Concise synthesis of (-)-steviamine and analogues and their glycosidase inhibitory activities

Article abstract of DOI:10.1039/c3ob40374b

A concise synthesis of (-)-steviamine is reported along with the synthesis of its analogues 10-nor-steviamine, 10-nor-ent-steviamine and 5-epi-ent-steviamine. These compounds were tested against twelve glycosidases (at 143 μg mL^-1 concentrations) and were found to have in general poor inhibitory activity against most enzymes. The 10-nor analogues however, showed 50-54% inhibition of α-l-rhamnosidase from Penicillium decumbens while one of these, 10-nor-steviamine, showed 51% inhibition of N-acetyl-β-d-glucosaminidase (from Jack bean) at the same concentration (760 μM). This journal is The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40374b

Organic &
Biomolecular Chemistry
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Concise synthesis of (−)-steviamine and analogues and
their glycosidase inhibitory activities†
Cite this: Org. Biomol. Chem., 2013, 11,
3826
Nadechanok Jiangseubchatveera,a,b Marc E. Bouillon,a Boonsom Liawruangrath,b
Saisunee Liawruangrath,c Robert J. Nashd and Stephen G. Pyne*a
A concise synthesis of (−)-steviamine is reported along with the synthesis of its analogues 10-nor-stevi-
amine, 10-nor-ent-steviamine and 5-epi-ent-steviamine. These compounds were tested against twelve
glycosidases (at 143 μg mL−1 concentrations) and were found to have in general poor inhibitory activity
against most enzymes. The 10-nor analogues however, showed 50–54% inhibition of α-L-rhamnosidase
from Penicillium decumbens while one of these, 10-nor-steviamine, showed 51% inhibition of N-acetyl-
β-D-glucosaminidase (from Jack bean) at the same concentration (760 μM).
Received 21st February 2013,
Accepted 24th April 2013
DOI: 10.1039/c3ob40374b
www.rsc.org/obc
Introduction
(−)-Steviamine 1 is the most recent member of the polyhy-
droxylated indolizidine natural products (Fig. 1). Steviamine
was isolated from the leaves of Stevia rebaudiana (Asteraceae)
and its absolute configuration was established by X-ray crystal-
lographic analysis of its hydrobromide salt.1,2 (−)-Steviamine 1
is the first polyhydroxylated indolizidine to have a methyl
group at C-5 and a hydroxymethyl group at C-3. This group of
alkaloids which includes, swainsonine 2, castanosperimine 3
and lentiginosine 4 (Fig. 1) have potential utility as antidia-
betic, antiviral, anticancer and immunoregulatory agents.3
Unlike swainsonine 2, steviamine 1 and its synthesised enan-
tiomer ((+)-steviamine), have shown relatively weak to modest
glycosidase inhibitory activity against a number of different
glycosidases.4 The most potent activity found in this study was
against β-galactosidase (from rat intestinal lactase), where ent-
steviamine had an IC50 value of 35 μM.4 While, ent-steviamine4
and some of its analogues, including 10-nor-steviamine 5 (and
some of its 1,2,3,8a-epimers),5 5-epi-ent-steviamine 74 and 1,3-
di-epi-10-(4-methoxyphenyl)steviamine6 have been synthesized
recently, (−)-steviamine 1 itself has not been previously pre-
pared. We report here a concise synthesis of (−)-steviamine 1
Fig. 1 Representative polyhydroxylated indolizidine natural products (1–4) and
synthetic analogues (5–7).
aSchool of Chemistry, University of Wollongong, Wollongong, New South Wales 2522,
Australia. E-mail: spyne@uow.edu.au
and the synthesis of three analogues, 10-nor-steviamine 5, 10-
nor-ent-steviamine 6 and 5-epi-ent-steviamine 7 (Fig. 1) and
their activities against a panel of twelve glycosidases.
bDepartment of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai
University, Chiang Mai, 50200, Thailand
cDepartment of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai,
50200, Thailand
dPhytoquest Limited, IBERS, Aberystwyth University, Plas Gogerddan, Aberystwyth
SY23 3EB, Ceredigion, Wales, UK
†Electronic supplementary information (ESI) available: Experimental details for
the synthesis of compounds 8 and 15. Copies of the 1H and 13C NMR spectra of
all new compounds, NOESY/ROSY spectra of compounds 14 and 19. See DOI:
10.1039/c3ob40374b
Results and discussion
The synthesis of (−)-steviamine 1 started with a Petasis
boronic acid Mannich reaction (PBAMR)7,8 between the known
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unreacted 11a by column chromatography. A ring-closing
metathesis reaction of diene 11a using 18 mol% Grubbs’
second generation catalyst, in the presence of Ti(Oi-Pr)4 (0.2
equivalents)8b,10 to deactivate the amino group in 11a, gave the
indolizidine 12a in 76% yield. Hydrogenation/hydrogenolysis
8c
of 12a, over PdCl2/H2 gave (−)-steviamine 1 in quantitative
yield after neutralization/purification by basic ion-exchange
chromatography.
The NMR spectroscopic data of synthetic 1, matched very
closely (1H NMR 0.1 ppm consistent differences, 13C NMR
0.04 ppm consistent differences) to those of the natural
product2 (see the ESI†). Further the specific rotation of the syn-
thetic material, [α]2D5 −23.8 (c 1.0, MeOH), was of the same sign
and close in magnitude to that of the natural product (lit.2
[α]2D0 −22 (c 1.0, MeOH). Thus the first synthesis of (−)-stevi-
amine 1 has been achieved in four synthetic steps from com-
pounds 8, 9a and 10. Since compound 8 was prepared in four
steps (45% overall yield (see ESI†)) from commercially available
β-L-ribofuranose-1,2,3,5-tetra-O-acetate, this synthesis rep-
resents an eight step total synthesis of steviamine 1 from com-
mercially available starting materials with an over yield of
17%. This concise strategy was further employed to prepare
the analogues 5, 6 and 7.
Treatment of a mixture of 8, 4-buten-1-amine·HCl 9b and
10 under the aforementioned PBAMR conditions gave the
amino diol 11b as a single diastereomer in 59% yield
(Scheme 1). This compound was converted to 10-nor-stevi-
amine 5, in an analogues fashion, in 30% overall yield
(Scheme 1). While this compound had a specific rotation of
[α]2D5 −7.7 (c 1.0, H2O), similar to that reported in the literature
([α]2D5 −8.7 (c 1.2, H2O),5 there were significant differences in
the 1H NMR spectral data recorded in D2O (see ESI†). The
most significant difference was the relative chemical shifts for
the protons H-1 and H-9a and H-9b in the range of δ ∼ 3.8–3.9.
In our sample the H9 protons were observed as dd resonances
(J = 12.0, 5.0–5.5 Hz) at δ 3.87 and δ 3.81 while the H-1 reson-
ance at δ 3.82 (apparent t, J = 6.0 Hz) was observed at a chemi-
cal shift in between those of the two H-9 resonances. The
literature, however, reported the H9 protons as dd resonances
(J = 12.2, 5.0–5.5 Hz) at δ 3.83 and δ 3.77 with the H-1 signal
being the most downfield of this group at δ 3.87 (apparent t,
J = 5.5 Hz). Further, H-8a resonated at δ 2.67–2.72 (m) in our
sample while the literature value for the chemical shift of this
proton was δ 2.97 (s). The 13C NMR chemical shifts were also
significantly different with chemical shift differences varying
from −1.9 to 0.2 ppm (see ESI†). Since NOESY and ROESY
NMR experiments on our sample of 5 were not unequivocal in
defining the stereochemistry of our compound, because of the
closeness of the individual resonances, and because the NMR
chemical shifts of these types of polyhydroxylated compounds
in D2O can vary with pH and concentration,8d,f we prepared 14,
the triacetate derivative of 5 (Scheme 1). ROESY NMR experi-
ments in CDCl3 clearly indicated the assigned stereochemistry
of 14. Significant cross peaks were observed between H-8a and
both H-5β and H-9, which clearly supported the relative syn-
stereochemical relationship between these three protons (Fig. 2).
