4-C-Me-DAB and 4-C-Me-LAB - Enantiomeric alkyl-branched pyrrolidine iminosugars - Are specific and potent α-glucosidase inhibitors; Acetone as the sole protecting group

Article abstract of DOI:10.1016/j.tetlet.2010.10.173

The syntheses of 4-C-Me-DAB [1,4-dideoxy-1,4-imino-4-C-methyl-d-arabinitol] from l-erythronolactone and of 4-C-Me-LAB [from d-erythronolactone] require only a single acetonide protecting group. The effect of pH on the NMR spectra of 4-C-Me-DAB [pK_a

Full text of DOI:10.1016/j.tetlet.2010.10.173

Tetrahedron Letters 52 (2011) 219–223
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
4-C-Me-DAB and 4-C-Me-LAB—enantiomeric alkyl-branched pyrrolidine
iminosugars—are specific and potent
the sole protecting group
a-glucosidase inhibitors; acetone as
Filipa P. da Cruz a, Scott Newberry a, Sarah F. Jenkinson a, Mark R. Wormald b, Terry D. Butters b,
Dominic S. Alonzi b, Shinpei Nakagawa c, Frederic Becq d, Caroline Norez d, Robert J. Nash e,
Atsushi Kato c, George W. J. Fleet a,
⇑
a Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
b Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
c Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
d Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS, 40 avenue du Recteur Pineau, 86022 Poitiers, France
e Phytoquest Limited, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, Wales, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The syntheses of 4-C-Me-DAB [1,4-dideoxy-1,4-imino-4-C-methyl-D-arabinitol] from L-erythronolactone
Received 29 September 2010
Revised 14 October 2010
Accepted 29 October 2010
Available online 11 November 2010
and of 4-C-Me-LAB [from D-erythronolactone] require only a single acetonide protecting group. The effect
of pH on the NMR spectra of 4-C-Me-DAB [pKa of the salt around 8.4] is discussed and illustrates the need
for care in the analysis of both coupling constants and chemical shift. 4-C-Me-DAB (for rat intestinal
sucrase Ki 0.89
0.95 M, IC50 0.66
for the -glucosidase inhibition by DAB, LAB, 4-C-Me-DAB, 4-C-Me-LAB and isoDAB—but not isoLAB—is
provided. Both are inhibitors of endoplasmic reticulum (ER) resident -glucosidase I and II.
l
M, IC50 0.41
l
M) is a competitive—whereas 4-C-Me-LAB (for rat intestinal sucrase Ki
l
lM) is a non-competitive—specific and potent
a
-glucosidase inhibitor. A rationale
a
a
Ó 2010 Elsevier Ltd. All rights reserved.
This letter describes the synthesis of the enantiomers 4-C-Me-
cystic fibrosis.8 N-Nalkylation of monocyclic iminosugars can en-
hance glycosidase inhibition by several orders of magnitude;9 such
modification of the alkyl-branched parent structures may access a
series of new bioactive compounds.
