Synthesis of 1,5-dideoxy-1,5-iminoribitol C-glycosides through a nitrone-olefin cycloaddition domino strategy: Identification of pharmacological chaperones of mutant human lysosomal β-galactosidase

Article abstract of DOI:10.1021/jo500328u

We report herein a newly developed domino reaction that facilitates the synthesis of new 1,5-dideoxy-1,5-iminoribitol iminosugar C-glycosides 7a-e and 8. The key intermediate in this approach is a six-membered cyclic sugar nitrone that is generated in situ and trapped by an alkene dipolarophile via a [2 + 3] cycloaddition reaction to give the corresponding isooxazolidines 10a-e in a one-pot protocol. The iminoribitol C-glycosides 7a-e and 8 were found to be modest β-galactosidase (bGal) inhibitors. However, compounds 7c and 7e showed pharmacological chaperone activity for mutant lysosomal bGal activity and facilitated its recovery in GM1 gangliosidosis patient fibroblasts by 2-6-fold.

Full text of DOI:10.1021/jo500328u

Article
pubs.acs.org/joc
Synthesis of 1,5-Dideoxy-1,5-iminoribitol C‑Glycosides through a
Nitrone−Olefin Cycloaddition Domino Strategy: Identification of
Pharmacological Chaperones of Mutant Human Lysosomal
β‑Galactosidase
Aloysius Siriwardena,*,‡ Dhiraj P. Sonawane,† Omprakash P. Bande,‡ Pramod R. Markad,†
Sayuri Yonekawa,§ Michael B. Tropak,*,§ Sougata Ghosh,⊥ Balu A. Chopade,⊥ Don J. Mahuran,§
and Dilip D. Dhavale*,†
†Department of Chemistry, Garware Research Centre, and ⊥Institute of Bioinformatics and Biotechnology, University of Pune, Pune
411007, India
‡Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources, Universite
80039, France
́
de Picardie Jules Verne, FRE 3517 CNRS, Amiens
§Programme in Genetics and Genome Biology, Sickkids, Toronto, Ontario M5G 1X8, Canada
S
* Supporting Information
ABSTRACT: We report herein a newly developed domino
reaction that facilitates the synthesis of new 1,5-dideoxy-1,5-
iminoribitol iminosugar C-glycosides 7a−e and 8. The key
intermediate in this approach is a six-membered cyclic sugar
nitrone that is generated in situ and trapped by an alkene
dipolarophile via a [2 + 3] cycloaddition reaction to give the
corresponding isooxazolidines 10a−e in a “one-pot” protocol.
The iminoribitol C-glycosides 7a−e and 8 were found to be
modest β-galactosidase (bGal) inhibitors. However, compounds
7c and 7e showed “pharmacological chaperone” activity for
mutant lysosomal bGal activity and facilitated its recovery in GM1
gangliosidosis patient fibroblasts by 2−6-fold.
5a, which are competitive inhibitors of bGal, also function as
INTRODUCTION
■
PCs in a mouse model of GM1 gangliosidosis (featuring the
Natural products such as 1-deoxynojirimycin (DNJ, 1; Figure
1) and its analogues have been found to inhibit various
carbohydrate-processing enzymes such as glycosidases1 and
glycosyltransferases.2 These promising activities have led to
them being developed as potential drugs for the treatment of
various diseases such as viral infections, diabetes, and lysosomal
storage disorders (LSDs). The LSDs result in GM1
gangliosidosis and Gaucher diseases3 due to the 10−20%
reduction of the acid β-galactosidase (bGal) and β-glucocere-
brosidase (GCase) activity, respectively, in the lysosome.4 It has
been demonstrated that the intracellular activity of a given
defective enzyme, leading to an LSD, can be increased using an
appropriate “pharmacological chaperone” (PC).5 Small mole-
cules such as iminosugar motifs continue to attract considerable
interest as potential PCs for the treatment for LSDs. For
example, N-butyl-1-deoxynojirimycin (2) (marketed under the
trade name Zavesca) is used in substrate reduction therapy of
Gaucher disease.5 A number of N-alkylated iminosugars such as
1-deoxygalactonojirimycin (DGJ, 3a) and N-nonyl-DGJ (3b)
are known as PCs able to rescue the intracellular activity of
mutant bGal in GM1 gangliosidosis patient fibroblasts.4a The
N-octyl-4-epi-β-valienamine (4) and a bicyclic DGJ analogue,
R201C point mutation).4a,6
A popular strategy for the design of a potential PC is to take
a natural inhibitor of the compromised enzyme of interest and
make a rational chemical modification to its structure, with a
view to accentuate its inhibitory activity and selectivity toward
its target enzyme. However, the inhibitory potency of an
iminosugar does not always correlate in a simple way with its
performance as a PC, and even rather poor inhibitors of a
particular mutant glycosidase have proved in several instances
to behave as PCs.6b Such findings underline the difficulties in
formulating reliable structure−activity relationships (SARs) for
PCs. Recently, our group as well as others have described
iminosugar C-glycosides 6a−c with interesting PC activities for
mutant GCase.7 In the continuation of our interest in this area,
we were curious to know how compounds analogous to those
described for GCase would behave as PCs toward mutant bGal.
In this respect, we report here a new domino strategy8 for the
synthesis of a small library of iminosugar C-glycosides 7a−e
Received: February 11, 2014
Published: April 15, 2014
© 2014 American Chemical Society
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Scheme 1. Retro-Synthetic Analysis of Iminosugar
C‑Glycosides
and upon being heated at 100 °C, the resulting mixture
afforded the ring-fused isooxazolidine 10a as the major isolable
product in 68% yield (Scheme 2 and Table 1, entry i). We
Scheme 2. Synthesis of Iminosugars 7a−e and 8
Figure 1. Iminosugars and iminosugar C-glycosides 7a−e and 8.
and 8 and a study of their glycosidase inhibition activities as
well as their effect as PCs of mutant bGal in GM1
gangliosidosis patient fibroblasts.
RESULTS AND DISCUSSION
■
Among the various synthetic approaches described to
iminosugars and their analogues,9 several that figure prom-
inently exploit sugar-derived nitrones and five-membered cyclic
sugar nitrone10 intermediates.11 Fewer reports exist however in
which the analogous six-membered nitrones are intermediates
in the syntheses of the corresponding piperidine, indolizidine,
and pyrrolidizine skeletons.12 Indeed, the use of a six-
membered sugar nitrone as a precursor in the synthesis of
iminosugar C-glycosides via a domino sequence,8 to the best of
our knowledge, is not known.
