Probing the active-site requirements of human intestinal N-terminal maltase-glucoamylase: Synthesis and enzyme inhibitory activities of a six-membered ring nitrogen analogue of kotalanol and its de-O-sulfonated derivative

Article abstract of DOI:10.1016/j.bmc.2010.09.059

In order to probe the active-site requirements of the human N-terminal subunit of maltase-glucoamylase (ntMGAM), one of the clinically relevant intestinal enzymes targeted for the treatment of type-2 diabetes, the syntheses of two new inhibitors are descr

Full text of DOI:10.1016/j.bmc.2010.09.059

Bioorganic & Medicinal Chemistry 18 (2010) 7794–7798
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Probing the active-site requirements of human intestinal N-terminal
maltase-glucoamylase: Synthesis and enzyme inhibitory activities
of a six-membered ring nitrogen analogue of kotalanol
and its de-O-sulfonated derivative
Sankar Mohan a, Lyann Sim b, David R. Rose b,c, B. Mario Pinto a,
⇑
a Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6
b Department of Medical Biophysics, University of Toronto and Division of Molecular and Structural Biology, Ontario Cancer Institute, Toronto, ON, Canada M5G 2M9
c Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to probe the active-site requirements of the human N-terminal subunit of maltase-glucoamylase
(ntMGAM), one of the clinically relevant intestinal enzymes targeted for the treatment of type-2 diabetes,
the syntheses of two new inhibitors are described. The target compounds are structural hybrids of
kotalanol, a naturally occurring glucosidase inhibitor with a unique five-membered ring sulfonium-
sulfate inner salt structure, and miglitol, a six-membered ring antidiabetic drug that is currently in
clinical use. The compounds comprise the six-membered ring of miglitol and the side chain of kotalanol
or its de-O-sulfonated derivative. Inhibition studies of these hybrid molecules with human ntMGAM
indicated that they are inhibitors of this enzyme with comparable Ki values to that of miglitol (kotalanol
Received 16 August 2010
Revised 22 September 2010
Accepted 23 September 2010
Available online 29 September 2010
Keywords:
Glucosidase inhibitors
Kotalanol
Nitrogen analogues
De-O-sulfonated analogues
Deoxynojirimycin
Miglitol
analogue: 2.3 0.6
l
M; corresponding de-O-sulfonated analogue: 1.4 0.5
lM; miglitol: 1.0 0.1 lM).
However, they are less active compared to kotalanol (Ki = 0.19 0.03
l
M). These results suggest that
the 3T2 enzyme-bound conformation of the five-membered thiocyclitol moiety of the kotalanol class of
compounds more closely resembles the 4H3 conformation of the proposed transition state for the forma-
tion of an enzyme–substrate covalent intermediate in the glycosidase hydrolase family 31 (GH31)-cata-
lyzed reaction.
Ammonium ions
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
isolated from S. reticulata extracts, by total synthesis.16 Four of
these compounds 2–5 strongly inhibited the action of the human
The plant extracts from Salacia reticulata, known as
kothalahimbutu in Singhalese, have been widely used in the an-
cient Ayurvedic system of medicine for treating type-2 diabe-
tes.1–6 S. reticulata is a large woody climbing plant widely found
in Sri Lanka and southern parts of India. Typically, water stored
overnight in a mug made from the roots of S. reticulata was given
to patients as an herbal remedy for type-2 diabetes. Extracts from
the other plants in the Salacia genus such as Salacia chinensis,
Salacia oblonga, and Salacia prinoides have also been used in this
herbal remedy.5,6 Attempts to identify the source of the antidia-
betic property possessed by these aqueous extracts have yielded
N-terminal subunit of maltase-glucoamylase (ntMGAM),18,19
a
OH
OH OH
OH OH OH
S
OSO3
S
OSO3
OH
S
O
OH
SO3
HO
HO
HO
HO
OH
HO
HO
OH
salacinol (2)
salaprinol (1)
ponkoranol (3)
OH OH OH
OH OH
OH OH OH
a
novel class of sulfonium-ion glucosidase inhibitors 1–5
OH
OH
(Fig. 1).6–10 The absolute stereostructures, as shown in Figure 1,
were eventually established through total synthesis.11–17 Very
recently, we have established the stereostructure of kotalanol 4
and de-O-sulfonated kotalanol 5, the two most active components
S
S
O
OH
SO3
HO
HO
X
HO
OH
HO
OH
de-O-sulfonated kotalanol (5)
kotalanol (4)
⇑
Corresponding author. Tel.: +1 778 782 4152; fax: +1 778 782 4860.
