Synthesis and glycosidase inhibitory activity of noeurostegine - A new and potent inhibitor of β-glucoside hydrolases

Article abstract of DOI:10.1039/b918576c

A new, stable hemi-aminal nor-tropane christened noeurostegine was synthesised in 22 steps from levoglucosan and tested for inhibitory activity against glycoside hydrolases. Sweet almond and Thermotoga maritimaβ- glucosidases, coffee bean α-galactosidase,

Full text of DOI:10.1039/b918576c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and glycosidase inhibitory activity of noeurostegine†—a new and
potent inhibitor of b-glucoside hydrolases‡
Tina Secher Rasmussen and Henrik Helligsø Jensen*
Received 8th September 2009, Accepted 19th October 2009
First published as an Advance Article on the web 23rd November 2009
DOI: 10.1039/b918576c
A new, stable hemi-aminal nor-tropane christened noeurostegine was synthesised in 22 steps from
levoglucosan and tested for inhibitory activity against glycoside hydrolases. Sweet almond and
Thermotoga maritima b-glucosidases, coffee bean a-galactosidase, and Asp. oryzae b-galactosidase were
inhibited in the low micromolar region but significant tightening of binding to Ki 50 nM for almond
b-glucosidase was found to occur after pre-incubation. Yeast a-glucosidase and E. coli b-galactosidase
were not inhibited at 1 mM.
undergo Amadori rearrangement.8,9 Structurally similar, naturally
occuring nor-tropane glycoside hydrolase inhibitor calystegine B2
(3, Fig. 1),10,11 however, has been shown to be stable, possibly due
Introduction
The medicinal potential of modulating glycoside hydrolase activity
by small molecules is commonly recognised,1 and valuable activity
to the bicyclic structure preventing iminium ion formation (Bredt’s
towards diseases like HIV, cancer, diabetes, influenza and lysomal
rule).
storage disorders have been found.2 Significant progress in design
and synthesis of new glycoside hydrolase inhibitors have occurred
and the field remains a highly active area of research. Among
the most potent compounds targeting these enzymes are synthetic
Calystegine B2 has recently been crystallographically shown
to bind to Thermotoga maritima of glycoside hydrolase family
1 (TmGH1) in a ‘noeuromycin binding mode’ rather than the op-
posite ‘1-deoxynojirimycin (4) binding mode’ placing the nitrogen
and naturally occurring piperidine carbohydrate mimics, referred
atom in the position expected to be occupied by the anomeric
carbon of the substrate.12 This allows for tight inhibitor binding
between the hemi-aminal oxygen and the protein but places a
hydroxyl group in a position occupied by the hydroxymethyl
substituent of both a substrate glucoside and noeuromycin (1).
We envisaged13 that a hybrid of noeuromycin (1) and calystegine
B2 (3), being noeurostegine,† (5, Fig. 1) would inherit the potency
of the former and stability of the latter inhibitor provided
that the ethylene bridge can be accommodated by the enzyme
active site. Changing a hydroxyl for a hydroxymethyl substituent
has previously been shown to result in a dramatic increase in
inhibitory potency for, e.g., isofagomine (2),14–16 being three orders
of magnitude better than piperidine triol 6 (Fig. 1).17
to as iminosugars or azasugars depending on the position of the
nitrogen atom.3
It has been shown that a large degree of transition state stabilis-
ation during enzymatic glycoside hydrolysis occurs through the
2-OH of the substrate.4–7 This stabilisation is exploited by one of
the strongest current glycoside hydrolase inhibitors, noeuromycin
(1, Fig. 1).8 A disadvantage of this hemi-aminal inhibitor is its
lack of stability for longer periods of time as it has been shown to
Synthesis
Calystegine B2 has been synthesised previously, but the ele-
gant ring-closing metathesis route by Boyer/Hanna18 and
Skaanderup/Madsen19 especially drew our attention for the syn-
thesis of noeurostegine. However, we decided to introduce nitrogen
as an azide instead of as a carbamate to achieve well resolved NMR
spectra for easy structure elucidation. A known key intermediate
820 was needed for this approach, which could be prepared in a
streamlined approach from levoglucosan (7) in eight steps.21 This
sequence of reactions could be carried out on a multigram scale
without the need for intermediate chromatographic purification.