Scheme 1 Synthesis of steviamine 1 and 10-nor-steviamine 5 and its triacetate
derivative 14.
L-β-ribofuranose derivative 89 ((3S,4R,5S)-4-(benzyloxy)-5-(ben-
zyloxymethyl)tetrahydrofuran-2,3-diol) and commercially avail-
able (R)-4-penten-2-amine·HCl 9a and E-styrylboronic acid 10
(Scheme 1). Stirring these three components in the presence
of triethylamine (to generate the free amine of 9a) in ethanol
at rt for 4 d, gave, after purification of the crude reaction
mixture by column chromatography, the amino alcohol 11 in
77% yield, as a single diastereomer. Shorter reaction times
(1–2 d) and other solvents (e.g. MeOH, CH2Cl2 and MeCN) gave
lower yields. The configuration at the newly created, amino
group bearing, stereogenic centre in 11a, that would become
C-8a in the final target 1, was assumed to the desired one
based on reports that the PBAMR normally provides 1,2-anti-
amino alcohol products via a boronate intermediate, similar to
A, as shown in Scheme 1.7,8 This assumption was later con-
firmed to be correct in the eventual execution of the synthesis
of 1. Treatment of 11a with 1.07 equivalents of methanesulfo-
nyl chloride and 3.5 equivalents of triethylamine,8g followed by
warming of the O-mesylate intermediate to 40–45 °C for 4 h
provided the fully substituted pyrrolidine 12a in 66% yield
after separation of small amounts of O,N-dimesylated 11a and
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NMR spectroscopic data of 5 and 6 and those of 14 and 19
were identical, allowing for slight spectrometer variations.
While the optical rotations of 5 and 6 were opposite in sign
they varied significantly in magnitude (see Experimental
section), however those of compounds 14 and 19, which could
be purified on silica gel using organic solvents, were essen-
tially equal and opposite in sign (14: [α]2D5 +9.4 (c 0.2, CHCl3);
19: [α]2D5 −9.2 (c 0.2, CHCl3)). These results suggested that the
samples of 5 or 6 may be different hydrates resulting in incor-
rect mass measurements. Repeated purifications of these
samples did not provide more closely matching specific
rotations.
Fig. 2 Significant ROESY cross-peaks of compound 14 (SPARTAN generated
structure using a DFT calculation (B3LYP/6-31G** level)).
Glycosidase inhibition studies
The results of our glycosidase inhibitor testing for (−)-stevi-
amine 1, its analogues 5–7 and swainsonine 2, ent-2 and casta-
nosperimine 3 against twelve glycosidases are shown in
Table 1. These mean % inhibition tests were determined for
each compound at 143 μg mL−1 according to previously pub-
lished protocols.11 In general compounds 1 and 5–7 were
found to have poor inhibitory activity against most enzymes.
None were as active as (−)-swainsonine 2 against α-L-rhamnosi-
dase (from Penicillium decumbens) or (+)-swainsonine (ent-2)
against α-D-mannosidase (from Jack bean) or castanosperi-
mine 3 against α-D-glycosidase (from Bacillus sterothermophi-
lus) and almond β-D-glycosidase. The 10-nor analogues, 5 and
6, however, showed 50–54% inhibition of α-L-rhamnosidase
from Penicillium decumbens while 10-nor-steviamine 5, showed
51% inhibition of N-acetyl-β-D-glucosaminidase (from bovine
kidney) at the same concentration (760 μM). It is interesting
that the enantiomeric compounds 5 and 6 give almost equal
inhibition of α-L-rhamnosidase whereas 7, with the extra
methyl group, is a much weaker inhibitor. Both 5 and 6 have
two equivalent hydroxyls to ent-swainsonine (ent-2) and swain-
sonine 2, respectively and yet they both follow ent-swainsonine
in inhibition of α-L-rhamnosidase and not α-mannosidase.
(−)-Steviamine 1 does not show significant inhibition of any
glycosidase tested; it could be that it has a biological function
in the source plant inhibiting a glycosidase we have not tested
against or it is clear that iminosugars can be functional
without glycosidase inhibition and in fact this lack of glycosi-
Scheme 2 Synthesis of 5-epi-ent-steviamine 7, 10-nor-ent-steviamine 6 and its
triacetate derivative 19.
For the synthesis of 5-epi-ent-steviamine 7 the known D-β-ribo- dase inhibition (or high selectivity) may make them more suit-
furanose derivative 159 ((3R,4S,5R)-4-(benzyloxy)-5-(benzyloxy- able as pharmaceutical products.12
methyl)tetrahydrofuran-2,3-diol) was treated with (R)-4-penten-
Interestingly, all compounds appeared to promote the
2-amine·HCl 9a, triethylamine and E-styrylboronic acid 10 in activities of certain enzymes. In particular, compounds 1 and
ethanol solution at rt for 4 d to give the amino diol 16a in 82% 6 seemed to significantly promote the activity of α-D-glucosi-
yield (Scheme 2). This compound was readily converted to dase (from Bacillus sterothermophilus). This promotion of
5-epi-ent-steviamine 7 in three efficient steps according to the activity could be due to enzyme stabilisation or improved
protocols developed in Scheme 1. The overall yield of 7 was folding of the enzyme via non-catalytic site binding.
38% from 15 or 24% from D-β-ribofuranose. The NMR spectro-
The inhibitory activity of compound 5, which was prepared
scopic data of 7 agreed well with those reported2 (see ESI†), previously,5 and had different NMR properties to ours, was
the specific rotation of 7 ([α]2D5 −4.6 (c 1.0, MeOH)) was of reported to have no inhibitory activity against two α-D-glucosi-
the same sign and of similar low magnitude to that reported dases (from Baker’s yeast and rice), one β-D-glucosidase (from
([α]2D0 −1.2 (c 1.0, MeOH)).2
sweet almonds) and a β-D-galactosidase (from bovine liver).