E-mail address: bpinto@sfu.ca (B.M. Pinto).
Figure 1. Sulfonium-ion glucosidase inhibitors isolated from Salacia species.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.059

S. Mohan et al. / Bioorg. Med. Chem. 18 (2010) 7794–7798
OH OH OH OH OH OH
7795
OH
OH
OH
HO
HO
OH
Cl
N
HO
HO
OH
SO3
OH
NH
O
NH OH
HO
HO
OH
OH
OH
OH
OH
8
miglitol (6)
7
OH OH
OH OH
Cl
NH OH
OH
NH
O
HO
HO
SO3
HO
HO
OH
OH
OH
9
10
Figure 2. Miglitol and related analogues.
family 31 glycoside hydrolase (GH31)20 and one of the clinically
relevant intestinal glucosidases targeted for the treatment of
type-2 diabetes.
static contacts in the ꢀ1 subsite, the polyhydroxylated side chain
makes several hydrogen bonding interactions in the +1 subsite
(Fig. 3B). In the case of miglitol (6), the interactions in the ꢀ1 subsite
are very similar to those observed with kotalanol, mainly hydrogen
bonding interactions between the hydroxyl groups present in the
six-membered iminocyclitol moiety, the head group of miglitol
(Fig. 3A). But, unlike kotalanol, the N-hydroxyethyl side chain of
miglitol does not make any interactions in the +1 subsite of
ntMGAM, presumably accounting for the lesser potency of miglitol
(6) relative to kotalanol (4). In addition, it was also suggested that
the ring conformations adopted by the thiocyclitol and iminocyclitol
moieties mimick two different points in the reaction trajectory of
Both kotalanol (4) and miglitol 621 (Fig. 2), an antidiabetic drug
that is currently in clinical use, are inhibitors of ntMGAM, with Ki
values of 0.19 lM and 1.0 l
M, respectively.19 The active-site of ntM-
GAM consists of a ꢀ1 and a +1 sugar-binding site.20 Recent crystals
structures of ntMGAM in complex with kotalanol (4) and miglitol (6)
indicated that kotalanol utilizes both subsites for binding, whereas
miglitol binds only in the ꢀ1 subsite (Fig. 3).19 While the five-mem-
bered thiocyclitol moiety with the permanent positive charge, the
head group of kotalanol (4), makes hydrogen bonding and electro-
Figure 3. Comparison of the interactions of (A) miglitol (6) and (B) kotalanol (4) with the active-site residues of ntMGAM. (C) Comparison of the enzyme-bound conformation
of the head group of miglitol to that of kotalanol in the ꢀ1 subsite. Ring carbon atoms of miglitol are numbered in pink and those of kotalanol are in green. Reproduced from
the respective PDB files (3L4 W, 3L4 V).

7796
S. Mohan et al. / Bioorg. Med. Chem. 18 (2010) 7794–7798
the enzyme catalyzed reaction.19 For the GH31 family, the proposed
reaction mechanism follows a 4C1–4H3–1S3 conformational itinerary
for the formation of an enzyme–substrate covalent intermediate.22
The thiocyclitol moiety of kotalanol (4) adopts a 3T2 (carbohydrate
numbering) conformation which closely resembles the proposed
4H3 conformation of the oxacarbenium-ion like transition state,
whereas the iminocyclitol moiety of miglitol (6) adopts a 4C1 (carbo-
hydrate numbering) conformation that resembles the substrate-
binding conformation (Fig. 3C).19
In order to probe this hypothesis further, it was of interest to syn-
thesize an inhibitor 7 in which the N-hydroxyethyl substituent of
miglitol was replaced with the polyhydroxylated side chain of kotal-
anol, as shown in Figure 2. The resulting hybrid molecule 7 was ex-
pected to show binding interactions in the +1 subsite as seen in the
case of kotalanol (4). The corresponding de-O-sulfonated compound
8 (Fig. 2) is also of interest, as our recent structure–activity relation-
ship (SAR) studies of kotalanol analogues consistently show that the
inhibitory activities against ntMGAM improve significantly upon
de-O-sulfonation.23 For example, de-O-sulfonated kotalanol (5)
K2CO3, as shown in Scheme 1. The coupled product 13 was found
to be unstable, as indicated by thin layer chromatography (TLC),
probably due to partial removal of the para-methoxybenzyl
(PMB) protecting groups. Hence, without any further characteriza-
tion, compound 13 was deprotected. Treatment with 80% trifluoro-
acetic acid (TFA) followed by hydrogenolysis using 10% Pd/C and
H2 at 100 psi in aqueous acetic acid gave the target compound 7
in 62% yield (two steps).
