography column
HRCIMS m/z found 347.1727, calc. for C17H23N4O4: 347.1719.
(EtOAc–Hex, 1 : 1) to give pure diol (85%). The 3,4-O-un-
(2S,3S,4R,5S)-2-Ethynyl-5-methylpyrrolidine-3,4-diol
(14). protected pyrrolidine (28 mg, 0.069 mmol) was dissolved in
Compound 11 (44.7 mg, 0.16 mmol) was dissolved in TFA– MeOH–HCl (1 M) (1 : 1, 3 mL), a catalytic amount of Pd/C
H2O 4 : 1 (0.5 mL) and the mixture was stirred at r.t. for 2.5 h. (10%) was added and the resulting mixture was hydrogenated
Evaporation of the solvent and chromatographic purification under atmospheric pressure for 2 h. The catalyst was filtered
on Dowex 50WX8 eluting with MeOH (50 mL), H2O (50 mL) over celite and the filtered solution was evaporated. The result-
and NH4OH 10%, afforded 14 (19.2 mg, 0.14 mmol, 87%) as a ing residue was purified by chromatography column on silica
yellow solid. [α]D26 −10.6 (c 0.83, MeOH); IR (ν cm−1) 3306 (OH, gel (DCM–MeOH–NH4OH, 5 : 1 : 0.05) affording 15 (10.4 mg,
NH), 1662, 1454, 1190, 1134, 844, 799, 724. 1H NMR (300 MHz, 0.038 mmol, 55%). [α]2D8 −24.2 (c 0.72, MeOH). IR (ν cm−1
)
MeOD, δ ppm, J Hz) δ 4.17 (dd, 1H, J3,2 = 6.8, J3,4 = 4.4, H-3), 3300 (OH), 1452, 1237, 1119, 764, 692. 1H NMR (500 MHz,
3.91 (t, 1H, J4,5 = 4.4, H-4), 3.77 (dd, 1H, J2,2′ = 2.3, H-2), CD3OD, δ ppm, J Hz) δ 8.37 (s, 1H, H-5″), 7.82–7.80 (m, 2H,
3.37–3.28 (m, 1H, H-5), 2.73 (d, 1H, H-2′), 1.16 (d, 3H, JMe,5
=
H-aromat.), 7.45–7.41 (m, 2H, H-aromat.), 7.36–7.32 (m, 1H,
2
6.7, Me). 13C NMR (75.4 MHz, MeOD, δ ppm) δ 85.3 (C-1′), 81.0 H-aromat.), 4.63 (dd, 1H, J1′a,1′b = 13.9, J1′a,5 = 4.3, H-1′a), 4.48
5902 | Org. Biomol. Chem., 2014, 12, 5898–5904
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(dd, 1H, J1′b,5 = 8.1, H-1′b), 3.99 (dd, 1H, J4,5 = 8.1, J4,3 = 4.1, 405 nm. Under these conditions, the p-nitrophenolate released
H-4), 3.81 (br. dd, 1H, J3,2 = 3.0, H-3), 3.64 (td, 1H, H-5), 3.18 led to optical densities linear with both reaction time and con-
(qd, 1H, JMe,2 = 6.7, H-2), 1.16 (d, 1H, Me). 13C NMR centration of the enzyme. Blank experiments with sodium
(125.7 MHz, CD3OD, δ ppm) δ 148.8 (C-4″), 131.8, 130.0, 129.3, ascorbate/CuSO4 and with each of the azides a–u were also
126.7 (C-aromat.), 123.0 (C-5″), 77.6 (C-4), 75.1 (C-3), 62.8 (C-5), carried out. An aqueous solution of sodium ascorbate
56.7 (C-2), 54.6 (C-1′), 14.4 (Me). HRCIMS m/z found 275.1508, (0.66 μM), CuSO4 (1 μM), a solution of CuSO4–sodium ascor-
calc. for C14H19O2N4: 275.1508.