10-Nor-ent-steviamine 6 and its triacetate derivative 19 were These results are consistent with our results shown in Table 1.
prepared in an analogous fashion from 15 (Scheme 2). The The earlier report showed that compound 5 was a more
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Table 1 The glycosidase inhibition of compounds 1, and 5–7 (Mean % Inhibition at 143 μg mL−1
)
Enzyme (source, pH)
1
7
5
6
ent-2
2
3
α-D-Glucosidase (Saccharomyces cerevisiae, 6.8)
α-D-Glucosidase (Bacillus sterothermophilus, 6.8)
α-D-Glucosidase (rice, 4.0)
β-D-Glucosidase (Almond (Prunus sp.), 5.0)
α-D-Galactosidase (Green coffee bean (Coffea sp.), 6.5)
β-D-Galactosidase (Bovine liver, 7.3)
α-D-Mannosidase (Jack bean (Canavalia ensiformis), 4.5)
β-D-Mannosidase (Cellullomonas fimi, 6.5)
α-L-Rhamnosidase (Penicillium decumbens, 4.0)
N-Acetyl-β-D-glucosaminidase (Bovine kidney, 4.25)
N-Acetyl-β-D-glucosaminidase (Jack bean, 5.0)
β-Glucuronidase (Bovine liver, 5.0)
0
0
18
−9
0
−15
6
0
0
20
0
30
0
0
5
0
0
12
0
−4
−3
11
2
100
13
3
3
15
2
2
1
0
80
6
−27
−6
0
0
0
11
−13
6
0
11
−5
22
−8
11
12
19
31
0
53
−9
51
0
−38
−6
0
0
20
9
90
−3
16
9
—
39
—
—
—
7
100
−5
4
5
24
—
0
50
−19
−17
0
—
significant inhibitor of α-L-rhamnosidase (from Penicillium Analytical TLC was performed with aluminium backed Merck
decumbens, IC50 35 μM)) and α-D-mannosidase (from Jack 254 sorbent silica gel. TLC plates were visualized by ultraviolet
F
bean, IC50 82 μM)) and a significantly weaker inhibitor of light or by treatment with acidified, aqueous solution of
α-L-fucosidase (from bovine kidney, IC50 593 μM)). Our com- ammonium molybdate and cerium(IV) sulfate, followed by
pound 5 also showed some, although very weak, activity development with a 1400 Watt heat gun. Chromatographic
against α-D-mannosidase (from Jack bean, only 31% inhibition purification of products was carried out by flash column
at 708, μM Table 1) and an IC50 of approximately 708 μM (53% chromatography on silica gel (70–230 mesh). Basic ion-
inhibition) against the α-L-rhamnosidase from Penicillium exchange chromatography was performed using Amberlyst
decumbens.
A-26(OH) resin. Infrared spectra were recorded as neat samples
on a MIRacle 10 Shimadzu Spectrophotometer. NMR spectra
were measured in CDCl3 (with TMS as internal standard) or
D2O (with MeOH as internal standard) on a Varian VNMRS
PS54-500 or a Varian INOVA-500 (1H at 500 MHz, 13C at
125 MHz) magnetic resonance spectrometer. Chemical shifts
(δ) are reported in ppm, and coupling constants (J) are in Hz.
The following abbreviations were used to explain the multipli-
cities: s = singlet, d = doublet, t = triplet, q = quartet, m = mul-
tiplet. Low-resolution mass spectra were obtained on a Waters
LCZ single quadrupole (ESI+). High-resolution mass spectra
(HRMS) were recorded on a Waters QTOF (ESI+), a Waters Xevo
(ESI+) or a Waters Xevo (ASAP). Polarimetry was carried out
using a JASCO P-2000 Digital Polarimeter and the measure-
ments were made at the sodium D-line with a 1 dm path
length cell. Concentrations (c) are given in grams per 100 mL.
(2S,3R,4R,E)-1,3-Bis(benzyloxy)-5-((R)-pent-4-en-2-ylamino)-
7-phenylhept-6-ene-2,4-diol (11a). To a solution of 8 (1.00 g,
3.03 mmol, see ESI† for synthesis details) in absolute ethanol
(25 mL) was added (R)-pent-4-en-2-amine·hydrochloride 9a
(368 mg, 3.03 mmol, a commercial sample from NetChem,
Inc. USA, >95% ee, [α]2D5 +4.0 (c 1.0, EtOH)) followed by Et3N
(0.42 mL, 3.03 mmol) and trans-2-phenylvinyl boronic acid (10)
(448 mg, 3.03 mmol, commercial sample from Aldrich). The
mixture was stirred at rt for 4 d, followed by the evaporation of
all volatiles in vacuo. The residue was dissolved in CH2Cl2
(10 mL) and washed with sat. aq. NaHCO3 (2 × 10 mL). The
organic layer was dried (MgSO4), filtered and concentrated in
vacuo to afford a brown foam. Purification by flash column
chromatography (increasing polarity from 0 : 100 to 20 : 80
MeOH–CH2Cl2 as eluent) afforded the title compound (1.16 g,
77%) as a yellow foam. Rf 0.48 (10 : 90 MeOH–CH2Cl2).
[α]2D5 +47.9 (c 2.0, CHCl3). IR (cm−1): 3289, 3072, 1452, 1072,
1028. 1H NMR (500 MHz, CDCl3) δ 7.41–7.20 (m, 15H), 6.47 (d,
Conclusions
In conclusion, a concise and efficient four step synthesis of
natural (−)-steviamine 1 has been developed from the readily
accessible L-β-ribofuranose derivative 8. This synthetic protocol
involves a highly anti-selective Petasis reaction, and efficient
ring-closing metathesis and O-mesylate cyclization reactions to
prepare the piperidine and pyrrolidine rings, respectively. This
synthetic protocol allowed for the synthesis of the (−)-stevi-
amine analogues 10-nor-steviamine, 10-nor-ent-steviamine and
5-epi-ent-steviamine. These compounds were tested against
twelve glycosidases (at 143 μg mL−1 concentrations) and were
found in general to have poor inhibitory activity against most
enzymes. The 10-nor analogues however, showed 50–54% inhi-
bition of α-L-rhamnosidase from Penicillium decumbens while
one of these, 10-nor-steviamine, showed 51% inhibition of
N-acetyl-β-D-glucosaminidase (from bovine kidney) at the same
concentration (457 μM).
Experimental section
General information
All reagents were used as received from commercial sources
without further purification. Solvents were purchased as
Analytical Reagents (AR) grade. Petrol refers to the hydro-
carbon fraction of bp 40–60 °C. Tetrahydrofuran was stored
over KOH pellets until needed, then distilled over sodium wire
under nitrogen, using benzophenone as indicator. Anhydrous
CH2Cl2 and MeOH were purchased from Aldrich. Reactions
were stirred using Teflon-coated magnetic stirring bars.
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J = 16.0 Hz, 1H), 6.16 (dd, J = 16.0, 9.0 Hz, 1H), 5.75–5.67 (m, the title compound (294 mg, 76%) as a brown oil. Rf 0.28 (1 : 4
1H), 5.05 (d, J = 16.0 Hz, 1H), 5.04 (d, J = 12.0 Hz, 1H), 4.63, petrol–EtOAc). [α]2D5 +38.6 (c 1.4, CHCl3). IR (cm−1): 3382, 3015,
1
4.54 (ABq, JAB = 12.0 Hz, 2H), 4.56, 4.48 (ABq, JAB = 11.5 Hz, 2928, 2874, 2316, 1496, 1451, 1152, 1055. H NMR (500 MHz,
2H), 4.06–4.03 (m, 1H), 3.95 (t, J = 4.5 Hz, 1H), 3.78 (dd, J = CDCl3) δ 7.34–7.21 (m, 10H), 5.79–5.76 (m, 1H), 5.53 (d, J =
9.0, 5.0 Hz, 1H), 3.75–3.66 (ma, 3H), 2.88–2.76 (m, 1H), 10.5 Hz, 1H), 4.93 (d, J = 9.5 Hz, 1H), 4.71, 4.45 (ABq, JAB
=
2.22–2.10 (ma, 2H), 1.04 (d, J = 6.5 Hz, 3H) [a indicates the over- 11.5 Hz, 2H), 4.69, 4.54 (ABq, JAB = 12.0 Hz, 2H), 4.12 (dd, J =
lapping of signals]. 13C NMR (125 MHz, CDCl3) δ 138.4, 138.2, 9.5, 4.5 Hz, 1H), 3.97 (dd, J = 9.5, 4.5 Hz, 1H), 3.91 (brs, 1H),
136.5, 135.1, 133.9, 128.7, 128.5, 128.0, 127.9, 127.7, 126.5, 3.58 (d, J = 9.0 Hz, 1H), 3.41 (dd, J = 9.5, 3.0 Hz, 1H), 3.34 (brd,
127.5, 117.9, 80.0, 74.0, 73.7, 73.0, 71.7, 69.1, 59.6, 49.2, 42.1, J = 9.5 Hz, 1H), 3.27–3.20 (m, 1H), 2.04–1.98 (m, 1H), 1.77–1.72
19.2. MS (ESI +ve) m/z 502.4 (M + H+, 100%). HRMS (ESI +ve) (m, 1H), 1.17 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3)
calculated for C32H40NO4 (M + H+) 502.2957, found 502.2938.