The structure of compound 7 was confirmed by 1D and 2D NMR
analyses. At neutral pH, the 1H NMR spectrum of 7 in D2O was ex-
tremely broad, however all the resonances were sharpened when
the NMR sample was made basic (pH >8) by the addition of small
amount of solid K2CO3. The broadening of the resonances at neutral
pH and sharpening at basic pH indicate that the product 7 exists as
an equilibrium mixture of ammonium salt and the corresponding
tertiary amine. In the 1H NMR spectrum, in addition to the desired
compound resonances, one also observed a singlet at 1.8 ppm
resulting from KOAc contamination; a similar observation was
noted in our previous studies.25 Compound 7 was then converted
into the corresponding de-O-sulfonated derivative 8 using 5%
methanolic HCl, followed by treatment with Amberlyst A-26 (chlo-
ride resin) in methanol (Scheme 1). Analysis of the 1H NMR cou-
pling constants revealed that the six-membered iminocyclitol
moiety in both compounds, 7 and 8, exists in a 4C1 (Fig. 4) confor-
mation in solution. It is important to note that this is also the bio-
active conformation of miglitol in the ntMGAM active-site.19
Finally, we comment on the glucosidase inhibitory activities of
compounds 7 and 8 against ntMGAM using maltose as a substrate.
Both compounds, 7 and 8, inhibited ntMGAM activity with similar
(Ki = 0.03
kotalanol 4 (Ki = 0.19
l
M) is a ꢁsevenfold better inhibitor of ntMGAM than
l
M) itself.19 In addition, the target compounds
7 and 8, in conjunction with the recent crystallographic studies,19
could also be used to probe the relative importance of transition
state mimicry by the kotalanol class of compounds. We recently
reported our first attempt to probe the active-site requirements of
ntMGAM using a 30-O-methyl-substituted ponkoranol analogue.24
We now report the synthesis of compounds 7 and 8 and their eval-
uation as glucosidase inhibitors against ntMGAM. It is noteworthy
that the miglitol analogue 9 containing the 4-carbon polyhydroxy-
lated chain present in salacinol (2) and the corresponding
de-O-sulfonated derivative 10, synthesized by us25 and others,26
Ki values, 2.3 0.6
son with the inhibitory activity of miglitol (Ki = 1.0 0.1
l
M and 1.4 0.5
l
M, respectively. In compari-
l
M),19
were found to be as active as salacinol (2) against rat intestinal
glucosidases (Fig. 2).26 In comparison with the inhibitory activity
of miglitol (Ki = 1.0 0.1
M),19 the target compounds 7 and 8 did
a
-
the target compounds did not show any significant improvement,
suggesting that the newly appended polyhydroxylated side chain,
contrary to our expectation, does not contribute appreciably to
binding in the +1 subsite of the ntMGAM active-site. Supporting
evidence can be realized from the fact that de-O-sulfonation (8)
also did not have any effect on the inhibitory activity, in contrast
to the results obtained to date with kotalanol and related ana-
logues.19,23 On the other hand, the decrease in inhibitory activities
l
not show any significant improvement.