bate (0.5 μM and 0.33 μM, respectively) and a solution of each
(2S,3S,4R,5S)-2-[1-((4-Benzyloxycarbonyl-5-methylfuran-2-yl)- azide (1.8 μM) were prepared in different wells. Each solution
methyl)-1H-1,2,3-triazol-4-yl]-5-methylpyrrolidine-3,4-diol was assayed towards α-fucosidase as described above and no
(14p). A solution of alkyne 14 (13.9 mg, 0.098 mmol) and inhibition was observed for all cases. For the highest inhi-
azide p (32.1 mg, 0.118 mmol) in a 2 : 1 mixture of tBuOH–H2O bition rate (triazole 14p) and for compounds 14, 14a and 15,
(3.3 mL) was treated with a catalytic amount of CuSO4 the IC50 value (concentration of inhibitor required for 50%
(0.54 mg, 3.37 × 10−3 mmol) followed by sodium ascorbate inhibition of the enzyme activity) and Ki towards α-fucosidase
(2 mg, 0.011 mmol). After 24 h at r.t., the mixture was concen- were calculated. IC50 values were calculated from plots of per-
trated under reduced pressure. The resulting residue was puri- centage of inhibition versus inhibitor concentration and the Ki
fied by chromatography column on silica gel (CH2Cl2–MeOH, values were determined from the Lineweaver–Burk plots (see
8 : 1) to give 14p (22 mg, 0.053 mmol, 54%) as a yellow solid. ESI† for details). All the experiments were performed by dupli-
[α]2D2 −33.2 (c 0.52, MeOH); IR (ν cm−1) 3346 (OH, NH), 2912, cate. In the case of the other ten enzymes, % of inhibition was
1711, 1218, 1076, 770, 623. 1H NMR (500 MHz, DMSO-d6, determined at 1 mM of inhibitor on the well with the appropri-
363 K, δ ppm, J Hz) δ 7.90 (s, 1H, H-5′), 7.41–6.76 (m, 5H, ate p-nitrophenyl glycosides as substrates. Commercially avail-
H-aromat.), 6.76 (s, 1H, H-3′′′), 5.56 (s, 2H, H-1″), 5.28 (s, 2H, able glycosidases from Sigma–Aldrich: β-galactosidase (EC
–CH2Ph), 4.13–4.12 (m, 2H, H-2, H-3), 3.83 (t, 1H, J4,3 = J4,5
=
3.2.1.23) from Aspergillus oryzae and from Escherichia coli,
4.0, H-4), 3.33–3.31 (m, 1H, H-5), 2.52 (s, 3H, Me), 1.07 (d, 3H, α-mannosidase (EC 3.2.1.24) from Jack beans, β-N-acetylglucos-
JMe,5 = 6.5, Me). 13C NMR (125.7 MHz, DMSO-d6, 363 K, δ ppm) aminidase (EC 3.2.1.30) from Jack beans, α-galactosidase (EC
162.1 (CvO), 158.8, 149.3, 146.8, 135.8, 127.9, 127.5, 127.3, 3.2.1.22) from coffee beans, amyloglucosidase (EC 3.2.1.3)
113.4 (C-aromat.), 121.3 (C-5′), 109.8 (C-3′′′), 78.1, 57.7 (C-2, from Aspergillus niger, α-glucosidase (EC 3.2.1.20) from rice,
C-3), 72.8 (C-4), 65.0 (–CH2Ph), 54.7 (C-5), 45.0 (C-1″), 14.7 α-glucosidase (EC 3.2.1.20) from Saccharomyces cerevisiae,
(Me), 12.9 (Me). HRCIMS m/z found 413.1822, calc. for β-glucosidase (EC 3.2.1.21) from almonds and β-mannosidase
C21H25O5N4: 413.1825.
(EC 3.2.1.25) from snail.
In situ screening towards α-fucosidase. Glycosidase inhibition
assays
t
Acknowledgements
To a solution of alkyne 14 (0.3 mL, 30 mM in BuOH–H2O
(2 : 1)) in an eppendorf, a solution of the corresponding azide
(a–u) was added (0.1 mL, 108 mM in tBuOH) followed by 25 μL
of an aqueous solution of sodium ascorbate (40 mM) and
25 μL of an aqueous solution of CuSO4 (12 mM). The final con-
centration of the alkyne in each eppendorf was 20 mM. The
resulting mixtures were left at room temperature for 24 h (the
reactions were monitored for completion by TLC). Then,
the reactions were diluted to the desired concentration in order
to use them in the enzymatic assays. In the preliminary screening
of the resulting crude (pyrrolidin-2-yl)triazoles, % of inhibition
towards α-fucosidase from bovine kidney (EC 3.2.1.51) was
determined in the presence of 0.5 µM of the inhibitor on the
well (concentration corresponding to the IC50 of the alkyne 14)
with p-nitrophenyl α-L-fucopyranoside (Sigma–Aldrich) as the
substrate. Each enzymatic assay (final volume 0.12 mL) con-
tained 0.01 to 0.5 unit mL−1 of the enzyme and 10 mM aqueous
solution of the appropriate p-nitrophenyl glycoside substrate
buffered to the optimal pH of the enzyme. The enzyme and
inhibitor were preincubated for 5 min at r.t., and the reaction
started by the addition of the substrate. After 20 min of incu-
bation at 37 °C, the reaction was stopped by the addition of
0.1 mL of sodium borate buffer (pH 9.8). The p-nitrophenolate
formed was measured by visible absorption spectroscopy at
This work was supported by the Ministerio de Economía y
Competitividad of Spain (CTQ2012-31247), the European
Community’s Seventh Framework Program FP7-2007-2013
(grant HEALTHF2-2011-256986) and the Junta de Andalucía
(FQM-345). P. E.-R. acknowledges the Spanish government
for a FPU fellowship. We thank A. Sghiouri for technical
assistance.
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Organic & Biomolecular Chemistry
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