Known anhydrosugar 8 underwent acid-promoted methan-
olysis followed by iodination to give halide 10 in 87% yield
over two steps (Scheme 1).22 Zinc-mediated fragmentation under
sonication in THF–H2O23 resulted in clean conversion to aldehyde
11, which was used directly in the following Barbier reaction
without purification. This resulted in a separable diastereoisomeric
Fig. 1
Department of Chemistry, Aarhus University, Langelandsgade 140, DK-
8000, Aarhus C, Denmark. E-mail: hhj@chem.au.dk; Tel: +4589423963
† We suggest the name noeurostegine for this new compound since it is a
hybrid between noeuromycin and calystegine B2.
‡ Electronic supplementary information (ESI) available: Experimental
section, NMR spectra, and Michaelis–Menten and Hanes plots. See DOI:
10.1039/b918576c
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Scheme 1 Synthesis of cycloheptene 14S from anhydrosugar 8.
1 : 1.7 mixture of homoallylic alcohols 12R/12S. Configuration
was established by NMR analysis at a later stage. The alcohol
function of the S-isomer was protected as a p-methoxybenzyl
(PMB) ether (13S). The following ring closing metathesis24 was
found to proceed smoothly to give cycloheptene 14S in 84% yield
but required that the homoallylic alcohol function was protected
(Scheme 1).
The cycloheptene (14S) was oxidized to an isomeric mixture of
secondary alcohols by a standard hydroboration/oxidative work-
up protocol in 86% yield. The desired regioisomer 16S was found
to be the major product as only one diastereoisomer in the ratio
of 3 : 1.25 The configuration of the undesired isomer (15S) was not
established, but was also found to be only one diastereoisomer.
Alcohol 16S was protected as a benzoyl ester with benzoyl chloride
(BzCl) in pyridine to afford 17S, which was reacted with DDQ26
to give 18S in 85% over two steps (Scheme 2).
The free secondary alcohol of 18S was substituted for azide
to give 19 with inversion of configuration using diphenyl phos-
phonic azide (DPPA) under Mitsunobu conditions in 76%
yield (Scheme 2). The benzoyl ester was removed by Zemple´n
deacylation27 and the alcohol (20) oxidized by Dess–Martin
periodinane28 (DMP) to ketone 21 in 80% over two steps
(Scheme 3).
spontaneously, making it necessary to raise the pH and impeding
product isolation.29 Accordingly, we chose to spend a further
synthetic step and found that Staudinger conditions30 indeed
caused clean and spontaneous cyclisation, as established by a
hemi-aminal 13C chemical shift of 92.9 ppm rather than a ketone
resonance around 209 ppm. Catalytic hydrogenolysis over Pearl-
man’s catalyst next yielded the desired noeuromycin/calystegine
B2-hybrid (Scheme 3).
It was next investigated whether the minor product from the
Barbier reaction (Scheme 1) could also be used for the synthesis
of noeurostegine 5. Minor homoallylic alcohol 12R was protected
as its PMB-ether (13R) and cyclised using the Hoveyda–Grubbs
catalyst (Scheme 4).24 Hydroboration/oxidation resulted in a
separable 2 : 1 mixture of regioisomers favouring 16R. Again, only
one diastereoisomer of the major isomer was formed, which, after
purification, underwent benzoylation, PMB-ether cleavage with
DDQ26 and oxidation to ketone 23 with DMP in 90% yield over
three steps. The ketone 23 was then reduced with lithium tri-tert-
butoxyaluminium hydride to predominantly give alcohol 18S in
85% yield.
Inhibition studies
To investigate hemi-aminal stability, noeurostegine (5) was dis-
solved in D2O and left for 10 d at room temperature without any
signs of degeneration by 1H-NMR analysis. The nor-tropane 5 was
then evaluated as an inhibitor of commercially available glycoside
hydrolases and the values compared to known compounds.
In an attempt to convert azido ketone 21 directly into the desired
deprotected noeurostegine 5, only the azide and not the benzyl
ethers was found to undergo reduction with H2 over Pearlman’s
catalyst (Pd(OH)2/C). As previously reported by others, we also
found that cyclisation onto the keto functionality did not occur
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Scheme 2 Synthesis of azido cycloheptane 19 from cycloheptene 14S.