A retrosynthetic analysis (Scheme 1) suggests that the
targeted iminosugar C-glycosides 7 and 8 could be obtained
from the isooxazolidine I upon deprotection of the acetonide
group and N−O bond cleavage. The key intermediate I might
in turn be obtained via an intermolecular 1,3-dipolar cyclo-
addition of an in situ generated six-membered sugar nitrone, II,
and an alkene. We anticipated that a D-ribose-derived
hemiacetal, III, bearing a 5-O-tosyl group as a leaving group
would give the required nitrone II in a single step.13
Furthermore, if conditions could be developed such that the
in situ formed sugar nitrone II were to undergo a [2 + 3]
cycloaddition with a preadded dipolarophile in a “one-pot”
domino pathway, then the need for isolation or purification of
the relatively unstable six-membered ring sugar nitrone
intermediate II would be obviated.
believe that the domino pathway involves first the formation of
a sugar oxime (from D-ribose) followed by its intramolecular N-
alkylation with departure of the O-tosyl group to give the
corresponding six-membered cyclic nitrone, which concom-
itantly undergoes an intermolecular 1,3-dipolar cycloaddition
Table 1. Domino Reaction of 9 with Different Alkenes
11a−e
time (h)/temp
yield
(%)
entry
alkene
11a
solvent
(°C)
product
i
neat
4/100
4/100
8/70
10a
10b
10c
10d
10e
68
73
69
67
66
The required key precursor, 2,3-O-isopropylidene-5-O-tosyl-
α-D-ribofuranose (9), was obtained from D-ribose in two steps
in an overall 86% yield as described earlier.14 In a trial reaction,
precursor 9 was mixed with hydroxylamine hydrochloride and
allyl alcohol (11a) (10 equiv) in the presence of triethylamine,
ii
iii
iv
v
11b
neat
11c (3.5 M)
11d (3.5 M)
11e (3.5 M)
ethanol
ethanol
toluene
10/70
11/80
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with allyl alcohol present to give the expected isooxazolidine
10a in a one-pot transformation.
facial” attack of the dipolarophile at the nitrone carbon, the
alternative “re-facial” attack expected to be disfavored due to
steric hindrance by the isopropylidene functionality as depicted
in Figure 2.
The [2 + 3] cycloaddition step in this domino sequence
proceeded with high stereoselectivity to give only one
diastereoisomeric isooxazolidine. In principle, four diaster-
eomers would be possible due to the exo/endo-selectivity as well
as the regioselective addition of allyl alcohol to nitrone in the
1,3-dipolar cycloaddition. Tufariello and co-workers15 reported
that the [2 + 3] cycloaddition reaction of allyl alcohol with a
six-membered cyclic nitrone occurs regioselectively and that the
nitrone oxygen attacks the internal carbon of the alkene moiety,
with an exo-selective addition of allyl alcohol, and moreover
explained that the stereochemical identity at the ring junction is
governed by the π-facial selectivity at the nitrone carbon. We
expected that the orientation of H-5 (axial or equatorial) at the
fused carbon C-5 in 10a with respect to H-4 (as shown in
stereostructures A and B, Figure 2) could in principle be
The assignment of the relative stereochemistry at the newly
generated carbon centers C-5 and C-7 was established using the
1H NMR chemical shift difference (δ) of H-6 methylene
protons wherein Δδ for cis-isooxazolidine is higher than for
trans-isooxazolidine.18 The negligible Δδ value observed for the
H-6 methylene protons is consistent with conformation A
formed via the exo-selective addition of the alkene at the
nitrone carbon to circumvent the unfavorable steric interactions
between the ring hydrogens with the alkyl/aryl group of alkene
existing in the “endo” addition mode.15 Thus, the overall 1,3-
dipolar cycloaddition of the alkene to the six-membered cyclic
nitrone was found to be exo with si-facial selectivity at the
nitrone carbon to give isooxazolidine 10a.
To establish the scope of the domino reaction, we next varied
the alkene component. Thus, homoallyl alcohol was reacted
with precursor 9 under the same reaction conditions that
afforded isooxazolidine 10b as a single diastereoisomer in high
yield (Table 1, entry ii). The application of the domino
sequence to alternate alkenes, including styrene (11c), Cbz-
protected allylamine (11d), and 1-decene (11e), required their
prior solution in mixtures of ethanol/toluene as a solvent. The
change in operational conditions to include a cosolvent rather
than a neat alkene component afforded the corresponding
cycloadducts 10c−e with high selectivity and good yields
(Table 1, entries iii−v).
Targeting the synthesis of iminosugar C-glycosides, the
individual isooxazolidines 10a−e were separately treated with
TFA−H2O for the hydrolysis of the acetonide group, affording
on reductive cleavage of the N−O bond using PdCl2 under
hydrogen pressure the corresponding 1,5-dideoxy-1,5-iminor-
ibitol C-glycosides 7a−e in 70−80% yield. In contrast,
treatment of 10c under the same conditions afforded two
compounds, one arising from the N−O bond cleavage (7c,
34%) and a second arising from both the N−O bond rupture
and the O−CHPh bond cleavage (8, 44%). No optimization
was attempted, and instead both analogues were subjected to
biological assays.
Figure 2. Facial approach of the alkene to the nitrone.