DAB 1D [1,4-dideoxy-1,4-imino-4-C-methyl-
C-Me-LAB 1L with only a single acetonide needed as a protecting
group, both of which are micromolar inhibitors of some -glucosi-
dases; in accord with Asano’s hypothesis,1 the
-iminosugar 1D is a
D-arabinitol] and 4-
a
D
The enantiomeric azidolactones 4L and 4D are key intermedi-
ates in the synthesis of 4-C-methyl pyrrolidines 1L and 1D
[Scheme 1]. The protected 2-C-methyl arabinono-lactone 5D can
be prepared by a Kiliani reaction10 on a protected 1-deoxyribu-
competitive inhibitor, whereas the enantiomer 1L is a non-compet-
itive inhibitor. Synthetic enantiomers of natural iminosugars are
frequently powerful glycosidase inhibitors.2 Natural and synthetic
iminosugars comprise a major family of glycosidase inhibitors.3
The introduction of an alkyl substituent into a sugar mimic usually
removes any significant glycosidase inhibition,4 however, intro-
lose11 derived from
D-erythronolactone 6D; 5D is a useful chiron
for the preparation of 20-C-methyl nucleosides12 and carbon-
branched ketoses.13 Reaction of 5D with triflic anhydride, followed
by treatment with sodium azide, results in nucleophilic displace-
ment at a tertiary centre with inversion to give the azidolactone
duction of a C6 methyl branch into the piperidine ring of
onine increases the inhibition of naringinase by an order of
magnitude5 in comparison to the parent indolizidine,
-swainso-
nine.6 The natural product DAB 3D is a good—but the enantiomer
LAB 3L is a more potent and more specific—inhibitor of -glucosi-
dases.7 The isomer isoDAB 2D is also a very good inhibitor of
-glu-
L-swains-
L
4D.14 The enantiomeric azido-2-C-methyl-
was prepared from
L
-arabinonolactone 4L
L
-erythronolactone 6L by similar procedures.
a
For the synthesis of 4-C-Me-DAB 1D, it was necessary to invert
the configuration at C4 of the azidolactone 4L [Scheme 2]. Hydro-
lysis of the isopropylidene protecting group in the 1,5-lactone 4L
by trifluoroacetic acid in aqueous 1,4-dioxane gave the 1,4-lactone
a
cosidases but the enantiomer 2L shows no inhibition of any
glycosidase. This letter provides a rationale for isoLAB 2L being
only one of the six simple pyrrolidines 1, 2 and 3 which does not
7L, {mp 76–78 °C; ½a D25
ꢁ132.9 (c, 1.06 in CH3OH)} in 87% yield.
ꢀ
inhibit
a-glucosidases; isoLAB 2L is the only one of the sugar mim-
Selective esterification of the primary hydroxy group in 7L with to-
syl chloride in pyridine afforded the tosylate 8L {mp 110–112 °C;
ics which partially rescues the defective F508del-CFTR function in
½
a 2D1
ꢀ
ꢁ107.7 (c, 1.07 in CH3CN)} in 57% yield. Reaction of
L-ribono-
lactone 8L with potassium hydroxide in aqueous 1,4-dioxane,
⇑
Corresponding author.
E-mail address: george.fleet@chem.ox.ac.uk (G.W.J. Fleet).
followed by treatment with an acidic ion exchange resin, resulted
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.10.173

220
F. P. da Cruz et al. / Tetrahedron Letters 52 (2011) 219–223
H
N
H
N
H
N
H
N
H
N
H
N
HOH2C
Me
HOH2C
HOH2C
HOH2C
Me
HOH2C
HO
HOH2C
HO
OH
OH
2D isoDAB
HO
OH
HO
3L LAB
L-sugars in red
OH
HO
OH
HO
OH
1D 4-C-Me-DAB
3D DAB
D-sugars in blue
2L isoLAB
1L 4-C-Me-LAB
O
O
O
O
O
O
O
O
O
O
O
O
N3
Me
Me
HO
Me
N3
O
O
O
O
O
HO
Me
HO
OH
HO
OH
O
O
O
4L
5L
6L
6D
5D
4D
Scheme 1. Structural relationship and synthesis of 4-C-methyl branched pyrrolidines.
OTs
O
H
O
O
O
HO
O
CH2OR
(vi)
O
CH2OR
N3 OH
7L R = H
HOH2C
Me
HO
(i)
HO
Me
4
N
4
4
N3
Me
Me
Me
O
N3 OH
N3 OH
11D
O
OH
(iii)
(v)
9D R = H
4L
1D
(ii)
(iv)
8L R = OTs
10D R = OTs
Scheme 2. Reagents and conditions: (i) CF3COOH/H2O/1,4-dioxane, 4:1:1, 87%; (ii) TsCl, pyridine, 57%; (iii) KOH, 1,4-dioxane; then Dowex, 87%; (iv) TsCl, pyridine, 66%; (v)
NaBH4, MeOH, 66%; (vi) H2, Pd/C, 1,4-dioxane: H2O (2:1), 100%.