Noeurostegine (5) was found to competitively inhibit a-
galactosidase with an inhibition constant (Ki) of 2.5 mM being
somewhat weaker than calystegine B2 (Ki 0.86 mM).31 Also Asp.
oryzae b-galactosidase was inhibited by noeurostegine (5) in the
micromolar region (Ki 23 mM) while E. coli b-galactosidase was
not inhibited at 1 mM (Table 1).
and furthermore is a potent, competitive inhibitor of almond
and Thermotoga maritima b-glucosidase. Yeast a-glucosidase,
however, was not inhibited at 1 mM, making noeurostegine a
highly selective inhibitor, which can possibly be traced back to its
congested bicyclic structure.
Noeuromycin (1) is known to be a very powerful inhibitor of
yeast a-glucosidase but neither calystegine B2 (3)31 nor noeuroste-
gine (5) inhibit this enzyme at 1 mM. This could be due to the
spatial requirement of the ethylene bridge making noeurostegine
(5) too large for the enzyme active site.
b-Glycosidase from Thermotoga maritima (TmGH1) was found
to be inhibited strongly by noeurostegine (5) with an inhibition
constant of 1.1 mM at enzyme optimum (Ki 0.14 mM at noeu-
romycin optimum)32 (Table 1). This is slightly better than what
has been observed for calystegine B2 (3), which suggests that a
hydroxymethyl substituent has a positive influence over a hydroxyl
substituent in inhibitor binding to TmGH1.
General experimental information
All reagents and enzymes, except otherwise stated were used
as purchased without further purification. Levoglucosan was
purchased from Carbosynth Ltd. and enzymes from Sigma-
Aldrich, except TmGH1, which was purchased from Megazyme.
Oven dried glassware (ca. 120 ◦C) was used for reactions carried
out under nitrogen or argon atmosphere. Solvents were dried
according to standard procedures prior to use (‘Purification
of Laboratory Chemicals, 3rd Edition’ D. D. Perrin, W. L. F.
Armarego, 1988, Butterworth-Heinemann Ltd). Dichloro-
methane was dried by distillation over CaH2, and diethyl ether was
dried over sodium wire. THF was dried over sodium and distilled
from benzophenone. Flash chromatography was performed with
Merck silica 60 (230-400 mesh) as stationary phase and TLC was
performed on silica-coated aluminium plates (Merck 60 F254).
TLC plates were first observed in UV-light and then visualised
with ceric sulfate/ammonium molybdate in 10% H2SO4 stain
or KMnO4 stain. 1H-NMR (400 MHz) and 13C-NMR (100
MHz) were recorded on a Varian Mercury 400 spectrometer.
CDCl3 (d 7.26 ppm (CHCl3) for proton and d 77.16 ppm for
carbon resonances) and D2O (d 4.79 ppm for proton) were used
as internal references. Spectra were assigned based on gCOSY,
gHMQC, and DEPT-135 experiments. MS spectra were recorded
on a Micromass LC-TOF instrument by using electrospray
ionization (ESI). High resolution spectra were recorded with
either of the following compounds as internal standard: (Boc-L-
alanine: C8H15NO4Na: 212.0899; BzGlyPheOMe: C19H20N2O4Na:
363.1321; BocSer(OBn)SerLeuOMe: C25H39N3O8Na: 532.2635;
erythromycin: C37H67NO13Na: 756.4510) Masses of standards
and analytes are calculated and reported in Daltons for un-
charged species. Melting points were measured on a Bu¨chi B-540
Initial experiments also established noeurostegine (5) as a
low micromolar inhibitor (Ki 1.5 mM) of almond b-glucosidase.
However, when measuring after pre-incubation of the enzyme with
the inhibitor for 30 min, as described for calystegine B2 (3),31
a
significant tightening of binding (Ki 50 nM) was observed. Slow
onset of inhibition of sweet almond b-glucosidase by nitrogen
containing carbohydrate mimics has previously been thoroughly
described. We speculate that this slow binding process could be
a result of slow almond b-glucosidase dynamics upon binding of
the rigid inhibitor 5. No such slow inhibition was observed for any
other of the enzymes tested in this study.