established from an exhaustive analysis of the NMR coupling
1
constant data for 10a. However, the H NMR spectrum of
compound 10a in CDCl3 features broad, overlapping sets of
signals due to equivalence of protons that appear at nearly
identical δ values.16 In an attempt to overcome this problem,
1
the H NMR spectrum of 10a was recorded in alternative
deuterated solvents in the hope that a solvent shift effect17
might come into play and give the appropriately resolved
spectrum. Although, in the case of acetone-d6 and MeOH-d4,
the signals for H-4 and H-5 remained overlapped (merged with
signals for H-2 and H-7), the spectrum of 10a in benzene-d6 at
30 °C was found to be better resolved. The resolution was
further improved when the spectrum was recorded at 50 °C in
benzene-d6 (see the Supporting Information, Figures 23S,
23.1S, and 23.2S), giving signals with good multiplicity from
which the assignments were made conveniently. In the
decoupling experiments, irradiation of the signal at δ 3.91
(H-3) converts the doublet of doublets at 3.69 (H-4) into a
doublet (J4,5 = 7.5 Hz) while irradiation of the signal at δ 2.85
(H-5) led to collapse of the multiplet at δ 3.69 (H-4) into a
doublet (J4,3 = 5.1 Hz). The large coupling observed for H-4
(J4,5 = 7.5 Hz) requires an axial−axial orientation of H-4 and H-
5. As H-4 is axially oriented in both stereostructures A and B
(Figure 2), H-5 must necessarily also be axially orientated, thus
placing the alkyl side chain equatorial with an absolute
configuration of 5R. The value of the axial−equatorial coupling
constant for H-4 (J4,3 = 5.1 Hz) suggests that the dihedral angle
between H-4 and H-3 is close to 10°. We ascribe this to a
distortion of the piperidine ring conformation possibly
provoked by its being part of a rather strained tricyclic
framework. The assignments are consistent with a preferred “si-
Conformational Assignments of 7a−e and 8. It is
known that the biological activity of iminosugar C-glycosides is
dependent on their preferred solution conformation (4C1 or
1C4), and it is the relative orientation of the C-alkyl substituent
that has a major influence on the conformational preference.19
Conformations adopted by 7a−e and 8 were established using
1
their H NMR data. Of particular significance was the value of
the coupling constant between the H-1 and H-2 protons (1H
NMR in D2O), which were in some cases evaluated with
recourse to decoupling experiments. The large coupling
constant values for 1Ha (J1a,2a = 11−12 Hz and J1a,1e = 11−
12 Hz) suggested that compounds 7a−e and 8 exist
predominantly in their 1C4 conformation (Figure 3). This
observation is in agreement with the previously reported C-
glycoside iminosugars.7b,d
Inhibitory Activity toward Selected Glycosidases. The
inhibitory activity of iminosugar C-glycosides 7a−e and 8 was
studied against lysosomal bGal, human α-galactosidase (haGal),
human α-glucosidase (haGlu), α-glucosidase (aGlu), α-
galactosidase (aGal), and α-mannosidase (aMan). The
observed values are shown in Table 2 (see also the
Experimental Section and Supporting Information). None of
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Figure 3. Conformations of 7a−e and 8.
Table 2. Inhibitory Activity (IC50, mM) of Synthetic
Compounds against Screened Glycosidases
a
b
c
d
e
f
compd
bGal
haGal
haGlu
aGlu
aGal
aMan
g
7a
7b
7c
7d
7e
8
0.66
0.66
NI
NI
NI
NI
NI
NI
NI
0.18
0.44
0.20
1.1
0.21
0.20
0.15
0.27
1.8
0.17
0.18
0.29
0.22
0.15
0.15
NI
NI
NI
NI
NI
0.075
0.38
0.044
0.11
0.16
0.20
0.20
a
b
c
Lysosomal β-galactosidase. Human α-galactosidase. Human α-
d
e
glucosidase. α-Glucosidase (Bacillus stearothermophilus). α-Galacto-
f
sidase (green coffee beans). α-Mannosidase (Canavalia ensiformis).
g
NI = no inhibition observed at the highest concentration tested (1
mM).
Figure 4. Intracellular bGal activity in compound-treated GM1
gangliosidosis patient fibroblasts: (A) mutant bGal activity, (B) wild-
type Hex activity controlling for cell number and toxicity. Average
values (N = 3) and standard deviations are shown.
the analogues 7a−e and 8 showed any inhibition of haGal and
haGlu even at high concentration (1 mM), although weak
inhibition of bGal, aGlu, aGal, and aMan was observed. This β-
selective recognition of bGal over haGal and haGlu, displayed
by the iminosugars, is considered a favorable characteristic for
compounds intended as PCs.6b,20 The IC50 values against
lysosomal enzyme bGal for compounds 7c and 7e were found
to be 75 and 44 μM, respectively. These values are large relative
to those reported for bicyclic analogue 5a/5b or DGJ (3a) (3.1
μM (pH 7.0), 0.5 μM (pH 7.0), and 25 μM (pH 5.0),
respectively) and much higher than those observed for 3b and
4 (0.12 and 0.02 μM, respectively), which are both reported as
PCs for bGal.6b No significant inhibition was observed with
compounds 7a−e and 8 when screened for the nonmammalian
orthologues aGlu, aGal, and aMan.
Treatment of GM1 Gangliosidosis Patient Fibroblasts.
We were curious to ascertain if any of the new compounds
behave as PCs and enhance the activity of mutant bGal in
patient cells, with the knowledge that relatively poor inhibitors
of glycosidase had nevertheless shown significant activity as
PCs in the past.6b Fibroblasts from a patient with GM1
gangliosidosis heterozygous for the mutations (p.R201H/
IVS14-2A>G) were used to test the efficacy of the compounds
on increasing the intracellular activity of the mutant bGal
enzyme (see the Experimental Section).
Only compounds 7c, 7e, and 8 with IC50 values of <100 μM
were examined against patient fibroblasts and were found to
enhance mutant bGal activity after 5 days of incubation (Figure
4A). Intracellular β-hexosaminidase (Hex) activity was used as a
control for cell number as well as to monitor toxicity. Although
compound 7e increased intracellular bGal activity approx-
imately 2-fold, it has a significant negative effect on the viability
of cells at concentrations greater than 100 μM as indicated by
the parallel decrease in bGal and Hex activity (Figure 4B). On
the other hand, compound 7c elicited a 6-fold increase in bGal
activity, an increase greater than the 4-fold increase recently
reported for N-nonyl-DGJ (3b) against the same bGal
mutant.4a
The difference in chaperoning efficacy of compound 7c
compared to 7e suggests that the nature of the C-aglycon is
important in dictating the PC activities of such iminosugar-
based analogues. The origin of the effect might relate to the
physicochemical properties of the pseudoaglycon function that
are known to have consequences on the bioavailability of the
compound, i.e., the ability of the PC to enter the cell and gain
access to the endoplasmic reticulum (ER) or other off-target
effects. This has been suggested previously to explain variations
observed in PC efficacy of a number of analogues directed
against GCase.7a−c,21,22
A comparison of the maximal increase in mutant bGal PC
activity vs bGal inhibitory activity (IC50) of compound 7c (IC50
= 75 μM; PC at 394 μM, (6.2 0.4)-fold) with corresponding
data for N-nonyl-DGJ (3b) (IC50 = 0.12 μM; PC at 1.1 μM,
4.5-fold) against the same patient fibroblasts (p.R201H/IVS14-
2A>G)4a is pertinent. IC50 values were determined using the
same substrate concentration (MU-bGal, 0.45 mM) and similar
enzyme preparation (human bGal (Km = 0.3 mM) concanavalin
A-enriched lysosomal enzyme preparation). The bicyclic DGJ
isothiourea analogue 5b maximally increases the intracellular
activity of bGal featuring the R201C mutation more than 6-fold
(at 240 μM) as compared to that achieved with analogue 4 (at
0.2 μM).6b That the PC activity of any iminosugar analogue,
toward a given mutant enzyme, often correlates very poorly
with its inhibitory potency of the wild-type enzyme has been
remarked upon in the past in the case of both Gaucher and
gangliosidosis GM1.6b,20 A notable example is a series of N-
substituted δ-lactams that were reported to be weak inhibitors
of human GCase.21 For all but one member of the latter series,
IC50 values of >100 μM were recorded. Nonetheless, several of
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evaporated at reduced pressure. The crude masses were subjected to
column chromatography to obtain compounds 10a and 10b.