δ 138.4, 137.5, 129.1, 128.4, 128.3, 128.2, 127.8, 127.6, 127.7,
(2R,3R,4S,5R)-4-(Benzyloxy)-5-(benzyloxymethyl)-1-((R)-pent- 78.2, 74.0, 71.5, 71.5, 70.6, 66.7, 55.7, 50.6, 26.1, 21.3. MS
4-en-2-yl)-2-styrylpyrrolidin-3-ol (12a). To a solution of 11a (ESI +ve) m/z 380.2 (M + H+, 100%). HRMS (ESI +ve) calculated
(1.02 g, 2.02 mmol) in anhydrous CH2Cl2 (6 mL) at 0 °C was for C24H30NO3 (M + H+) 380.2226, found 380.2214.
added Et3N (0.99 mL, 7.08 mmol) under an atmosphere of N2.
(1R,2S,3R,5R,8aR)-3-(Hydroxymethyl)-5-methyloctahydro-
The mixture was then cooled to −10 °C followed by the indolizine-1,2-diol((−)-steviamine) (1). To a solution of 13a
addition of a 0.13 M solution of methanesulfonyl chloride in (183.5 mg, 0.484 mmol) in MeOH (10 mL) was added PdCl2
anhydrous CH2Cl2 (17.0 mL, 2.18 mmol MeSO2Cl). After com- (171.5 mg, 0.967 mmol). The mixture was stirred at rt under an
plete addition the reaction mixture was gradually warmed to atmosphere of H2 (balloon) for 3 h. The mixture was filtered
40 °C over 3 h and stirred for further 30 min with heating through a pad of Celite and the solids were washed with
under a gentle reflux. The solution was subsequently concen- MeOH. The combined filtrates were evaporated in vacuo and
trated in vacuo to afford a brown oil. Purification by flash the residue was dissolved in water (10 mL) and applied to a
column chromatography (increasing polarity from 10 : 90 to column of Amberlyst A-26 (OH−) resin (3 cm). Elution with
20 : 80 EtOAc–petrol and 20 : 80 MeOH–CH2Cl2 as eluent) water followed by evaporation in vacuo afforded the title com-
afforded the title compound (642.2 mg, 66%) as a yellow oil. pound (98.0 mg, 100%) as a brown oil. [α]2D5 −23.8 (c 1.0,
Rf 0.62 (3 : 7 EtOAc–petrol). [α]2D5 −29.4 (c 1.0, CHCl3). IR MeOH) (lit.2 [α]D25 −22.0 (c 1.0, MeOH)). IR (cm−1): 3329, 2929,
(cm−1): 3393, 3062, 3027, 2906, 2869, 1496, 1452, 1365, 1178, 2855, 1631, 1441, 1379, 1315, 1214, 1137, 1097, 1079, 1036,
1138, 1116, 1098, 1055, 1026. 1H NMR (500 MHz, CDCl3) 1006.
δ 7.35–7.19 (m, 15H), 6.52 (d, J = 16.0 Hz, 1H), 5.97 (dd, J =
1H NMR (500 MHz, D2O) δ 4.33 (t, J = 7.5 Hz, 1H, H-2), 3.96
16.0, 9.0 Hz, 1H), 5.84–5.76 (m, 1H), 5.03 (d, J = 17.0 Hz, 1H), (dd, J = 12.5, 5.5 Hz, 1H, H-9), 3.91 (dd, J = 12.0, 3.5 Hz, 1H,
4.97 (d, J = 10.0 Hz, 1H), 4.76, 4.48 (ABq, JAB = 12.0 Hz, 2H), H-9′), 3.80 (t, J = 7.0 Hz, 1H, H-1), 3.52 (dd, J = 9.5, 6.5 Hz, 1H,
4.62 (d, J = 10.5 Hz, 1H), 4.59, 4.55 (ABq, JAB = 12.0 Hz, 2H), H-3), 2.85–2.82 (m, 1H, H-5), 2.67–2.64 (m, 1H, H-8a), 2.00
4.15 (t, J = 5.5 Hz, 1H), 3.97 (dd, J = 10.5, 5.5 Hz, 1H), 3.88 (d, (brd, J = 12.5 Hz, 1H, H-8), 1.81 (brd, J = 13.0 Hz, 1H, H-7),
J = 9.0 Hz, 1H), 3.62–3.56 (ma, 3H), 3.08–3.02 (m, 1H), 1.74 (brd, J = 13.0 Hz, 1H, H-6), 1.42–1.34 (m, 1H, H-7′),
2.40–2.34 (m, 1H), 2.17–2.11 (m, 1H), 1.03 (d, J = 6.5 Hz, 1H) 1.21–1.12 (ma, 5H, H-6′, H-8′ and CH3) [a = overlapping of
a
[
indicates the overlapping of signals]. 13C NMR (125 MHz, signals]. 13C NMR (125 MHz, D2O) δ 74.1 (C-1), 69.3 (C-2), 66.9
CDCl3) δ 138.5, 137.6, 137.3, 137.2, 132.5, 130.5, 128.7, 128.5, (C-8a), 61.5 (C-3), 56.7 (C-9), 52.8 (C-5), 33.6 (C-6), 29.5 (C-8),
128.4, 127.9, 127.7, 127.4, 126.4, 115.7, 77.9, 74.9, 73.8, 71.4, 23.9 (C-7), 19.4 (CH3). MS (ESI +ve) m/z 202.0 (M + H+, 100%).
70.0, 68.0, 58.9, 51.4, 39.6, 17.9. MS (ESI +ve) m/z 484.3 HRMS (ESI +ve) calculated for C10H20NO3 (M + H+) 202.1443,
(M + H+, 100%). HRMS (ESI +ve) calculated for C32H38NO3 found 202.1465.
(M + H+) 484.2852, found 484.2832.