Conclusion
A new nor-tropane (noeurostegine, 5, Fig. 1) has been synthesised
in 22 steps from levoglucosan through a cycloheptene prepared by
Hoveyda–Grubbs catalyst-mediated ring closing metathesis. We
have demonstrated that the target compound possessing hydroxyl
groups in the 2, 3, 4 and 6 positions corresponding to the enzyme
substrates, is fully stable in aqueous solution for several days
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Table 1 Ki values in nM at pH 6.8. Inhibition was found to be competitive
a-Galctosidase (Green coffee beans)
b-Galactosidase (E. coli)
—
—
860a,b
—
2500
>106
23000
>106
140
b-Galactosidase (Asp. oryzae)
a-Glucosidase (Yeast)
—
—
22b
37
>106
1250
4000c
1200a,b
b-Glucosidase (T. maritima)
88c
69b
1100c
1500
50a
b-Glucosidase (Sweet almonds)
a Pre-incubation (30 min). b pH-value unknown. c pH 5.8.
instrument and are uncorrected. Optical rotation was measured
on a PE-241 polarimeter and reported in units of deg cm2 g-1.
Concentrations are reported in g/100 mL. Sonication was con-
ducted by use of a Branson 1510 ultrasonic bath. Microwave
experiments were carried out on a Biotage Initiator (Biotage,
Sweden). Reaction times listed refer to ‘hold time’ at the specified
temperature.
30 min prior to addition of substrate and monitoring of reaction
rates. KM and K¢M values were obtained by fitting to the equation
v = (Vmax·[S])/(KM+[S]) using the program Sigma Plot 11.0.
Competitive inhibition was established from Hanes plots. Ki values
were calculated as Ki = [I]/((KM¢/KM) - 1) having [I] ª Ki. For
Michaelis–Menten and Hanes plots, see the ESI.‡
Organic synthesis
Inhibition studies
Methyl 3,4-di-O-benzyl-2-C-benzyloxymethyl-2-deoxy-a/b-D-
glucopyranoside (9). Anhydrosugar 8 (2.50 g, 5.59 mmol) was
dissolved in dry methanol containing 10 v/v% sulfuric acid
(70 mL) and the reaction mixture heated to 40 ◦C under a nitrogen
atmosphere for 24 h. The reaction mixture was then poured into
a saturated aq. solution of NaHCO3 (150 mL) and the aqueous
phase extracted with EtOAc (3 ¥ 170 mL). The combined organic
layers were washed with brine (2 ¥ 200 mL), dried over Na2SO4,
filtered and concentrated. The obtained anomeric mixture of 9
was used directly for the next reaction, but could be purified by
flash column chromatography (pentane–EtOAc 5 : 1→4 : 1) to give
the product 9 (2.47 g, 92%) as a 3 : 1 (determined by 13C NMR)
mixture of a/b anomers. LRMS (ES+) calcd. for C29H34O6Na:
501.2, found 501.4. 13C NMR (100 MHz, CDCl3): d (ppm) a:
138.6, 138.4, 138.3 (ArC), 128.6-127.7 (m, ArC), 99.2 (C1), 79.6,
Inhibition constants (Kis) were determined by measuring initial
rates (<10% of substrate conversion) using 2,4-dinitrophenyl,
o-nitrophenyl or p-nitrophenyl glycosides (p-nitrophenyl b-D-
glucopyranoside for almond b-glucosidase, p-nitrophenyl a-
D-glucopyranoside for yeast a-glucosidase, o-nitrophenyl b-D-
galactopyranoside for E. coli b-galactosidase, p-nitrophenyl b-D-
galactopyranoside for Asp. oryzae b-galactosidase, p-nitrophenyl
a-D-galactopyranoside for green coffee bean a-galactosidase, 2,4-
dinitrophenyl b-D-glucopyranoside for TmGH1 b-glucosidase)
at six concentrations ranging from 1 –4 times KM monitoring
4
at 400 nm with A < 1 using a Varian Cary 100 Bio uv-vis
spectrophotometer. Measurements were conducted in 50 mM
phosphate buffer (pH 6.8 or pH 5.8) at 25 ◦C for 2 min. For
experiments with pre-incubation inhibitor and enzyme were mixed
Scheme 3 Synthesis of noeurosegine 5 from cycloheptane 19 via Staudinger reduction.
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Scheme 4 Synthesis sequence for the preparation of 18S from 12R.