the latter N-substituted δ-lactams were reported to display
significant PC activity.
Data for (3aR,4R,8S,9aS,9bS)-8-(hydroxymethyl)-2,2-dimethyl-
hexahydro-3aH-[1,3]dioxolo[4,5-c]isoxazolo[2,3-a]pyridin-4-ol
(10a): Rf = 0.3 in EtOAc:acetone = 9:1; column purification in
CONCLUSION
■
EtOAc:acetone = 94:6; yield 0.48 g, 68%; mp 121−123 °C; [α]D27.1
=
In conclusion, we have devised a new domino strategy for the
synthesis of 1,5-dideoxy-1,5-iminoribitol C-glycosides 7a−e and
8. Our methodology involves treatment of 2,3-O-isopropyli-
dine-5-O-tosyl-α-D-ribofuranose with hydroxylamine hydro-
chloride to give a six-membered cyclic nitrone trapped by an
alkene dipolarophile in a [2 + 3] cycloaddition reaction to give
the corresponding isooxazolidines 10a−e in one pot. Two of
these analogues, 7c and 7e, have been shown to inhibit only
modestly the target lysosomal bGal activity (IC50 < 100 μM),
but none of the analogues 7a−e and 8 inhibited either the
haGal or the corresponding haGlu activities. The high β/α-
selectivity shown by these new analogues is notable. Although
weak inhibitors of bGal, compounds 7c and 7e were found to
facilitate the recovery of mutant lysosomal bGal activity in
GM1 gangliosidosis patient fibroblasts by 2−6-fold.
−54.4 (c = 0.10 CHCl3); IR (neat; ν, cm−1) 3252 (br), 2980, 1640,
1
1448, 1375, 1219, 1030; H NMR (300 MHz, CDCl3 + 1 drop of
D2O) δ 1.38 (s, 3H), 1.53 (s, 3H), 2.04−2.24 (m, 1H), 2.35 (ddd, J =
4.8, 7.2, 11.7 Hz, 1H), 2.50−2.64 (m, 2H), 3.32−3.46 (m, 1H), 3.56
(dd, J = 4.8, 12 Hz, 1H), 3.76 (dd, J = 1.8, 12 Hz, 1H), 4.02−4.30 (m,
3H), 4.33 (t, J = 4.8 Hz, 1H); 13C NMR (75 MHz, acetone-d6) δ 26.1,
28.1, 35.9, 56.4 (w), 64.0, 66.4 (w), 76.2, 77.4, 78.0, 78.4, 109.6;
HRMS (ESI-TOF) m/z [M + H]+ calcd for C11H20NO5 246.1342,
found 246.1341.
Data for (3aR,4R,8S,9aS,9bS)-8-(2-hydroxyethyl)-2,2-dimethyl-
hexahydro-3aH-[1,3]dioxolo[4,5-c]isoxazolo[2,3-a]pyridin-4-ol
(10b): Rf = 0.4 in EtOAc:acetone = 9:1; column purification in
EtOAc:acetone = 95:5; yield 0.55 g, 73%; [α]2D8.7 = −155.2 (c = 0.139
CHCl3); IR (neat; ν, cm−1) 3367 (br), 2937, 1647, 1382, 1244, 1039;
1H NMR (300 MHz, CDCl3 + 1 drop of D2O) δ 1.39 (s, 3H), 1.53 (s,
3H), 1.78−1.90 (m, 2H), 2.14−2.24 (m, 2H), 2.72−2.92 (br m, 2H),
3.35−3.5 (m, 1H), 3.75 (t, J = 5.7 Hz, 2H), 4.05−4.17 (m, 2H), 4.28−
4.34 (m, 2H); 13C NMR (75 MHz, acetone-d6) δ 25.7, 27.7, 38.5, 38.8,
56.1 (w), 58.9, 65.6, 66.0 (w), 74.4, 75.7, 77.1, 109.3; HRMS (ESI-
TOF) m/z [M + H]+ calcd for C12H22NO5 260.1499; found 260.1499.
General Procedure for Compounds 10c−e. To mixtures of
compound 9 (1 g, 2.91 mmol, 1 equiv) and alkenes 11c−e (3.5 M
solution in ethanol/toluene, 29.1 mmol, 10 equiv) were added
NH2OH·HCl (0.8 g, 11.64 mmol, 4 equiv) and Et3N (2.03 mL, 14.55
mmol, 5 equiv) simultaneously. The reaction mixtures were stirred for
45 min and on TLC analysis showed consumption of starting material.
Then the reaction mixtures were refluxed for 8−11 h, and the solvent
was evaporated at reduced pressure. The crude masses were subjected
to column chromatography to obtain compounds 10c−e.