in inversion of configuration at C4 to form the
D
-lyxonolactone 9D
{oil; ½a 2D1
ꢀ
+48.4 (c, 0.74 in MeOH)} in 87% yield. Esterification of 9D
with tosyl chloride in pyridine yielded the primary tosylate 10D
{oil; ½a 2D0
ꢀ
+13.2 (c, 0.85 in CH3CN)} (66% yield), which on reduction
with sodium borohydride in methanol formed the azidotriol 11D,
(oil, 66% yield); it was thus possible to reduce the hindered lactone
10D to the triol 11D without any competing formation of epoxide.
Hydrogenation of the unprotected azide 11D in the presence of
palladium on carbon in 1,4-dioxane gave the corresponding amine
which spontaneously cyclised to form 4-C-Me-DAB 1D, in quanti-
tative yield. The tosylate salt of 1D could be converted into the free
base 1D by ion exchange chromatography with Dowex 1-X2
(OHꢁ form) with water as eluent {free base: oil, ½a 2D5
ꢁ20.1 (c,
ꢀ
0.57 in H2O); hydrochloride salt: ½a D25
ꢁ5.22 (c, 1.07 in H2O)}.
ꢀ
The enantiomer 4-C-Me-LAB 1L was prepared by an identical route
from
D
-erythronolactone 6D {free base: oil, ½a D25
ꢀ
+21.3 (c, 0.71 in
H2O); hydrochloride salt: ½a D25
ꢀ
+4.10 (c, 1.0 in H2O)}.15 It is note-
worthy that only one isopropylidene group is used throughout this
sequence.
Since the pKa of the salts of imino sugars is around 7–8, the 1H
NMR spectrum is strongly dependent on the pH. This is illustrated
for 4-C-Me-DAB 1D at six different pH values with CH3CN as the
internal standard [Fig. 1]. The pKa of the hydrochloride salt of 1D
was determined as 8.38 from the change in the chemical shift of
the C-methyl group with pH [Fig. 2]. The full resonance assign-
ments of the salt at pH 2.4 and of the free base at pH 11.1 of 1D
are given in Tables 1 and 2, respectively.
LAB 3L is a more potent and specific inhibitor of
than its naturally occurring enantiomer DAB 3D [Table 3],16 whilst
isoDAB 2D is a more potent inhibitor of -glucosidases than DAB
a-glucosidases
a
3D and is completely specific; in contrast, isoLAB 2L does not show
any inhibition of these enzymes.8 4-C-Me-DAB 1D and 4-C-Me-LAB
1L were both found to be specific and potent (
glucosidases. In particular, these compounds showed strong inhibi-
tion of rat intestinal sucrase with IC50 values of 0.41 and 0.66 M,
lM) inhibitors of a-
l
respectively. The kinetic analysis showed that 4-C-Me-DAB 1D is a
competitive inhibitor of rat intestinal sucrase with a Ki value of
Figure 1. 1H NMR (500 MHz) of 4-C-Me-DAB 1D in D2O at different pHs.
0.89
l
M, whereas the enantiomer 4-C-Me-LAB 1L is a non-compet-
M) of the enzyme. Although DAB
3D has been reported to be a good inhibitor of glycogen phosphor-
ylase and has been investigated as a potential therapeutic agent for
itive inhibitor (Ki value of 0.95
l

F. P. da Cruz et al. / Tetrahedron Letters 52 (2011) 219–223
221
layed with glucose in the same way as 3D, with the inhibitor
methyl group in the position of the glucose C5 axial proton
[Fig. 3(b)]. IsoDAB 2D could be overlayed with glucose in the same
way, with the inhibitor CH2OH group in the position of the glucose
C4 axial proton [Fig. 3(c)]. This CH2OH group in 2D might be able to
interact with groups which normally bind to the glucose C6 OH,
depending on the glucose C5–C6 torsion angle in the bound form.