((2S,3R,4R,E)-1,3-Bis(benzyloxy)-5-(but-3-enylamino)-7-phenyl-
((1R,2S,3R,5R,8aR)-2-(Benzyloxy)-3-(benzyloxymethyl)-5-methyl- hept-6-ene-2,4-diol (11b) and its enantiomer 16b. To a solu-
1,2,3,5,6,8a-hexahydroindolizin-1-ol (13a). To a solution of 12a tion of 8 (30.4 mg, 0.09 mmol) in absolute EtOH (0.75 mL) was
(490.7 mg, 1.02 mmol) in anhydrous CH2Cl2 (36 mL) was added 3-butenylamine.hydrochloride (9b) (9.7 mg, 0.09 mmol)
added via syringe
a
solution of Ti(Oi-Pr)4 (0.06 mL, followed by Et3N (0.013 mL, 0.09 mmol) and trans-2-phenylvi-
0.203 mmol) in anhydrous CH2Cl2 (11 mL). The above solution nyl boronic acid (10) (13.3 mg, 0.09 mmol). The mixture was
was stirred at rt for 0.5 h, then added Grubbs II catalyst stirred at rt for 4 d, followed by the evaporation of all volatiles
(155.3 mg, 0.183 mmol). The reaction mixture was heated at in vacuo. The residue was dissolved in CH2Cl2 (4 mL) and
reflux at 45 °C for 2.5 h, when TLC analysis showed complete washed with sat. aq. NaHCO3 (2 × 4 mL). The organic layer was
consumption of 12a. The reaction mixture was then diluted dried (MgSO4), filtered and concentrated in vacuo to afford a
with CH2Cl2 (125 mL) and washed with sat. aq. NaHCO3 brown foam. Purification by flash column chromatography
(87 mL). The aqueous layer was further extracted with CH2Cl2 (increasing polarity from 0 : 100 to 20 : 80 MeOH–CH2Cl2 as
(125 mL). The organic layers were dried (MgSO4) and concen- eluent) afforded the title compound (26.3 mg, 59%) as a brown
trated in vacuo to afford a dark brown oil as a crude product. foam. Rf 0.45 (10 : 90 MeOH–CH2Cl2). [α]2D5 +51.5 (c 1.3,
Purification by flash column chromatography (increasing CHCl3). IR (cm−1): 3376, 3029, 1452, 1088, 1072, 1028.
polarity from 50 : 50 to 0 : 100 petrol–EtOAc as eluent) afforded 1H NMR (500 MHz, CDCl3) δ 7.39–7.19 (m, 15H), 6.51 (d,
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J = 16.0 Hz, 1H), 6.23 (dd, J = 16.0, 9.0, 1H), 5.77–5.67 (m, 1H), sat. aq. NaHCO3 (11 mL). The aqueous layer was further
5.07 (d, J = 18.0 Hz, 1H), 5.03 (d, J = 10.5 Hz, 1H), 4.64, 4.56 extracted with CH2Cl2 (17 mL). The organic layers were dried
(ABq, JAB = 12.5 Hz, 2H), 4.55, 4.47 (ABq, JAB = 11.5 Hz, 2H), (MgSO4) and concentrated in vacuo to afford a dark brown oil.
4.04–4.00 (ma, 2H), 3.75–3.68 (ma, 3H), 3.65 (dd, J = 7.0, Purification by flash column chromatography (increasing
4.5 Hz, 1H), 2.76–2.71 (m, 1H), 2.60–2.55 (m, 1H), 2.26 (q, J = polarity from 20 : 80 to 10 : 90 petrol–EtOAc and 20 : 80 MeOH–
7.0 Hz, 2H) [a = overlapping of signals]. 13C NMR (125 MHz, CH2Cl2 as eluent) afforded the title compound (30.9 mg, 62%)
CDCl3) δ 138.5, 138.0, 136.4, 135.7, 134.6, 128.8, 128.5, 128.1, as a brown oil. Rf 0.28 (1 : 4 petrol: EtOAc). [α]2D5 +108.9 (c 0.2,
128.0, 127.9, 127.7, 126.8, 126.8, 117.1, 79.7, 73.8, 73.5, 72.9, CHCl3). MS (ESI +ve) m/z 366.3 (M + H+, 100%). HRMS (ESI
71.7, 68.8, 62.8, 46.0, 33.8. MS (ESI +ve) m/z 488.5 (M + H+, +ve) calculated for C23H28NO3 (M + H+) 366.2069, found
100%). HRMS (ESI +ve) calculated for C31H38NO4 (M + H+) 366.2053. IR (cm−1): 3259, 2922, 2854, 2364, 1731, 1631, 1452,
1
488.2801, found 488.2784.
1362, 1143, 1084, 1025. H NMR (500 MHz, CDCl3) δ 7.36–7.25
Its enantiomer (16b) was prepared as described above using (m, 10H), 5.84–5.82 (m, 1H), 5.62 (d, J = 9.0 Hz, 1H), 4.68, 4.54
15 (see ESI† for syntheses details) as a starting material (ABq, JAB = 11.5 Hz, 2H),4.62, 4.53 (ABq, JAB = 11.5 Hz, 2H),
(0.605 mmol scale). Compound 16b was obtained as a brown 4.15 (dd, J = 7.5, 4.0 Hz, 1H), 3.96 (brs, 1H), 3.77 (brs, 1H), 3.63
foam (167.9 mg, 57%). [α]2D5 −58.7 (c 1.0, CHCl3).
(dd, J = 9.5, 4.5 Hz, 1H), 3.55 (dd, J = 9.5, 3.0 Hz, 1H),
(2R,3R,4S,5R)-4-(Benzyloxy)-5-(benzyloxymethyl)-1-(but-3-enyl)- 3.29–3.27 (m, 1H), 3.04–3.00 (ma, 2H), 2.24–2.22 (m, 1H), 1.77
2-styrylpyrrolidin-3-ol (12b) and its enantiomer 17b. To a solu- (d, J = 17.5 Hz, 1H). [a = overlapping of signals] 13C NMR
tion of 11b (80.0 mg, 0.1641 mmol) in anhydrous CH2Cl2 (125 MHz, CDCl3) δ 138.2, 137.9, 128.5, 128.4, 128.4, 128.1,
(9 mL) at 0 °C was added Et3N (0.023 mL, 0.164 mmol) under 127.9, 127.8, 127.7, 127.2, 78.6, 73.9, 73.4, 72.6, 68.8, 64.2,
an atmosphere of N2. The mixture was then cooled to −10 °C 60.9, 44.6, 19.3.
followed by the addition of a 0.13 M solution of methanesulfo-
Its enantiomer (18b) was prepared from 17b (0.182 mmol
nyl chloride in anhydrous CH2Cl2 (1.51 mL, 0.197 mmol scale) as described as above. Compound 18b was obtained as a
MeSO2Cl). After complete addition the reaction mixture was brown oil (41.8 mg, 63%). [α]2D5 −114.5 (c 0.2, CHCl3).
gradually warmed to 40 °C over 3 h and stirred for further
(1R,2S,3R,8aR)-3-(Hydroxymethyl)octahydroindolizine-1,2-
30 min under gentle reflux. The solution was subsequently diol (10-nor-steviamine) (5) and its enantiomer 6. To a solu-
concentrated in vacuo to afford a brown oil. Purification by tion of 13b (28.6 mg, 0.078 mmol) in MeOH (1.7 mL) was
flash column chromatography (increasing polarity from 10 : 90 added PdCl2 (20.8 mg, 0.117 mmol). The mixture was stirred at
to 20 : 80 EtOAc–petrol and 20 : 80 MeOH–CH2Cl2 as eluent) rt under an atmosphere of H2 (balloon) for 3 h. The mixture
afforded the title compound (63.9 mg, 83%) as a yellow oil. Rf was filtered through a pad of Celite and the solids were
0.52 (3 : 7 EtOAc–petrol). [α]2D5 −18.2 (c 0.7, CHCl3). MS (ESI washed with MeOH. The combined filtrates were evaporated in
+ve) m/z 470.4 (M + H+, 100%). HRMS (ESI +ve) calculated for vacuo and the residue was dissolved in water (1.5 mL) and
C31H36NO3 (M + H+) 470.2695, found 470.2674. IR (cm−1): applied to a column of Amberlyst A-26 (OH−) resin (3 cm).