Data for (3aR,4R,8S,9aS,9bS)-2,2-dimethyl-8-phenylhexahydro-
3aH-[1,3]dioxolo[4,5-c]isoxazolo[2,3-a]pyridin-4-ol (10c): Rf = 0.5 in
petroleum ether:EtOAc = 1:1; column purification in petroleum
ether:EtOAc = 76:24; yield 0.58 g, 69%; [α]2D7.3 = −85.2 (c = 0.10
CHCl3); IR (neat; ν, cm−1) 3392 (br), 2986, 1647, 1455, 1381, 1219,
1048, 866, 699; 1H NMR (300 MHz, CDCl3) δ 1.40 (s, 3H), 1.54 (s,
3H), 2.39−2.58 (m, 3H; after D2O exchange this multiplet integrated
for 2 protons), 2.82−3.10 (m, 2H), 3.57−3.65 (m, 1H), 4.13−4.30
(m, 2H), 4.38 (t, J = 4.8 Hz, 1H), 5.07 (dd, J = 6, 8.7 Hz, 1H), 7.20−
7.42 (m, 5H); 13C NMR (75 MHz, CDCl3) δ 25.9, 27.8, 41.4, 55.5
(w), 65.9 (w), 66.0, 74.7, 76.7, 78.6, 109.8, 126.4 (s), 127.9, 128.5 (s),
140.9; HRMS (ESI-TOF) m/z [M + H]+ calcd for C16H22NO4
292.1550, found 292.1546.
Data for benzyl (((3aR,4R,8S,9aS,9bS)-4-hydroxy-2,2-dimethyl-
hexahydro-3aH-[1,3]dioxolo[4,5-c]isoxazolo[2,3-a]pyridin-8-yl)-
methyl)carbamate (10d): Rf = 0.5 in EtOAc; column purification in
petroleum ether:EtOAc = 52:48; yield 0.74 g, 67%; [α]2D8.7 = +97.5 (c =
0.096 CHCl3); IR (neat; ν, cm−1) 3347 (br), 2925, 2854, 1705, 1539,
1455, 1381, 1253, 1055, 866, 698; 1H NMR (300 MHz, CDCl3) δ 1.38
(s, 3H), 1.51 (s, 3H), 2.08−2.24 (m, 2H), 2.65−2.95 (m, 2H), 3.22−
3.50 (m, 3H), δ 3.95−4.22 (m, 3H), 4.28 (t, J = 4.8 Hz, 1H), 5.07 (br
s, 2H), 7.14−7.25 (m, 5H); 13C NMR (75 MHz, CDCl3) δ 25.8, 27.6,
29.7, 36.0, 44.1, 56.1 (w), 65.1 (w), 66.0, 66.8, 74.5, 75.7, 77.2, 109.8,
128.11, 128.16, 128.5, 136.3, 156.5; HRMS (ESI-TOF) m/z [M + H]+
calcd for C19H27N2O6 379.1879, found 379.1867.
Data for (3aR,4R,8R,9aS,9bS)-2,2-dimethyl-8-octylhexahydro-
3aH-[1,3]dioxolo[4,5-c]isoxazolo[2,3-a]pyridin-4-ol (10e): Rf = 0.6
in petroleum ether:EtOAc = 1:1; column purification in petroleum
ether:EtOAc = 78:22; yield 0.63 g, 66%; [α]2D8.4 = −68.0 (c = 0.112
CHCl3); IR (neat; ν, cm−1) 3360 (br), 2926, 2855, 1650, 1378, 1246,
1071; 1H NMR (300 MHz, CDCl3 + 1 drop of D2O) δ 0.87 (t, J = 7.2
Hz, 3H), 1.15−1.70 (m, 20H), 2.0−2.17 (m, 2H), 2.55−2.85 (br m,
2H), 3.40−3.52 (m, 1H), 4.02−4.20 (m, 3H), 4.32 (t, J = 4.8 Hz, 1H);
13C NMR (75 MHz, acetone-d6) δ 14.3, 23.2, 26.2, 26.6, 28.3, 29.0,
EXPERIMENTAL SECTION
■
General Methods. Melting points were recorded with a melting
point apparatus and are uncorrected. IR spectra were recorded with an
FTIR instrument as a thin film or using KBr pellets, and the data are
expressed in inverse centimeters. 1H NMR (300 MHz) and 13C NMR
(75 MHz) spectra were recorded using CDCl3, acetone-d6, or D2O as
the solvent(s). Chemical shifts are reported in δ (parts per million)
with reference to TMS as an internal standard, and J values are given
in hertz. Optical rotations were measured using a polarimeter. High-
resolution mass spectrometry (HRMS) was performed on an electron
spray ionization time-of-flight (ESI-TOF) instrument. Thin-layer
chromatography was performed on precoated plates (0.25 mm, silica
gel 60 F254). Column chromatography was carried out with silica gel
(100−200 mesh). The reactions were carried out in oven-dried
glassware under a dry N2 atmosphere. Methanol, acetone, and ethanol
were purified and dried with the method reported in Vogel’s textbook
of practical organic chemistry. The petroleum ether (PE) that was
used was a distillation fraction between 40 and 60 °C. PdCl2 was
purchased from a chemical supplier. The purity of the compounds was
determined to be >95% by elemental microanalysis.
2,3-O-Isopropylidene-5-O-tosyl-α-D-ribofuranose (9).14 To a
suspension of D-ribose (10 g, 66.64 mmol) in dry acetone (60 mL)
was added dropwise concd H2SO4 (6 mL) at room temperature. The
mixture was stirred at rt for 3 h. The reaction mixture was neutralized
using solid NaHCO3 and filtered, and the filtrate was evaporated in
vacuo. The residue was purified by column chromatography by eluting
with petroleum ether−EtOAc (55:45) to give a thick liquid (11.7 g,
93%).
A solution of TsCl (3.31 g, 17.35 mmol, 1.1 equiv) in pyridine (5
mL) was added dropwise to a mixture of the above compound (3.0 g,
15.77 mmol, 1 equiv) in pyridine (5 mL) at −10 °C. The resulting
mixture was stirred at 0 °C for 1/2 h and then at 25 °C for 4 h. Ethyl
acetate (50 mL) was added, and the resulting solution was washed
with 10% H2SO4 (4 × 10 mL). The organic layer was further washed
with NaHCO3 (3 × 8 mL) and dried (Na2SO4), and the solvent was
evaporated under vacuum. The residue was purified by column
chromatography by eluting with petroleum ether−EtOAc (85:15) to
give 9 (4.3 g, 80%) as a white solid, mp =95−96 °C. The spectroscopic
data of 9 were identical to those previously reported.14
General Procedure for Compounds 10a and 10b. To mixtures
of compound 9 (1.0 g, 2.91 mmol, 1 equiv) and alkenes 11a,b (29.1
mmol, 10 equiv) as the solvent for reaction were added NH2OH·HCl
(0.8 g, 11.64 mmol, 4 equiv) and Et3N (2.03 mL, 14.55 mmol, 5
equiv) simultaneously. The reaction mixtures were stirred for 30 min
and on TLC analysis showed consumption of starting material. Then
the reaction mixtures were refluxed for 4 h, and the solvent was
29.9, 30.2, 32.5, 35.9, 39.4, 56.1 (w), 66.1, 66.2 (w), 76.3, 77.4, 77.7,
4402
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Article
109.7; HRMS (ESI-TOF) m/z [M + H]+ calcd for C18H34NO4
328.2489, found 328.2500.