In contrast, the best match between LAB 3L and glucose places the
C2 and C3 OHs of 3L overlaying the C2 and C3 OHs of glucose,
respectively [Fig. 3(d)]. This placed the CH2OH group of 3L close
to the C4 OH of glucose and the ring nitrogen of 3L close to the ring
oxygen of glucose. Again, in this orientation 3L mimicked three of
the glucose OH groups. 4-C-Methyl-LAB 1L was overlayed with
glucose in the same way as 3L, with the methyl group of 1L in
the position of the glucose C4 axial proton [Fig. 3(e)]. If the inactive
isoLAB 2L is overlayed in the same manner as 3L, then the CH2OH
group in 2L would be in the position of the glucose C3 axial proton,
Figure 2. Chemical shift of the methyl peak in 1D versus pH. The points are the
experimental values; the line is the fitted curve, pKa of 8.38.
on the C2OH/C4OH face of glucose. For a terminal a-linked glucose,
Table 1
NMR [1H (500 MHz) 13C (125 MHz) D2O] assignments of the HCl salt of 1D, pH 2.4
the rest of the glycan was on the C2OH/C4OH face of the glucose
residue (indicated by the large spheres in Fig. 3). Alternatively,
2L could be overlayed with the C2 and C3 OHs of 2L overlaying
the C3 and C2 OHs of glucose, respectively, with the CH2OH group
in 2L in the position of the glucose C2 axial proton, on the opposite
face [Fig. 3(f)]. In either case, the inhibitor could only mimic two of
the four glucose OH groups.
There are several possible explanations for the absence of activ-
ity of isoLAB 2L compared to the other five pyrrolidines, including:
(i) the inhibitor must mimic at least three of the glucose hydroxy
groups for effective binding, or (ii) a hydroxy group near the posi-
tion of the glucose C4 hydroxy is required for binding, or (iii) an
additional axial group at the positions of C2 or C3 in glucose cannot
be tolerated, whereas it can at positions C4 or C5. The differential
Label
pH 2.24
1H
13C
d (ppm)
d (ppm)
mult
3JHH (Hz)
C1
3.664
3.202
4.401
4.014
—
3.797
3.688
1.332
dd
dd
ddd
d
—
d
12.7/6.8
12.7/5.0
6.8/5.0/5.0
5.0
—
12.3
47.83
C2
C3
C4
C5
74.26
77.36
69.48
63.75
d
s
12.3
na
CH3
15.12
Table 2
NMR [1H (500 MHz) 13C (125 MHz) D2O] assignments of the free base of 1D, pH 11.1
inhibition of different
a-glucosidases by the various pyrrolidines
Label
pH 11.1
1H
13C
showed that the remainder of the substrates can significantly af-
fect the potency of inhibition. Whatever the explanation, it is
remarkable to find five such structurally simple pyrrolidines with
d (ppm)
d (ppm)
mult
3JHH (Hz)
C1
3.170
2.648
4.181
3.801
—
3.485
3.443
1.010
dd
dd
ddd
d
—
d
12.1/7.3
12.1/6.2
7.3/6.2/6.0
6.0
—
11.6
48.39
potent and specific
The effect of 1D and 1L on cellular endoplasmic reticulum (ER)
resident -glucosidase I and II activity in cells was studied using a
a-glucosidase inhibition.