3405, 3061, 3028, 2863, 1640, 1599, 1495, 1452, 1363, 1099, Elution with water followed by evaporation in vacuo afforded
1055. 1H NMR (500 MHz, CDCl3) δ 7.35–7.20 (m, 15H), 6.56 (d, the title compound (14.6 mg, 100%) as a brown oil. [α]D25 −7.7
J = 16.0 Hz, 1H), 6.03 (dd, J = 16.0, 9.0 Hz, 1H), 5.79–5.72 (m, (c 0.6, H2O), [α]2D5 −11.4 (c 0.6, MeOH) (lit.5 [α]D22 −8.7 (c 1.2,
1H), 4.99 (d, J = 17.0 Hz, 1H), 4.95 (d, J = 10.0 Hz, 1H), 4.76, H2O)). IR (cm−1): 3324, 2929, 1636, 1596, 1445, 1141, 1105,
4.51 (ABq, JAB = 12.0 Hz, 2H), 4.60, 4.57 (ABq, JAB = 12.0 Hz, 1083, 1049, 1007. 1H NMR (500 MHz, D2O) δ 4.40 (t, J =
2H), 4.18 (t, J = 7.0 Hz, 1H), 4.01 (brs, 1H), 3.69–3.61 (ma, 3H), 6.5 Hz, 1H, H-2), 3.87 (dd, J = 12.0, 5.5 Hz, 1H, H-9), 3.82
3.43 (d, J = 8.0 Hz, 1H), 2.78–2.66 (m, 2H), 2.25–2.14 (m, 2H) (t, J = 6.0 Hz, 1H, H-1), 3.81 (dd, J = 12.0, 5.0 Hz, 1H, H-9′),
a
[
indicates the overlapping of signals]. 13C NMR (125 MHz, 3.30 (dd, J = 12.5, 5.5 Hz, 1H, H-3), 2.96–2.93 (m, 1H, H-5α),
CDCl3) δ 138.4, 137.6, 137.1, 136.9, 131.8, 130.3, 128.7, 128.6, 2.76–2.74 (m, 1H, H-8a), 2.72–2.67 (m, 1H, H-5β), 1.87–1.84
128.5, 128.0, 127.9, 127.8, 127.6, 126.5, 115.6, 77.6, 74.7, 73.8, (m, 1H, H-8), 1.80–1.76 (m, 1H, H-7), 1.62–1.58 (m, 1H, H-6),
73.4, 71.8, 66.4, 61.4, 47.9, 33.1.
1.54–1.46 (m, 1H, H-6′), 1.41–1.32 (m, 1H, H-7′), 1.29–1.21
Its enantiomer (17b) was prepared from 16b (0.3341 mmol (m, 1H, H-8′). 13C NMR (125 MHz, D2O) δ 74.5 (C-1), 70.1 (C-2),
scale) as described above. Compound 17b was obtained as a 64.0 (C-3), 63.8 (C-8a), 58.5 (C-9), 47.3 (C-5), 27.2 (C-8), 23.1
yellow oil (90.5 mg, 58%). [α]2D5 +35.4 (c 0.8, CHCl3).
(C-6), 22.7 (C-7). MS (ESI +ve) m/z 188.2 (M + H+, 100%). HRMS
(1R,2S,3R,8aR)-2-(Benzyloxy)-3-(benzyloxymethyl)-1,2,3,5,6,8a- (ESI +ve) calculated for C9H18NO3 (M + H+) 188.1287, found
hexahydroindolizin-1-ol (13b) and its enantiomer 18b. To a 188.1288.
solution of 12b (63.9 mg, 0.136 mmol) in anhydrous CH2Cl2
Its enantiomer 6 (10-nor-ent-steviamine) was prepared from
(4.8 mL) was added via syringe a solution of Ti(Oi-Pr)4 18b (0.102 mmol scale) as described above. Compound 6 was
(0.008 mL, 0.027 mmol) in anhydrous CH2Cl2 (1.6 mL). The obtained as a brown oil (19.2 mg, 100%). [α]2D5 +23.8 (c 0.6,
above solution was stirred at rt for 0.5 h, then Grubbs II cata- MeOH).
lyst was added (13.84 mg, 0.016 mmol). The reaction mixture
(1R,2S,3R,8aR)-3-(Acetoxymethyl)octahydroindolizine-1,2-
was heated at reflux at 45 °C for 2.5 h, when TLC analysis diyldiacetate (14) and its enantiomer 19. To a solution of 5
showed complete consumption of 12b. The reaction mixture (3.0 mg, 0.0160 mmol) in dry pyridine (0.06 mL, 0.68 mmol)
was then diluted with CH2Cl2 (17 mL) and washed with was added Ac2O (0.06 mL, 0.64 mmol). The mixture was stirred
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at rt for 18 h followed by the evaporation of all volatiles. The was added Et3N (0.23 mL, 1.68 mmol) under an atmosphere of
oily residue was purified by flash column chromatography N2. The mixture was then cooled to −10 °C followed by the
(increasing polarity from 100 : 0 to 0 : 100 petrol–EtOAc and addition of a 0.13 M solution of methanesulfonyl chloride in
20 : 80 MeOH–CH2Cl2 as eluent) to afford the title compound anhydrous CH2Cl2 (4.10 mL, 0.529 mmol MeSO2Cl). After com-
(4.3 mg, 86%) as a yellow oil. Rf 0.38 (1 : 1 petrol: EtOAc). plete addition the reaction mixture was gradually warmed to
[α]2D5 +9.4 (c 0.2, CHCl3). MS (ESI +ve) m/z 314.3 (M + H+, 40 °C over 3 h and stirred for further 30 min under gentle
100%). HRMS (ESI +ve) calculated for C15H23NO6 (M + H+) reflux. The solution was subsequently concentrated in vacuo to
314.1604, found 314.1595. IR (cm−1): 2935, 2855, 1738, 1440, afford a brown oil. Purification by flash column chromato-
1369, 1220, 1148, 1131, 1091, 1038. 1H NMR (500 MHz, graphy (increasing polarity from 10 : 90 to 20 : 80 EtOAc–petrol
CDCl3) δ 5.44 (t, J = 6.5 Hz, 1H, H-2), 4.98 (dd, J = 6.5, 4.0 Hz, and 20 : 80 MeOH–CH2Cl2 as eluent) afforded the title com-
1H, H-1), 4.26 (dd, J = 11.5, 6.5 Hz, 1H, H-9), 4.09 (dd, J = pound (161 mg, 69%) as a brown oil. Rf 0.62 (3 : 7 EtOAc–
11.5, 5.5 Hz, 1H, H-9′), 3.59 (dd, J = 13.0, 6.0 Hz, 1H, H-3), petrol). [α]2D5 +20.0 (c 0.5 CHCl3). IR (cm−1): 3384, 3062,
3.04 (d, J = 12.5 Hz, 1H, H-5α), 2.93 (dt, J = 11.5, 4.0 Hz, 3028, 2931, 2869, 1725, 1640, 1495, 1452, 1174, 1140, 1087,
1
1H, H-8a), 2.75–2.69 (m, 1H, H-5β), 2.07 (s, 3H, Ac), 2.05 1054, 1026. H NMR (500 MHz, CDCl3) δ 7.37–7.20 (m, 15H),
(s, 6H, 2Ac), 1.82–1.75 (ma, 2H, H-6, H8), 1.49–1.42 (ma, 6.51 (d, J = 16.5 Hz, 1H), 5.98 (dd, J = 16.0, 9.0 Hz, 1H),
2H, H-7), 1.36–1.26 (m, 1H, H-6′), 1.23–1.15 (m, 1H, H-8′) 5.81–5.72 (m, 1H), 4.97 (d, J = 15.5 Hz, 1H), 4.95 (d, J =
a
[
indicates the overlapping of signals]. 13C NMR (125 MHz, 11.5 Hz, 1H), 4.77, 4.49 (ABq, JAB = 12.0 Hz, 2H), 4.71 (d, J =
CDCl3) δ 170.8 (CvO), 170.4 (CvO), 169.9 (CvO), 75.3 10.0 Hz, 1H), 4.61, 4.57 (ABq, JAB = 11.5 Hz, 2H), 4.19 (dd,
(C-1), 70.2 (C-2), 62.8 (C-8a), 61.7 (C-9), 59.6 (C-3), 47.7 (C-5), J = 8.0, 5.5 Hz, 1H), 3.99 (dd, J = 8.0, 6.5 Hz, 1H), 3.91 (d, J =
28.3 (C-8), 23.9 (C-6), 23.1 (C-7), 21.1 (CH3), 20.9 (CH3), 9.0 Hz, 1H), 3.62–3.54 (ma, 3H), 3.06–2.99 (m, 1H), 2.43–2.38
20.7 (CH3).