NMR (75 MHz, D2O) δ 14.4, 23.7, 26.8, 30.4, 30.7, 30.8, 33.0, 39.2,
39.8, 46.0, 53.1, 69.6, 69.8, 72.9, 73.7; HRMS (ESI-TOF) m/z [M +
H]+ calcd for C15H32NO4 290.2332, found 290.2332. Anal. Calcd for
C15H31NO4: C, 62.25; H, 10.80; N, 4.84. Found: C, 62.48; H, 10.84;
N, 4.94.
General Procedure for Compounds 7a−e and 8. Solutions of
compounds 10a−e (0.2 g, 1 equiv) in 4 mL of TFA−water (3:2) were
stirred for 2 h at 0 °C to rt. TFA was coevaporated with toluene at
reduced pressure. The crude reaction masses were taken in methanol,
and PdCl2 (0.2 equiv) was added. The reaction mixtures were
hydrogenated at balloon pressure for 6 h. The reaction mixtures were
filtered through Celite and washed with methanol, and the solvent was
evaporated at reduced pressure. Purification by column chromatog-
raphy (chloroform−methanol−25% aq NH3) gave compounds 7a−e
and 8.
Data for (2S,3S,4R,5R)-2-Phenethylpiperidine-3,4,5-triol (8): Rf =
0.6 in CHCl3:MeOH (1 drop of 25% aq NH3) = 4:1; column
purification in CHCl3:MeOH:aq NH3 (25% solution) = 92:7:1; yield
0.07 g, 44%; mp 151−153 °C; [α]2D6.8 = −46.5 (c = 0.11 MeOH); IR
(neat; ν, cm−1) 3728, 3271 (br), 841, 707; 1H NMR (300 MHz, D2O)
δ 1.51−1.66 (m, 1H), 2.02−2.18 (m, 1H), 2.70 (t, J = 11.4 Hz, 1H),
2.58−2.92 (m, 4H), 3.34 (dd, J = 3.0, 10.2 Hz, 1H), 3.69−3.78 (ddd, J
= 2.4, 4.8, 10.8 Hz, 1H), 4.08 (br s, 1H), 7.22−7.52 (m, 5H); 13C
NMR (75 MHz, D2O) δ 31.1, 32.9, 43.8, 52.9, 68.0, 71.3, 71.8, 126.0,
128.4 (s), 128.6 (s), 142.4; HRMS (ESI-TOF) m/z [M + H]+ calcd
for C13H20NO3 238.1444, found 238.1445. Anal. Calcd for
C13H19NO3: C, 65.80; H, 8.07; N, 5.90. Found: C, 65.61; H, 7.83;
N, 5.91.
Glycosidase Inhibition Assay. Glycosidase inhibition assay of the
derivatives was carried out by mixing a 0.1 unit/mL concentration each
of α-galactosidase, α-mannosidase, and α-glucosidase with the samples,
and the resulting mixtures were incubated for 1 h at 37 °C. Enzyme
action for α-galactosidase was initiated by addition of 10 mM p-
nitrophenyl α-D-galactopyranoside (pNPG) as a substrate in 200 mM
sodium acetate buffer followed by incubation for 10 min at 37 °C and
stopped by adding 2 mL of 200 mM borate buffer of pH 9.8. α-
Mannosidase activity was initiated by addition of 10 mM p-nitrophenyl
α-D-mannopyranoside as a substrate in 100 mM citrate buffer of pH
4.5. The reaction was incubated at 37 °C for 10 min and stopped by
adding 2 mL of 200 mM borate buffer of pH 9.8. Initiation of α-
glucosidase activity was carried out by addition of 10 mM p-
nitrophenyl α-D-glucopyranoside in 100 mM phosphate buffer of pH
6.8 and stopped by adding 2 mL of 0.1 M Na2CO3 after incubation for
10 min at 37 °C. α-Glycosidase activity was determined by measuring
the absorbance of the p-nitrophenol released from pNPG at 420 nm
using a spectrophotometer.
Purified recombinant preparations of the lysosomal enzyme haGlu
were utilized. For bGal and haGlu a concanavalin binding fraction
from human placenta lysate enriched in lysosomal enzymes was used
as the enzyme source.7d The enzyme activities of bGal, haGlu, and
haGal were monitored using the fluorogenic substrates [final
concentration] 4-methylumbelliferyl β-D-galactopyranoside (MU-
bGal) [0.45 mM], 4-methylumbelliferyl α-D-glucopyranoside (MU-
aGlu) [0.7 mM], and 4-methylumbelliferyl α-D-galactopyranoside
(MU-aGal) [0.5 mM], respectively, as previously described.4a,7d,e The
substrates were purchased from a chemical supplier. All enzyme
reactions were performed at 37 °C in McIlvaine citrate phosphate
buffer (100 mM), pH 4.5. The reactions were terminated with a 5-fold
excess of 0.1 M MAP, pH 10.5, and fluorescence was detected using an
excitation/emission wavelength of 365 nm/450 nm on a spectro-
fluorometer. To determine the inhibitory activity of the compounds,
serial 3-fold dilutions of the compounds in DMSO in triplicate were
incubated with the diluted lysosomal enzymes prior to initiation of the
reaction with addition of the appropriate fluorogenic substrate. IC50
values were determined using built-in equations within PRISM
Graphpad v5.2.