2. Results and discussion
The required benzyl-protected deoxynojirimycin derivative 11
was prepared using literature methods.27 The other coupling part-
ner, the heptitol-derived cyclic sulfate 12, was prepared from
of 7 and 8 compared to kotalanol (Ki = 0.19 0.03 l
M)19 suggests
the importance of the five-membered ring thiocyclitol moiety with
a permanent positive charge found in the kotalanol class of
compounds. These results corroborate our recent conclusion from
D
-perseitol, as reported earlier.16 The coupling reaction of 11 with
the cyclic sulfate 12 was performed in acetone in the presence of
PMBO
NH
OPMB OPMB
PMBO
OPMB OPMB
H
N
OSO3
OBn
O
O
BnO
BnO
BnO
acetone, K2CO3
60 oC
Ph
O
S
O
O
O
O
BnO
OBn
O
OBn
OBn
Ph
11
12
13
1) 80% TFA/CH2Cl2
2) H2/Pd, 80% AcOH
OH OH
OH
OH OH
OH
Cl
1) 5% methanolic HCl
2) Amberlyst-A26/MeOH
OSO3
OH
OH OH
OH
OH
OH OH
NH
NH
HO
HO
HO
HO
OH
OH
7
8
Scheme 1. Synthesis of target compounds 7 and 8.

S. Mohan et al. / Bioorg. Med. Chem. 18 (2010) 7794–7798
7797
was performed with Silica Gel 60 (230–400 mesh). High resolution
mass spectra were obtained by the electrospray ionization method,
using an Agilent 6210 TOF LC/MS high resolution magnetic sector
mass spectrometer.
H
N
OH
OH OH
OH
6
4
5
R
1
HO
HO
R =
2
3
OH
OSO3 OH OH
7
3.2. Enzyme kinetics
Figure 4. Conformation of iminocyclitol moiety in solution.
Activity of recombinant N-terminal domain of maltase-gluco-
amylase (ntMGAM) was determined using the glucose oxidase as-
say18 to follow the production of glucose from maltose upon
addition of the enzyme (0.8 nM). A no-inhibitor control and five
different inhibitor concentrations were used in combination with
7 different maltose concentrations (ranging from 1.5 to 24 mM).
A reaction time of 60 min at 37 °C was employed. Reactions were
linear within this time frame. The weights of compounds 7 and 8
were adjusted for the presence of KOAc. Values of Ki and standard
deviations were determined by the program GraFit 4.0.14 (Eritha-
cus Software)18 which employs nonlinear fitting of the data for
each inhibitor concentration to the Michaelis–Menten equation.
Competitive inhibition was confirmed by examination of the
Lineweaver–Burk plot. Ki values were determined by the equation
Ki = [I]/[(Kmobs/Km) + 1], where Kmobs is the Km in the presence of
inhibitor.
OH OH OH
OH OH OH
OH
OH
Cl
X
OH
O
X
OH OH
OH
HO
HO
SO3
HO
OH
HO
4, X = S; Ki = 0.19 µM
14, X = NH; Ki = 90 µM
15, X = Se; Ki = 0.08 µM
5, X = S; Ki = 0.03 µM
16, X = NH; Ki = 0.06 µM
17, X = Se; Ki = 0.02 µM
Figure 5. Kotalanol and de-O-sulfonated kotalanol and their nitrogen and selenium
analogues.
X-ray crystallographic studies that the 3T2 enzyme-bound confor-
mation of the five-membered thiocyclitol moiety of the kotalanol
class of compounds more closely resembles the 4H3 conformation
of the proposed transition state for the formation of an enzyme–
substrate covalent intermediate in the glycosidase hydrolase
family 31 (GH31)-catalyzed reaction (see Fig. 3C).19
3.3. 70-[(1,5-Dideoxy-1,5-imino-
D
-glucitol)-5-N-ammonium]-70-
deoxy-
D
-perseitol-50-sulfate (7)
The cyclic sulfate 1216 (403.5 mg, 0.56 mmol) and the benzyl-
protected deoxynojirimycin derivative 1127 (266 mg, 0.51 mmol)
were dissolved in acetone (2.5 mL), and anhydrous K2CO3
(20 mg) was added. The mixture was stirred in a sealed tube in
an oil bath at 60 °C for 5 days. The solvent was removed under re-
duced pressure, and the product was purified through a short silica
column with EtOAc/MeOH (95:5) as eluent to yield the protected
ammonium salt (13, 202 mg, 73% yield, based on the recovered
150 mg of unreacted starting material 11). However, the coupled
product 13 was found to be unstable, probably due to partial
deprotection of the PMB protecting groups, as indicated by thin
layer chromatography (TLC). Hence, without any further character-