78.9, 75.1, 75.0, 73.2, 71.3, 67.9, 62.2, 55.1, 47.0, b: 138.7, 138.5,
138.4 (ArC), 128.6-127.7 (m, ArC), 102.0 (C1), 79.8, 79.2, 75.4,
75.3, 73.3, 68.4, 64.8, 62.3, 57.3, 48.8.
10.0 Hz, H7), 3.64 (dd, 1 H, J2,7¢ = 6.0 Hz, Jgem = 10.0 Hz, H7¢),
2.89 (dq, 1 H, J1,2 = 1.2 Hz, J2,3;2,7;2,7¢ = 6.0 Hz, H2).
(3R,4R,5S,6R/S)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-6-hy-
droxy-1,8-nonadiene (12). To a stirred solution of the intermedi-
ate aldehyde 11 (2.22 g, 5.16 mmol) in 50% aqueous THF (32 mL)
was added indium dust (0.90 g, 7.84 mmol, 1.5 eq) and allyl
bromide (1.10 mL, 13.0 mmol, 2.5 eq). The reaction mixture was
stirred overnight at rt. before a saturated aq. solution of NH4Cl
was added and the aqueous phase neutralised with a saturated aq.
solution of NaHCO3. The resulting mixture was extracted with
CH2Cl2 (4 ¥ 80 mL) and the combined organic phases dried over
MgSO4 and concentrated. The crude product was purified by flash
column chromatography (CH2Cl2) to give the product 12 (2.04 g,
75% (two steps)) as a 4 : 5 mixture of R- and S-diastereoisomers.
Methyl 3,4-di-O-benzyl-2-C-benzyloxymethyl-2,6-dideoxy-6-
iodo-a/b-D-glucopyranoside (10). To a solution of a/b mixture
9 (2.47 g, 5.17 mmol) in dry toluene (100 mL) was added
triphenylphosphine (3.40 g, 12.95 mmol, 2.5 eq), iodine (1.44 g,
5.65 mmol, 1.1 eq) and imidazole (0.71 g, 10.43 mmol, 2.0 eq)
under a nitrogen atmosphere. The reaction mixture was heated
to reflux for 90 min before the yellow mixture became colourless
and turned clear. The reaction mixture was then cooled to rt
and 5% aqueous sodium thiosulfate (50 mL) was added, and
the organic layer washed with brine (2 ¥ 50 mL). The combined
organic phases were dried over MgSO4, filtered and concentrated.
The crude product was purified by flash column chromatography
(pentane–EtOAc 20 : 1) to give the product 10 (2.64 g, 87%) as a
3 : 1 (determined by 13C NMR) mixture of a/b anomers. NMR
data was in accordance with previously reported data.22b
(3R,4R,5S,6R)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-6-hy-
droxy-1,8-nonadiene (12R). Colourless oil (0.81 g, 30%). [a]2D0 -12
(c 1.0, CHCl3). Rf (CH2Cl2) = 0.29. 1H NMR (400 MHz, CDCl3):
d (ppm) 7.37-7.26 (m, 15 H, ArH), 5.98 (m, 1 H, H2), 5.72 (m, 1
H, H8), 5.35 (m, 2 H, H1), 5.02 (m, 2 H, H9), 4.76 (d, 1 H, Jgem
=
(2R,3R,4R)-3,4-Di-O-benzyl-2-C-benzyloxymethyl-2,5,6-tri-
deoxy-hex-5-enal (11). To a solution of iodide 10 (0.56 g,
0.96 mmol) in THF (25 mL) was added pre-activated Zn dust33
(0.69 g, 10.54 mmol, 11.0 eq) and water (2.8 mL). The resulting
suspension was sonicated at 40 ◦C until TLC-analysis showed
full conversion (1 h). The reaction mixture was then diluted with
Et2O (40 mL) and water (15 mL) and the resulting mixture filtered
through a pad of Celite and the organic phase washed with water
(15 mL), brine (15 mL), and dried over Na2SO4 and concentrated.