Data for (2S,3S,4R,5R)-2-((S)-2,3-dihydroxypropyl)piperidine-
3,4,5-triol (7a): Rf = 0.4 in CHCl3:MeOH (1 drop of 25% aq NH3)
= 2:3; column purification in CHCl3:MeOH:aq NH3 (25% solution) =
71:28:1; yield 0.13 g, 77%; [α]2D8.4 = −88.0 (c = 0.1 MeOH); IR (neat;
1
ν, cm−1) 3389 (br); H NMR (300 MHz, D2O) δ 1.42 (ddd, J = 2.4,
9.6, 13.0 Hz, 1H), 1.90 (ddd, J = 3.0, 10.4, 13.0 Hz, 1H), 2.73 (t, J =
11.9 Hz, 1H), 2.80−2.98 (m, 2H), 3.36 (dd, J = 1.9, 10.0 Hz, 1H),
3.53 (dd, J = 6.6, 11.7 Hz, 1H), 3.61 (dd, J = 4.5, 11.7 Hz, 1H), 3.70−
3.82 (m, 1H), 3.86−3.98 (m, 1H), 4.12 (br s, 1H); 13C NMR (75
MHz, D2O) δ 34.5, 43.7, 50.3, 65.8, 68.0, 68.3, 71.2, 71.9; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C8H18NO5 208.1186, found
208.1184. Anal. Calcd for C8H17NO5: C, 46.37; H, 8.27; N, 6.76.
Found: C, 46.43; H, 8.24; N, 6.69.
Data for (2S,3S,4R,5R)-2-((S)-2,4-dihydroxybutyl)piperidine-3,4,5-
triol (7b): Rf = 0.5 in CHCl3:MeOH (1 drop of 25% aq NH3) = 2:3;
column purification in CHCl3:MeOH:aq NH3 (25% solution) =
74:25:1; yield 0.14 g, 80%; [α]2D8.4 = −285.9 (c = 0.154 MeOH); IR
(neat; ν, cm−1) 3100 (br); 1H NMR (300 MHz, D2O) δ 1.45 (ddd, J =
1.8, 9.0, 11.8 Hz, 1H), 1.67−1.82 (m, 2H), 1.90 (ddd, J = 2.4, 9.6, 11.8
Hz, 1H), 2.70 (t, J = 11.1 Hz, 1H), 2.78−2.99 (m, 2H), 3.33 (dd, J =
2.4, 9.9 Hz, 1H), 3.63−3.82 (m, 3H), 3.90−4.02 (m, 1H), 4.08 (br s,
1H); 13C NMR (75 MHz, D2O) δ 38.4, 39.1, 43.6, 50.5, 58.4, 65.0,
67.8, 71.1, 71.8; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C9H20NO5 222.1342, found 222.1342. Anal. Calcd for C9H19NO5: C,
48.86; H, 8.66; N, 6.33. Found: C, 48.82; H, 8.89; N, 6.23.
Data for (2S,3S,4R,5R)-2-((S)-2-hydroxy-2-phenylethyl)piperidine-
3,4,5-triol (7c): Rf = 0.4 in CHCl3:MeOH (1 drop of 25% aq NH3) =
4:1; column purification in CHCl3:MeOH:aq NH3 (25% solution) =
88:11:1; yield 0.06 g, 34%; mp 158−160 °C; [α]2D8.3 = −47.2 (c =
0.121 MeOH); IR (neat; ν, cm−1) 3321 (br), 883, 697; 1H NMR (300
MHz, D2O) δ 1.68 (ddd, J = 3.4, 9.1, 13.2 Hz, 1H), 2.24 (ddd, J = 3.0,
9.5, 13.2 Hz, 1H), 2.68 (t, J = 11.8 Hz, 1H), 2.82 (dd, J = 5.1, 11.8 Hz,
1H), 2.85−2.92 (td, J = 2.4, 9.8 Hz, 1H), 3.34 (dd, J = 2.4, 9.8 Hz,
1H), 3.65−3.76 (m, 1H), 4.09 (br s, 1H), 4.95 (dd, J = 3.4, 9.5 Hz,
1H), 7.30−7.55 (m, 5H); 13C NMR (75 MHz, D2O) δ 40.3, 43.8,
50.7, 68.0, 70.5, 71.3, 72.0, 125.8, 127.8 (s), 128.7 (s), 144.0; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C13H20NO4 254.1393, found
254.1393. Anal. Calcd for C13H19NO4: C, 61.64; H, 7.56; N, 5.53.
Found: C, 61.66; H, 7.42; N, 5.52.
Data for (2S,3S,4R,5R)-2-((S)-3-amino-2-hydroxypropyl)-
piperidine-3,4,5-triol (7d): Rf = 0.2 in MeOH (1 drop of 25% aq
NH3); column purification in MeOH:aq NH3 (25% solution) = 99:1;
yield 0.085 g, 78%; [α]2D7.7 = −8.7 (c = 0.19 MeOH); IR (neat; ν,
1
cm−1) 3593−3300 (br); H NMR (300 MHz, D2O) δ 1.32−1.44 (m,
1H), 1.78−1.95 (m, 1H), 2.60−2.94 (m, 5H), 3.30 (dd, J = 2.1, 10.2
Hz, 1H), 3.66−3.78 (m, 1H), 3.78−3.92 (m, 1H), 4.09 (br s, 1H); 13C
NMR (75 MHz, D2O) δ 36.1, 43.8, 46.3, 50.2, 67.6, 68.3, 71.3, 72.2;
HRMS (ESI-TOF) m/z [M + H]+ calcd for C8H19N2O4 207.1346,
found 207.1340. Anal. Calcd for C8H18N2O4: C, 46.59; H, 8.80; N,
13.58. Found: C, 46.45; H, 9.00; N, 13.27.
Data for (2S,3S,4R,5R)-2-((R)-2-hydroxydecyl)piperidine-3,4,5-
triol (7e): Rf = 0.8 in CHCl3:MeOH (1 drop of 25% aq NH3) =
4:1; column purification in CHCl3:MeOH:aq NH3 (25% solution) =
95:4:1; yield 0.13 g, 73%; mp 136−138 °C; [α]2D6.6 = −36.5 (c = 0.12
MeOH); IR (neat; ν, cm−1) 3277 (br); 1H NMR (300 MHz, D2O) δ
0.87 (br t, J = 6.0 Hz, 3H), 1.30−1.60 (m, 15H), 1.77−1.94 (m, 1H),
2.72 (t, J = 11.4 Hz, 1H), 2.80−2.97 (m, 2H), 3.35 (dd, J = 2.4, 10 Hz,
1H), 3.68−3.78 (m, 1H), 3.78−3.92 (m, 1H), 4.09 (br s, 1H); 13C
Treatment of Patient Fibroblasts. Unaffected human skin
fibroblasts or those derived from GM1 gangliosidosis (p.R201H/
IVS14-2A>G)4a prior to treatment were seeded at (1−2) × 104 cells/
well into 96-well tissue culture plates and grown in αMEM medium
with 10% fetal calf serum at 37 °C in a CO2 humidified incubator.