ization, the protected compound 13 (200 mg, 0.16 mmol) was stir-
red in a mixture of 80% trifluoroacetic acid (3 mL) and CH2Cl2
(1 mL) at room temperature for 3 h. When starting material 13
had been fully consumed, all of the volatile components were re-
moved under reduced pressure, the crude material was dissolved
in 80% acetic acid (20 mL), and the solution was stirred with 10%
Pd/C catalyst (400 mg) under H2 at 100 psi for 4 days. The catalyst
was then removed by filtration and all of the volatile components
were removed under reduced pressure. The crude compound was
purified by column chromatography (EtOAc/MeOH/H2O 6:3:1) to
give the desired compound 7 as a pale-yellow foam (65 mg, con-
taining 8 wt % of KOAc by 1H NMR, 85% yield after correcting for
The effect of heteroatom on inhibitory activities in the
five-membered ring series also deserves comment. We note that
the five-membered ring nitrogen analogue (14, Fig. 5)23of kotalanol
showed less inhibitory activity (Ki = 90 lM against ntMGAM) than
the corresponding sulfur (kotalanol, 4) or selenium (15, Fig. 5)23
congeners, and even less compared to the six-membered ring
nitrogen analogue (7). The shorter C–N bond length compared to
C-S and C-Se bond lengths must position 14 unfavorably in the
active-site. Removal of the sulfate moiety allows repositioning of
the de-O-sulfonated nitrogen analogue (16, Fig. 5), as with the sul-
fur (5) and selenium (17) congeners (Fig. 5) to allow better electro-
static contacts with the positively charged heteroatom and the
catalytic aspartate residue, as well as more intimate contacts with
the polyhydroxylated chain,19 and gives a Ki value that is of similar
magnitude to those for 5 and 17.23 We contend, therefore, that the
conformation of all of the five-membered ring compounds resem-
bles the proposed 4H3 transition state for the formation of an en-
zyme–substrate covalent intermediate in the GH31-catalyzed
reaction. This is apparently not the case for the six-membered ring
ground-state mimics (miglitol, 619 and the nitrogen analogues 7
and 8 reported here), or the analogous six-membered ring, sulfur
and selenium congeners which show higher Ki values.18
acetate content).
½
a 2D3
ꢂ
= +4.1 (c 0.25, H2O). 1H NMR (D2O,
3. Experimental section
3.1. General methods
0
0
600 MHz, pH >8 by adding K2CO3): d 4.59 (1H, br d, J3 ,2 = 6.0 Hz,
H-30), 4.28 (1H, ddd, J2 ,1 a = 3.6, J2 ,1 b = 9.0 Hz, H-20), 4.04 (1H, dd,
0
0
0
0
0
0
J6a,6b = 12.6, J6a,5 = 3.0 Hz, H-6a), 4.0 (1H, br d, J4 ,5 = 10.2 Hz,
H-40), 3.99 (1H, br dd, H-60), 3.86 (1H, dd, J6b,5 = 3.6 Hz, H-6b),
Optical rotations were measured at 23 °C and reported in
deg dmꢀ1 gꢀ1 cm3. 1H and 13C NMR spectra were recorded at 600
and 150 MHz, respectively. All assignments were confirmed with
the aid of two-dimensional 1H, 1H (COSY) and/or 1H, 13C (HSQC)
experiments using standard pulse programs. Processing of the
spectra was performed with MestRec and/or MestReNova software.
Analytical thin layer chromatography (TLC) was performed on alu-
minum plates precoated with Silica Gel 60F-254 as the adsorbent.
The developed plates were air-dried, exposed to UV light and/or
sprayed with a solution containing 1% Ce(SO4)2 and 1.5% molybdic
acid in 10% aqueous H2SO4, and heated. Column chromatography
3.78 (1H, dd, J5 ,6 = 1.2 Hz, H-50), 3.70 (2H, m, H-70a and H-70b),
3.62 (1H, ddd, J2,1a = 4.8, J2,1b = 10.8, J2,3 = 9.0 Hz, H-2), 3.39 (1H,
dd, J4,3 = 9.0, J4,5 = 10.2 Hz, H-4), 3.31 (1H, dd, H-3), 3.24 (1H, dd,
0
0
J1 a,1 b = 15.0, H-10a), 3.17 (1H, dd, J1a,1b = 12.0, H-1a), 2.78 (1H, dd,
H-10b), 2.50 (1H, ddd, H-5), 2.43 (1H, dd, H-1b). 13C NMR (D2O,
150 MHz, pH >8 by adding K2CO3): d 167.8 K2CO3, 79.2 (C-30),
78.2 (C-3), 70.0 (C-4), 69.8 (C-60), 68.9 (C-50), 68.8 (C-20), 68.6 (C-
40), 68.5 (C-2), 65.9 (C-5), 63.3 (C-70), 57.9 (C-6), 56.1 (C-1), 53.2
(C-10). HRMS calcd for C13H27NO13SNa (M+Na): 460.1095. Found:
460.1092.