The isolated aldehyde 11 (0.41 g) was used directly in the next
reaction without further purification. 1H crude NMR (400 MHz,
CDCl3): d (ppm) 9.75 (s, 1 H, CHO), 7.35-7.23 (m, 15 H, ArH),
5.90 (ddd, 1 H, J4,5 = 7.6 Hz, Jcis = 10.6 Hz, Jtrans = 18.0 Hz, H5),
5.32 (m, 2 H, H6), 4.69 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.57
(d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.56 (d, 1 H, Jgem= 11.6 Hz,
OCH2Ph), 4.45 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.39 (d, 1 H,
Jgem= 11.6 Hz, OCH2Ph), 4.28 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph),
11.6 Hz, OCH2Ph), 4.64 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.52
(d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.43 (d, 1 H, Jgem= 11.6 Hz,
OCH2Ph), 4.33 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.32 (d, 1 H,
Jgem= 11.6 Hz, OCH2Ph), 4.10 (dd, 1 H, J3,4 = 4.0 Hz, J2,3
=
7.6 Hz, H3), 3.86 (m, 2 H, H4, H10), 3.88 (dd, 1 H, J5,10¢ = 3.6 Hz,
Jgem = 9.6 Hz, H10¢), 3.71 (m, 1 H, H6), 2.23 (m, 2 H, H7,H7¢),
2.09 (qu, 1 H, J4,5;5,6;5,10;5,10¢ = 3.6 Hz, H5). 13C NMR (100 MHz,
CDCl3): d (ppm) 138.5, 138.1, 137.7 (ArC) 136.1 (C2), 135.4 (C8),
128.5-127.6 (m, ArC), 119.0 (C1), 116.9 (C9), 80.9 (C4), 80.7 (C3),
74.9 (OCH2Ph), 73.5 (OCH2Ph), 71.5 (C6), 70.4 (OCH2Ph), 67.7
(C10), 43.5 (C7), 40.3 (C5). HRMS (ES+): calcd. for C31H36O4Na:
495.2511, found 495.2512.
(3R,4R,5S,6S)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-6-hy-
droxy-1,8-nonadiene (12S). Colourless oil (1.23 g, 45%). [a]D20
-15 (c 1.0, CHCl3). Rf (CH2Cl2–EtOAc 100 : 1) = 0.47. 1H NMR
(400 MHz, CDCl3): d (ppm) 7.30-7.20 (m, 15 H, ArH), 5.76 (m,
2 H, H2, H8), 5.29-5.21 (m, 2 H, H1/H9), 5.05-5.00 (m, 2 H,
H1, H9), 4.87 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.60 (d, 1 H,
4.05 (dd, 1 H, J3,4 = 4.8 Hz, J2,3 = 6.0 Hz, H3), 3.95 (dd, 1 H, J3,4
4.8 Hz, J4,5 = 7.6 Hz, H4), 3.88 (dd, 1 H, J2,7 = 6.0 Hz, Jgem
=
=
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Jgem= 11.6 Hz, OCH2Ph), 4.56 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph),
4.43 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.37 (d, 1 H, Jgem= 11.6 Hz,
OCH2Ph), 4.32 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.05 (m, 1 H,
4.28 (m, 1 H, H3), 4.08 (m, 1 H, H6), 3.82 - 3.70 (m, 6 H, H4, H8,
H8¢, OCH3), 2.42 (m, 2 H, H5, H7), 2.31 (m, 1 H, H7¢). 13C NMR
(100 MHz, CDCl3): d (ppm) 159.2, 139.0, 138.9, 138.8 (ArC),
132.4 (C2), 131.0 (ArC), 129.3-127.5 (m, ArC), 116.7 (C1), 113.8
(ArC), 81.6 (C3), 78.9 (C4), 73.8, 73.2 (OCH2Ph), 73.0 (C6), 72.7,
70.5 (OCH2Ph), 68.6 (C8), 55.4 (OCH3), 48.5 (C5), 31.9 (C7).
HRMS (ES+): calcd. for C37H40O5Na: 587.2773, found 587.2772.