Serially diluted compounds were evaluated in triplicate and diluted
100-fold into the medium to ensure that the DMSO solvent was
maintained at 1%. Following 5 days of treatment, the growth medium
was removed, and adherent cells were washed with two changes of
PBS. The cells were lysed using 75 μL of McIlvaine citrate phosphate
buffer containing 0.4% Triton X-100 at 4 °C for a minimum of 30 min.
Lysosomal enzyme activities in the lysates (25 μL) were evaluated
using the 4-methylumbelliferone-based fluorogenic substrates de-
scribed above. Human bGal and β-N-acetylhexosaminidase activities
4403
dx.doi.org/10.1021/jo500328u | J. Org. Chem. 2014, 79, 4398−4404

The Journal of Organic Chemistry
Article
were monitored using 0.45 mM MU-bGal and 1.6 mM 4-
methylumbelliferyl β-N-acetylglucosaminide, respectively. Following
1 h of incubation at 37 °C, the reaction was terminated with a 5-fold
excess of 0.1 M MAP, pH 10.5, and fluorescence was detected using an
excitation/emission wavelength of 365 nm/450 nm on a spectro-
fluorometer. Activity values obtained for compound-treated cells were
normalized to those of cells treated with solvent (DMSO, 1%) only.
(6) (a) Suzuki, Y.; Ichinomiya, S.; Kurosawa, M.; Matsuda, J.; Ogawa,
S.; Iida, M.; Kubo, T.; Tabe, M.; Itoh, M.; Higaki, K.; Nanba, E.;
Ohno, K. Mol. Genet. Metab. 2012, 106, 92−98. (b) Takai, T.; Higaki,
K.; Aguilar-Moncayo, M.; Mena-Barragan, T.; Hirano, Y.; Yura, K.; Yu,
L.; Ninomiya, H.; Garcia-Moreno, M. I.; Sakakibara, Y.; Ohno, K.;
Nanba, E.; Ortiz Mellet, C.; Garcia Fernandez, J. M.; Suzuki, Y. Mol.
Ther. 2013, 21, 526−532. (c) Schitter, G.; Steiner, A. J.; Pototschnig,
G.; Scheucher, E.; Thonhofer, M.; Tarling, C. A.; Withers, S. G.;
Fantur, K.; Paschke, E.; Mahuran, D. J.; Rigat, B. A.; Tropak, M. B.;
Illaszewicz, C.; Saf, R.; Stutz, A. E.; Wrodnigg, T. M. ChemBioChem
2010, 11, 2026−2033.
ASSOCIATED CONTENT
* Supporting Information
Characterization data of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
(7) (a) Zhu, X.; Sheth, K. A.; Li, S.; Hui-Hwa, C.; Fan, J.-Q. Angew.
Chem., Int. Ed. 2005, 44, 7450−7453. (b) Compain, P.; Martin, O. R.;
Boucheron, C.; Godin, G.; Yu, L.; Ikeda, K.; Asano, N. ChemBioChem
2006, 7, 1356−1359. (c) Oulaidi, F.; Front-Deschamps, S.; Gallienne,
E.; Lesellier, E.; Ikeda, K.; Asano, N.; Compain, P.; Martin, O. R.
ChemMedChem 2011, 6, 353−361. (d) Goddard-Borger, E. D.;
Tropak, M. B.; Yonekawa, S.; Tysoe, C.; Mahuran, D. J.; Withers, S.
G. J. Med. Chem. 2012, 55, 2737−2745. (e) Hill, T.; Tropak, M. B.;
Mahuran, D.; Withers, S. G. ChemBioChem 2011, 12, 2151−2154.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: aloysius.siriwardena@u-picardie.fr.
*E-mail: mb.tropak@utoronto.ca.
*E-mail: ddd@chem.unipune.ac.in.
■
Notes
(8) (a) Pellissier, H. Chem. Rev. 2013, 113, 442−524. (b) Domling,
̈
The authors declare no competing financial interest.
A.; Wang, W.; Wang, K. Chem. Rev. 2012, 112, 3083−3135.
(9) Compain, P.; Chagnault, V.; Martin, O. R. Tetrahedron:
Asymmetry 2009, 20, 672−711.
ACKNOWLEDGMENTS
■
(10) (a) Goti, A.; Cicci, S.; Fedi, V.; Nannelli, L.; Brandi, A. J. Org.
Chem. 1997, 62, 3119−3125. (b) Yu, C. Y.; Huang, M. H. Org. Lett.
2006, 8, 3021−3024. (c) Peer, A.; Vasella, A. Helv. Chim. Acta 1999,
82, 1044−1065.
(11) (a) Bande, O. P.; Jadhav, V. H.; Puranik, V. G.; Dhavale, D. D.
Tetrahedron: Asymmetry 2007, 18, 1176−1182. (b) Bande, O. P.;
Jadhav, V. H.; Puranik, V. G.; Dhavale, D. D.; Lombardo, M.
Tetrahedron Lett. 2009, 50, 6906−6908. (c) Karanjule, N. S.; Markad,
S. D.; Sharma, T.; Sabharwal, S. G.; Puranik, V. G.; Dhavale, D. D. J.
Org. Chem. 2005, 70, 1356−1363. (d) Closa, M.; Wightman, R. H.
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We are thankful to the Department of Science and Technology,
New Delhi (Project File No. SR/S1/OC-20/2010) for
providing financial support. D.P.S. and P.R.M. are thankful to
the University Grants Commission (UGC) and Council of
Scientific and Industrial Research (CSIR), New Delhi, for
providing senior research fellowships. O.P.B. and A.S. thank the
Centre Franco-Indien pour la Promotion de la Recherche
́
Avancee (CEFIPRA) and the Centre National de la Recherche
Scientifique (CNRS) for funding. M.B.T. and D.J.M. acknowl-
edge support from grants provided by the Canadian Institutes
of Health Research (CIHR) and helpful discussions with Dr.
Brigitte Rigat and others.
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Holmes, A. B. Org. Lett. 2008, 10, 4227−4229. (b) Coldham, I.; Jana,
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(c) Coldham, I.; Jana, S.; Watson, L.; Martin, N. G. Org. Biomol. Chem.
2009, 7, 1674−1679.
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