0
0

7798
S. Mohan et al. / Bioorg. Med. Chem. 18 (2010) 7794–7798
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3.4. 70-[(1,5-Dideoxy-1,5-imino-
deoxy-D-perseitol chloride (8)
D
-glucitol)-5-N-ammonium]-70-
Council of Sri Lanka: Colombo, 1981. p 77.
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Compound 7 (50 mg, 0.11 mmol) was stirred in 5% methanolic
HCl (3 mL) at room temperature for 3.5 h. The solvent was evapo-
rated and the residue was treated with Amberlyst A-26 resin
(60 mg, chloride form) in MeOH (1 mL). After stirring for 2.5 h,
the resin was removed by filtration and the solvent was evaporated
to give compound 8 as a pale-yellow foam in quantitative yield
7. Yoshikawa, M.; Murakami, T.; Shimada, H.; Matsuda, H.; Yamahara, J.; Tanabe,
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(42 mg). ½a 2D3
ꢂ
= +8.1 (c 0.5, H2O). 1H NMR (D2O, 600 MHz, pH >8
by adding K2CO3): d 4.05 (1H, dd, J6a,6b = 12.6, J6a,5 = 3.0 Hz, H-6a),
4.01 (1H, ddd, J2 ,1 a = 4.8, J2 ,1 b = 7.2, J2 ,3 = 7.8 Hz, H-20), 3.99 (1H,
0
0
0
0
0
0
9. Ozaki, S.; Oe, H.; Kitamura, S. J. Nat. Prod. 2008, 71, 981.
10. Muraoka, O.; Xie, W.; Tanabe, G.; Amer, M. F. A.; Minematsu, T.; Yoshikawa, M.
Tetrahedron Lett. 2008, 49, 7315.
br dd, J6 ,7 a = J6 ,7 b = 6.0 Hz, H-60), 3.89 (1H, d, J4 ,5 = 9.6 Hz, H-40),
3.88 (1H, dd, J6b,5 = 3.0 Hz, H-6b), 3.81 (1H, d, H-30), 3.70 (2H, br
d-like, H-70a and H-70b), 3.66 (1H, br d, H-50), 3.60 (1H, ddd,
J2,1a = 4.8, J2,1b = 10.8, J2,3 = 9.0 Hz, H-2), 3.39 (1H, dd, J4,3 = 9.0,
0
0
0
0
0
0
11. Yuasa, H.; Takada, J.; Hashimoto, H. Tetrahedron Lett. 2000, 41, 6615.
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0
0
J4,5 = 9.6 Hz, H-4), 3.31 (1H, dd, H-3), 3.23 (1H, dd, J1 a,1 b = 14.4,
H-10a), 3.20 (1H, dd, J1a,1b = 12.0, H-1a), 2.68 (1H, dd, H-10b), 2.45
(1H, ddd, H-5), 2.40 (1H, dd, H-1b). 13C NMR (D2O, 150 MHz, pH
>8 by adding K2CO3): d 164.9 K2CO3, 78.2 (C-3), 72.0 (C-30), 70.2
(C-60), 69.9 (C-4), 69.2 (C-50), 68.4 (C-2, C-40), 68.1 (C-20), 66.1
(C-5), 63.3 (C-70), 57.6 (C-6), 56.2 (C-1), 53.9 (C-10). HRMS calcd
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for C13H28NO10 (Mꢀ Clꢀ): 358.1713. Found: 358.1720.
þ
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We are grateful to the Canadian Institutes for Health Research
(CIHR) and the Heart and Stroke Foundation of Ontario Grant
# NA-6305 for financial support of this work.
Supplementary data
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