(m, 1H), 1.98–1.92 (m, 1H), 1.12 (d, J = 7.0 Hz, 1H) [a indicates
Its enantiomer 19 was prepared from 6 (0.027 mmol scale) the overlapping of signals]. 13C NMR (125 MHz, CDCl3)
as described above. Compound 19 was obtained as a yellow oil δ 138.5, 137.5, 137.3, 137.3, 132.5, 130.6, 128.7, 128.6, 128.5,
(5.3 mg, 63%)). [α]2D5 −9.2 (c 0.2, CHCl3).
128.0, 127.9, 127.7, 127.5, 126.4, 116.0, 77.8, 74.9, 73.8, 71.5,
(2R,3S,4S,E)-1,3-Bis(benzyloxy)-5-((R)-pent-4-en-2-ylamino)-7- 70.5, 68.1, 59.8, 52.1, 40.4, 18.5. MS (ESI +ve) m/z 484.3
phenylhept-6-ene-2,4-diol (16a). To a solution of 15 (200 mg, (M + H+, 100%). HRMS (ESI +ve) calculated for C32H38NO3
0.605 mmol) in absolute ethanol (5 mL) was added (R)-pent-4- (M + H+) 484.2852, found 484.2837.
en-2-amine.hydrochloride (9a) (73.6 mg, 0.605 mmol) followed
(1S,2R,3S,5R,8aS)-2-(Benzyloxy)-3-(benzyloxymethyl)-5-methyl-
by Et3N (0.084 mL, 0.605 mmol) and trans-2-phenylvinyl 1,2,3,5,6,8a-hexahydroindolizin-1-ol (18a). To a solution of 17a
boronic acid (10) (89.6 mg, 0.605 mmol). The mixture was (62.5 mg, 0.129 mmol) in anhydrous CH2Cl2 (4.4 mL) was
stirred at rt for 4 d, followed by the evaporation of all volatiles added via syringe
a
solution of Ti(Oi-Pr)4 (0.008 mL,
in vacuo. The residue was dissolved in CH2Cl2 (5 mL) and 0.026 mmol) in anhydrous CH2Cl2 (1.5 mL). The above solu-
washed with sat. aq. NaHCO3 (2 × 5 mL). The organic layer was tion was stirred at rt for 0.5 h, then Grubbs II catalyst was
dried (MgSO4), filtered and concentrated in vacuo to afford a added (19.8 mg, 0.023 mmol). The reaction mixture was
brown foam. Purification by flash column chromatography heated at reflux at 45 °C for 2.5 h, when TLC analysis showed
(increasing polarity from 0 : 100 to 20 : 80 MeOH–CH2Cl2 as complete consumption of 17a. The reaction mixture was then
eluent) afforded the title compound (248 mg, 82%) as a brown diluted with CH2Cl2 (16 mL) and washed with sat. aq. NaHCO3
oil. Rf 0.45 (10 : 90 MeOH–CH2Cl2). [α]2D5 −35.7 (c 0.8, CHCl3). (11 mL). The aqueous layer was further extracted with CH2Cl2
IR (cm−1): 3366, 3062, 3029, 2925, 2863, 1641, 1599, 1495, (16 mL). The organic layers were dried (MgSO4) and concen-
1
1452, 1373, 1092, 1072. H NMR (500 MHz, CDCl3) δ 7.39–7.16 trated in vacuo to afford a dark brown oil. Purification by flash
(m, 15H), 6.48 (d, J = 16.0 Hz, 1H), 6.24 (dd, J = 16.0, 9.0 Hz, column chromatography (increasing polarity from 20 : 80 to
1H), 5.78–5.69 (m, 1H), 5.08 (d, J = 11.5 Hz, 1H), 5.07 (d, J = 10 : 90 petrol–EtOAc as eluent) afforded the title compound
14.5 Hz, 1H), 4.63, 4.55 (ABq, JAB = 12.5 Hz, 2H), 4.52, 4.45 (23.8 mg, 83%) as a brown oil. Rf 0.28 (1 : 4 EtOAc–petrol). [α]D25
(ABq, JAB = 11.0 Hz, 2H), 4.05–4.02 (m, 1H), 3.95 (t, J = 4.0 Hz, −7.7 (c 0.9, CHCl3). IR (cm−1): 3376, 3030, 2925, 2869, 2358,
1H), 3.82 (dd, J = 8.5, 4.0 Hz, 1H), 3.76–3.69 (ma, 2H), 3.64 (dd, 1636, 1453, 1375, 1265, 1207, 1087, 1060, 1027. 1H NMR
J = 7.5, 4.5 Hz, 1H), 2.82–2.78 (m, 1H), 2.25–2.20 (m, 1H), (500 MHz, CDCl3) δ 7.36–7.25 (m, 10H), 5.73–5.70 (m, 1H),
2.12–2.07 (m, 1H), 1.06 (d, J = 6.5 Hz, 3H) [a indicates the over- 5.60 (dd, J = 10.0, 1.0 Hz, 1H), 4.68, 4.57 (ABq, JAB = 12.0 Hz,
lapping of signals]. 13C NMR (125 MHz, CDCl3) δ 138.4, 138.0, 2H), 4.68, 4.60 (ABq, JAB = 12.0 Hz, 2H), 4.13 (dd, J = 8.0, 4.0
136.5, 134.6, 133.8, 128.7, 128.5, 128.4, 128.0, 127.9, 127.5, Hz, 1H), 3.96 (brs, 1H), 3.71 (brs, 1H), 3.63 (dd, J = 9.5, 5.0 Hz,
126.7, 126.5, 127.6, 118.0, 79.5, 73.8, 73.7, 72.8, 71.6, 68.6, 1H), 3.55 (brd, J = 8.5 Hz, 1H), 3.25–3.21 (m, 1H), 3.04 (brs,
60.2, 49.4, 39.5, 21.2. MS (ESI +ve) m/z 502.3 (M + H+, 100%). 1H), 2.37–2.32 (m, 1H), 1.63 (m, 1H), 1.18 (d, J = 7.0 Hz, 3H).
HRMS (ESI +ve) calculated for C32H40NO4 (M + H+) 502.2957, 13C NMR (125 MHz, CDCl3) δ 138.4, 138.2, 128.5, 128.4, 127.9,
found 502.2941.