C2
C3
C4
C5
77.61
80.67
63.41
66.98
a
free oligosaccharide assay.19 HL-60 cells were treated at concentra-
tions of 1D and 1L as indicated for 24 h [Fig. 4] and were compared
with the effect of 1-deoxynojirimycin (DNJ); free oligosaccharides
(FOS) were isolated from cells, fluorescently labelled, and followed
by separation using NP-HPLC. Peak areas for Glc1Man4GlcNAc1 and
Man4GlcNAc1 (non-glucosylated control species) were determined
and expressed as a ratio. The presence of Glc1Man4GlcNAc1 indi-
d
s
11.6
na
CH3
17.60
the treatment of diabetes,17 LAB 3L, isoDAB 2D, isoLAB 2L, 4-C-Me-
DAB 1D and 4-C-Me-LAB 1L showed no significant inhibition of this
enzyme.
cated inhibition of a-glucosidase II; at 500 lM DNJ is five times a
more potent inhibitor than 1L and forty times more potent than
In order to rationalise the glucosidase inhibition results, it was
assumed that these inhibitors mimic the glucoside substrate. The
six pyrrolidines were overlayed with glucose to: (i) position the
ring nitrogen close to the ring oxygen of glucose so that, in the pro-
tonated form, the positive charge can mimic the partial positive
charge of the transition state during hydrolysis, and (ii) match as
many hydroxy positions as possible, to retain any specific binding
interactions in the active sites.18 The different glucosidases will
recognise the terminal glucose residue of the substrates in differ-
ent ways. Thus it is not surprising that these inhibitors show
slightly different patterns of inhibition for the different glucosi-
dases, however, some general principles can be suggested.
The best match between DAB 3D and glucose placed the C2 and
C3 hydroxy (OH) groups of 3D to overlay the C3 and C4 OHs of glu-
cose, respectively [Fig. 3(a)]. This positioned the CH2OH group of
3D close to the C6 OH of glucose and the ring nitrogen of 3D close
to the ring oxygen of glucose. In this orientation the inhibitor mim-
icked three of the glucose OH groups. 4-C-Me-DAB 1D was over-
1D. The decline in Glc1Man4GlcNAc1 at concentrations of DNJ high-
er than 500 lM is due to inhibition of a-glucosidase I and, there-
fore, production of Glc3Man5GlcNAc1 species at the expense of
Glc1Man4GlcNAc1 species. In the presence of 2.5 mM branched
imino sugars, cells do not produce any Glc3Man5GlcNAc1 indicating
that compounds are either unable to inhibit this enzyme, that is,
are specific
concentrations have been reached to inhibit the
step in the pathway. In summary, both 4-C-methyl branched imino
sugars of 1D and 1L are weak inhibitors of -glucosidase II in cells;
a
-glucosidase II inhibitors, or most likely, insufficient
a-glucosidase I
a
this is in contrast to the behaviour of the hydroxymethyl branched
pyrrolidines 2D and 2L, neither of which showed any inhibition of
ER glucosidases.8 Both DAB 3D and LAB 3L are presumed inhibitors
of processing glucosidases; their N-butyl analogues are weak
inhibitors (IC50, 319 lM and 769 lM, respectively) of a-glucosi-
dase I using an in vitro assay and are consequently ineffective at
inhibiting glucosidase activity in cellular assays at concentrations
of 1 mM or less.20

222
F. P. da Cruz et al. / Tetrahedron Letters 52 (2011) 219–223
Table 3
Concentration of iminosugars giving 50% inhibition of various glycosidases and glycogen phosphorylase
Enzyme IC50
(l
M)
HOH2C
H
N
H
N
H
N
H
N
H
N
H
N
HOH2C
HOH2C
Me
HOH2C
Me
HOH2C
HO
HOH2C
HO
HO
OH
OH
OH
HO
OH
HO
OH
HO
OH
DAB 3D
isoDAB 2D
4-C-Me-DAB 1D
LAB 3L
isoLAB 2L
4-C-Me-LAB 1L
a
-Glucosidase
Rice
Yeast
250
0.15
55
5.8
16
41
NI
24
20
15
7.1
1.9
0.74
3.4
0.41
3.2
70
0.93
0.36
1.0
NI
NI
NI
NI
NI
5.8
NI
2.4
5.1
0.66
Rat intestinal maltase
Rat intestinal isomaltase
Rat intestinal sucrase
b-Glucosidase
Almond
Bovine liver
250
638
756
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Rat intestinal cellobiase
a
-Galactosidase
Coffee beans
Human lysosom
NIa
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
b-Galactosidase
Bovine liver
Rat intestinal lactase
NI
323
NI
NI
NI
NI
NI
415
NI
NI
NI
NI
a
-Mannosidase
Jack beans
320
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
38
NI
NI
NI
NI
NI
75
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
a
-Mannosidase
Snail
a
-
L
-Rhamnosidase
P. decumbens
NI
a-L-Fucosidase
Bovine epididymis
NI
Trehalase
Rat instestinal trehalase
61
Glycogen phosphorylase
Rabbit muscle
0.33
a
NI: No inhibition (less than 50% inhibition at 1000 lM).
Figure 3. Overlay of the six inhibitors, (a) DAB 3D, (b) 4-C-Me-DAB 1D, (c) isoDAB 2D, (d) LAB 3L, (e) 4-C-Me-LAB 1L and (f) isoLAB 2L, with an
a-glucoside. The glucose
residues are in grey, with the oxygens in slightly darker grey, and the inhibitors are in colour. Only the hydroxy protons are shown, for clarity. The large sphere shows the
position of the rest of the glycan.
N-Butyl-DNJ21 [an
a
-glucosidase inhibitor] and isoLAB 2L8
cue of the defective F508del-CFTR function and thus might have po-
[which has no -glucosidase inhibition] both show significant res-
a
tential in the treatment of cystic fibrosis.22 Neither of the 4-C-methyl

F. P. da Cruz et al. / Tetrahedron Letters 52 (2011) 219–223
223
5. Hakansson, A. E.; van Ameijde, J.; Horne, G.; Nash, R. J.; Wormald, M. R.; Kato,
A.; Besra, G. S.; Gurcha, S.; Fleet, G. W. J. Tetrahedron Lett. 2008, 49, 179–184.
6. (a) Hakansson, A. E.; van Ameijde, J.; Guglielmini, L.; Horne, G.; Nash, R. J.;
Evinson, E. L.; Kato, A.; Fleet, G. W. J. Tetrahedron: Asymmetry 2007, 18, 282–
289; (b) Davis, B.; Bell, A. A.; Nash, R. J.; Watson, A. A.; Griffiths, R. C.; Jones, M.
G.; Smith, C.; Fleet, G. W. J. Tetrahedron Lett. 1996, 37, 8565–8568; (c) Bell, A. A.;
Pickering, L.; Watson, A. A.; Nash, R. J.; Griffiths, R. C.; Jones, M. G.; Fleet, G. W. J.
Tetrahedron Lett. 1996, 37, 8561–8564.
7. (a) Fleet, G. W. J.; Nicholas, S. J.; Smith, P. W.; Evans, S. V.; Fellows, L. E.; Nash, R.
J. Tetrahedron Lett. 1985, 26, 3127–3130; (b) Scofield, A. M.; Fellows, L. E.; Nash,
R. J.; Fleet, G. W. J. Life Sci. 1986, 39, 645–650; (c) Fleet, G. W. J.; Smith, P. W.
Tetrahedron 1986, 42, 5685–5692; (d) Behling, J. R.; Campbell, A. L.; Babiak, K.
A.; Ng, J. S.; Medich, J.; Farid, P.; Fleet, G. W. J. Tetrahedron 1993, 49, 3359–3366.
8. Best, D.; Jenkinson, S. F.; Saville, A. W.; Alonzi, D. S.; Wormald, M. R.; Butters, T.
D.; Norez, C.; Becq, F.; Bleriot, Y.; Adachi, I.; Kato, A.; Fleet, G. W. J. Tetrahedron
Lett. 2010, 51, 4170–4174.
9. (a) Chang, C. F.; Ho, C. W.; Wu, C. Y.; Chao, T. A.; Wong, C. H.; Lin, C. H. Chem.