127.8, 127.7, 125.3, 78.4, 73.8, 73.2, 72.6, 69.3, 62.3, 60.6, 48.8,
(2S,3S,4R,5S)-4-(Benzyloxy)-5-(benzyloxymethyl)-1-((R)-pent- 25.6, 20.0. MS (ESI +ve) m/z 380.2 (M + H+, 100%). HRMS (ESI
4-en-2-yl)-2-styrylpyrrolidin-3-ol (17a). To a solution of 16a +ve) calculated for C24H30NO3 (M + H+) 380.2226, found
(241 mg, 0.481 mmol) in anhydrous CH2Cl2 (1.6 mL) at 0 °C 380.2227.
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(1S,2R,3S,5R,8aS)-3-(Hydroxymethyl)-5-methyloctahydro-
Notes and references
indolizine-1,2-diol (7). To
a solution of 18a (37.0 mg,
0.098 mmol) in MeOH (2 mL) was added PdCl2 (34.6 mg,
0.195 mmol). The mixture was stirred at rt under an atmos-
phere of H2 (balloon) for 3 h. The mixture was filtered through
a pad of Celite and the solids were washed with MeOH. The
combined filtrates were evaporated in vacuo and the residue
was dissolved in water (1.5 mL) and applied to a column of
Amberlyst A-26 (OH−) resin (3 cm). Elution with water
followed by evaporation in vacuo afforded the title compound
(19.6 mg, 100.0%) as a brown oil. [α]2D5 −4.6 (c 1.0, MeOH), lit.4
[α]2D5 −1.2 (c 1.0, MeOH). IR (cm−1): 3352, 2931, 2863, 1629,
1596, 1455, 1381, 1339, 1121, 1096, 1059, 1028. 1H NMR
(500 MHz, D2O) δ 4.37 (t, J = 6.0 Hz, 1H, H-2), 4.09 (dd, J =
10.5, 5.5 Hz, 1H, H-1), 3.77 (dd, J = 11.0, 9.5 Hz, 1H, H-9),
3.60 (dd, J = 11.0, 5.5 Hz, 1H, H-9′), 3.29–3.25 (m, 1H, H-3),
3.00–2.97 (m, 1H, H-8a), 2.55–2.51 (m, 1H, H-5), 1.77–1.75
(m, 1H, H-8), 1.71–1.63 (ma, 2H, H-6, H-8′), 1.56–1.52 (m, 1H,
H-7), 1.45–1.37 (m, 1H, H-7′), 1.16–1.08 (m, 1H, H-6′), 1.02 (d,
J = 6.0 Hz, 3H, CH3) [a indicates the overlapping of signals].
13C NMR (125 MHz, D2O) δ 70.6 (C-1), 69.8 (C-2), 66.2 (C-3),
60.4 (C-9), 58.5 (C-8a), 55.1 (C-5), 32.0 (C-6), 22.7 (C-8), 20.4
(CH3), 18.1 (C-7). MS (ESI +ve) m/z 202.1 (M + H+, 100%).
HRMS (ESI +ve) calculated for C10H20NO3 (M + H+) 202.1443,
found 202.1450.
1 A. L. Thompson, A. Michalik, R. J. Nash, F. X. Wilson,
R. van Well, P. Johnson, G. W. J. Fleet, C.-Y. Yu, X.-G. Hu,
R. I. Cooper and D. J. Watkin, Acta Crystallogr., Sect. E:
Struct. Rep. Online, 2009, 65, o2904–o2905.
2 A. Michalik, J. Hollinshead, L. Jones, G. W. J. Fleet,
C.-Y. Yu, X.-G. Hu, R. van Well, G. Horne, F. X. Wilson,
A. Kato, S. F. Jenkinson and R. J. Nash, Phytochem. Lett.,
2010, 3, 136–138.
3 N. Asano, in Modern Alkaloids: Structure, Isolation, Synthesis
and Biology, ed. E. Fattorusso and O. Taglialatela-Scafati,
Wiley-VCH Verlag, Weinheim, 2008, pp. 111–138.
4 X.-G. Hu, B. Bartholomew, R. J. Nash, F. X. Wilson, G. W. J.
Fleet, S. Nakagawa, A. Kato, Y.-M. Jia, R. van Well and
C.-Y. Yu, Org. Lett., 2010, 12, 2562–2565.
5 L. Gómez, X. Garrabou, J. Joglar, J. Bujons, T. Paralla,
C. Vilaplana, P. J. Cardona and P. Clapés, Org. Biomol.
Chem., 2012, 10, 6309–6321.
6 A. Chronowska, E. Gallienne, C. Nicolas, A. Kato, I. Adachi
and O. R. Martin, Tetrahedron Lett., 2011, 52, 6399–6402.
7 N. A. Petasis and I. A. Zavialov, J. Am. Chem. Soc., 1998, 120,
11798–11799.
8 For our previous use of the Petasis boronic acid Mannich
reaction in alkaloid synthesis see: (a) A. S. Davis, S. G.
Pyne, B. W. Skelton and A. H. White, J. Org. Chem., 2004,
69, 3139–3143; (b) C. W. G. Au and S. G. Pyne, J. Org.
Chem., 2006, 71, 7097–7099; (c) A. D. Davis, T. Ritthiwigrom
and S. G. Pyne, Tetrahedron, 2008, 64, 4868–4879; (d) T.
Ritthiwigrom and S. G. Pyne, Org. Lett., 2008, 10, 2769–
2771; (e) T. Machan, A. S. Davis, B. Liawruangrath and
S. G. Pyne, Tetrahedron, 2008, 64, 2725–2732; (f) T.
Ritthiwigrom, A. C. Willis and S. G. Pyne, J. Org. Chem.,
2010, 75, 815–824; (g) C. W. G. Au, R. J. Nash and S. G.
Pyne, Chem. Commun., 2010, 46, 713–715.
Glycosidase inhibition assay11
All enzymes and para-nitrophenyl substrates were purchased
from Sigma, with the exception of β-mannosidase which came
from Megazyme. Enzymes were assayed at 27 °C in 0.1 M citric
acid/0.2 M disodium hydrogen phosphate buffers at the
optimum pH for the enzyme. The incubation mixture con-
sisted of 10 μL enzyme solution, 10 μL of 1 mg mL−1 aqueous
solution of test compound and 50 μL of the appropriate 5 mM
para-nitrophenyl substrate made up in buffer at the optimum
pH for the enzyme. The reactions were stopped by addition of
9 S. Bachir-Lesage, P. Godé, G. Goethals, P. Villa and
P. Martin, J. Carbohydr. Chem., 2003, 22, 35–46.
70 μL 0.4 M glycine (pH 10.4) during the exponential phase of 10 Q. Yang, W.-J. Xiao and Z. Yu, Org. Lett., 2005, 7, 871–874.
the reaction, which had been determined at the beginning 11 A. A. Watson, R. J. Nash, M. R. Wormald, D. J. Harvey,
using uninhibited assays in which water replaced inhibitor.
Final absorbances were read at 405 nm using a Versamax
microplate reader (Molecular Devices). Assays were carried out
S. Dealler, E. Lees, N. Asano, H. Kizu, A. Kato, R. C.
Griffiths, A. J. Cairns and G. W. J. Fleet, Phytochemistry,
1997, 46, 255–259.
in triplicate, and the values given are means of the three repli- 12 R. J. Nash, A. Kato, C.-Y. Yu and G. W. J. Fleet, Future Med.
cates per assay.
Chem., 2011, 3, 1513–1521.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 3826–3833 | 3833