Biol. 2004, 11, 1301–1306; (b) Wu, C. Y.; Chang, C. F.; Chen, J. S. Y.; Wong, C. H.;
Lin, C. H. Angew. Chem., Int. Ed. 2003, 42, 4661–4664; (c) Ho, C.-W.; Popat, S. D.;
Liu, T. A.; Tsai, K.-C.; Ho, M. J.; Chen, W.-H.; Yang, A.-S.; Lin, C.-H. ACS Chem. Biol.
2010, 5, 489–497; (d) Rawlings, A. J.; Lomas, H.; Pilling, A. W.; Lee, M. J.-R.;
Alonzi, D. S.; Rountree, J. S. S.; Jenkinson, S. F.; Fleet, G. W. J.; Dwek, R. A.; Jones,
J. H.; Butters, T. D. ChemBioChem 2009, 10, 1101–1105.
4-C-Me-LAB 1L
4-C-Me-DAB 1D
DNJ
9
8
7
6
5
4
3
2
1
0
0
500
1000
1500
2000
2500
µ
Concentration ( M)
10. (a) Hotchkiss, D. J.; Soengas, R.; Simone, M. I.; van Ameijde, J.; Hunter, S.;
Cowley, A. R.; Fleet, G. W. J. Tetrahedron Lett. 2004, 45, 9461–9464; (b) Soengas,
R.; Izumori, K.; Simone, M. I.; Watkin, D. J.; Skytte, U. P.; Soetaert, W.; Fleet, G.
W. J. Tetrahedron Lett. 2005, 46, 5755–5759.
Figure 4. Glc1Man4GlcNAc1 following
a-glucosidase inhibition in FOS assay.
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Tetrahedron Lett. 2006, 47, 315–318; (b) Booth, K. V.; da Cruz, F. P.; Hotchkiss,
D. J.; Jenkinson, S. F.; Jones, N. A.; Weymouth-Wilson, A. C.; Clarkson, R.; Heinz,
T.; Fleet, G. W. J. Tetrahedron: Asymmetry 2008, 19, 2417–2424.
12. Jenkinson, S. F.; Jones, N. A.; Moussa, A.; Stewart, A. J.; Heinz, T.; Fleet, G. W. J.
Tetrahedron Lett. 2007, 48, 4441–4445.
13. Rao, D.; Yoshihara, A.; Gullapalli, P.; Morimoto, K.; Takata, G.; da Cruz, F. P.;
Jenkinson, S. F.; Wormald, M. R.; Dwek, R. A.; Fleet, G. W. J.; Izumori, K.
Tetrahedron Lett. 2008, 49, 3316–3321.
pyrrolidines 1D and 1L had any corrector effect on CFTR function in
CF-KM4 cells,23 as assessed by single-cell fluorescence imaging;24
thus, isoLAB 2L—the only one of the DAB analogues that is not an
a-glucosidase inhibitor—is the sole pyrrolidine to have chloride
channel rescue properties.
In summary, only a single acetonide protecting group is used in
the synthesis of the first examples of potent glycosidase inhibitors
by C-alkyl branching of the carbon chain of iminosugars. Of the
three pairs of enantiomers 1, 2 and 3, five structurally simple pyr-
14. da Cruz, F. P.; Horne, G.; Fleet, G. W. J. Tetrahedron Lett. 2008, 49, 6812–6815.
15. Data for free base 1D: ½a D25
ꢁ20.1 (c 0.57, H2O); dH (400 MHz, D2O) 1.01 (3H, s,
0 0 0
ꢀ
CH3), 2.65 (1H, dd, H1, J1,1 12.1, J1,2 6.2), 3.17 (1H, dd, H10, J1 ,1 12.1, J1 ,2 7.3),
3.44 (1H, d, H5, J5,5 11.6), 3.49 (1H, d, H50, J5 ,5 11.6), 3.80 (1H, d, H3, J3,4 6.0),
0
0
rolidines are potent (lM) and specific a-glucosidase inhibitors;
4.18 (1H, ddd, H2, J2,1 7.3, J2,1 6.2, J2,3 6.0); dC (100.6 MHz, D2O) 17.6 (C40), 48.4
0
(C1), 63.4 (C4), 67.0 (C5), 77.6 (C2), 80.7 (C3). Data for HCl salt of 1D: ½a D25
ꢀ
isoLAB 2L does not inhibit any glycosidase but is the only one to
exhibit significant rescue of the defective F508del-CFTR function.
Only DAB 3D shows any inhibition of glycogen phosphorylase.
ꢁ5.22 (c 1.07, H2O); mmax (thin film, Ge): 3356 (br s, OH, NH); dH (400 MHz,
D2O) 1.33 (3H, s, CH3), 3.20 (1H, dd, H1, J1,1 12.7, J1,2 5.0), 3.66 (1H, dd, H10, J1 ,1
0
0
12.7, J1 ,2 6.8), 3.69 (1H, d, H5, J5,5 12.3), 3.80 (1H, d, H50, J5 ,5 12.3), 4.01 (1H, d,
0
0
0
0
H3, J3,4 5.0), 4.40 (1H, ddd, H2, J2,1 6.8, J2,1 5.0, J2,3 5.0); dC (100.6 MHz, D2O)
15.1 (C40), 47.8 (C1), 63.7 (C5), 69.5 (C4), 74.3 (C2), 77.4 (C3); HRMS (FI+) Calcd.
Acknowledgements
for C6H13NO3 [MÅ]: 147.0895. Found: 147.0895.
16. For details of assays, see: (a) Mercer, T. B.; Jenkinson, S. F.; Nash, R. J.; Miyauchi,
S.; Kato, A.; Fleet, G. W. J. Tetrahedron: Asymmetry 2009, 20, 2368–2373; (b)
Best, D.; Wang, C.; Weymouth-Wilson, A. C.; Clarkson, R. A.; Wilson, F. X.; Nash,
R. J.; Miyauchi, S.; Kato, A.; Fleet, G. W. J. Tetrahedron: Asymmetry 2010, 21,
311–319.
17. (a) Andersen, B.; Rassov, A.; Westergaard, N.; Lundgren, K. Biochem. J. 1999,
342, 545–550; (b) Fosgerau, K.; Westergaard, N.; Quistorff, B.; Grunnet, N.;
Kristiansen, M.; Lundgren, K. Arch. Biochem. Biophys. 2000, 15, 274–284; (c)
Minami, Y.; Kuriyama, C.; Ikeda, K.; Kato, A.; Takebayashi, K.; Adachi, I.; Fleet,
G. W. J.; Kettawan, Q.; Okamoto, T.; Asano, N. Bioorg. Med. Chem. 2008, 16,
2734–2740.
Financial support [to F.P.C.] provided by the Fundação para a
Ciência e Tecnologia, Portugal is gratefully acknowledged. Part of
this work was supported by Grant Number R01CA125642 from
the National Cancer Institute (T.D.B., D.S.A.) and by grants from
the French association ‘Vaincre la Mucoviscidose’ C.N., F.B.). We
thank Dextra Laboratories Limited, Reading, UK for generous gifts
of
D-erythronolactone and L-erythronolactone.
References and notes
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derived from
a CF patient homozygous for the F508del mutation, see:
Kammouni, W.; Moreau, B.; Becq, F.; Saleh, R.; Pavirani, A.; Figarella, C.;
Merten, M. D. Am. J. Respir. Cell Mol. Biol. 1999, 20, 684–691.
24. CFTR Clꢁ channel activity was assayed by single-cell fluorescence imaging
using the potential-sensitive probe, bis-(1,3-diethylthiobarbituric acid)
trimethine oxonol. For experimental details, see Ref. 21(a).
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