H3), 3.88 (m, 1 H, H4), 3.82 (m, 1 H, H6), 3.68 (dd, 1 H, J5,10
5.2 Hz, Jgem = 9.6 Hz, H10), 3.59 (dd, 1 H, J5,10¢ = 7.2 Hz, Jgem
=
=
9.6 Hz, H10¢), 2.66 (bs, 1 H, OH), 2.23 (m, 2 H, H7, H7¢), 2.06
(m, 1 H, H5). 13C NMR (100 MHz, CDCl3): d (ppm) 138.7, 138.4
(ArC), 135.5, 135.2 (C2, C8), 128.4-127.6 (m, ArC), 119.4, 117.4
(C1, C9), 83.4 (C3), 80.3 (C4), 74.4 (OCH2Ph), 73.3 (OCH2Ph),
71.4 (C6), 70.6 (OCH2Ph), 68.3 (C10), 44.3 (C5), 39.9 (C7). HRMS
(ES+): calcd. for C31H36O4Na: 495.2511, found 495.2523.
(1R/S,3R,4R,5S,6S)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-
6-O-p-methoxybenzyl-cycloheptane-1-ol (15S) and (1R,2R,3R,
4S,5S)-2,3-di-O-benzyl-4-C-benzyloxymethyl-5-O-p-methoxy-
benzyl-cycloheptane-1-ol (16S). BH3·THF complex (1 M solu-
tion in THF, 6.0 mL, 6.0 mmol, 2.0 eq) was added in a drop wise
fashion to a stirred solution of 14S (1.69 g, 3.00 mmol) in dry
THF (60 mL) at 0 ◦C under an atmosphere of nitrogen. After
stirring for 2 h at 0 ◦C, 2 M NaOH (6 mL) and 35% aqueous
H2O2 (12 mL) were added. The reaction mixture was allowed to
reach rt and stirred for additional 2 h before diluted with Et2O
(100 mL). The organic phase was washed with water (3 ¥ 60 mL),
brine (2 ¥ 60 mL), dried over MgSO4, filtered and concentrated.
The crude product was purified by flash column chromatography
(pentane–EtOAc 4 : 1→2 : 1→0 : 1) to give regioisomers 15S and
16S (1.50 g, 86%) as a 1 : 3 mixture which were easily separated.
(3R,4R,5S,6S)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-6-O-p-
methoxybenzyl-1,8-nonadiene (13S). Sodium hydride (60%,
0.44 g, 11.08 mmol, 2.0 eq) was added to a cooled (0 ◦C) and
stirred solution of 12S (2.492 g, 5.28 mmol) in dry DMF (20 mL)
◦
under nitrogen atmosphere. After stirring for 10 min at 0 C, p-
methoxybenzyl chloride (1.44 mL, 10.6 mmol, 2.0 eq) was added.
The reaction mixture was warmed to rt and stirred overnight
before being quenched by addition of n-butylamine (6 mL). The
reaction mixture was diluted with EtOAc (100 mL) before the
organic layer was washed with water (3 ¥ 70 mL), dried over
MgSO4, filtered and concentrated. The crude product was purified
by flash column chromatography (heptane/EtOAc 30 : 1) to give
the product 13S (2.89 g, 92%) as a clear oil. [a]2D0 4 (c 1.0, CHCl3).
Rf (pentane–EtOAc 16 : 1) = 0.38. 1H NMR (400 MHz, CDCl3):
d (ppm) 7.30-7.13 (m, 17 H, ArH), 6.77-6.74 (m, 2 H, ArH), 5.78
(m, 2 H, H2, H8), 5.24 (m, 2 H, H1/H9), 4.99 (m, 2 H, H1/H9),
4.74 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.55 (d, 1 H, Jgem= 12.0 Hz,
OCH2Ph), 4.49 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.43 (d, 1 H,
Jgem= 10.8 Hz, OCH2Ph), 4.33 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph),
(1R/S,3R,4R,5S,6S)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-
6-O-p-methoxybenzyl-cycloheptane-1-ol (15S). Colourless oil
(0.45 g, 26%). [a]2D0 29 (c 1.0, CHCl3). Rf (pentane–EtOAc 2 : 1) =
1
0.18. H NMR (400 MHz, CDCl3): d (ppm) 7.35-7.20 (m, 17 H,
ArH), 6.83 (m, 2 H, ArH), 4.71 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph),
4.62 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.57 (d, 1 H, Jgem= 11.2 Hz,
OCH2Ph), 4.52 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.50 (d, 1 H,
Jgem= 11.2 Hz, OCH2Ph), 4.45 (d, 1 H, Jgem= 12.4 Hz, OCH2Ph),
4.40 (d, 1 H, Jgem= 12.4 Hz, OCH2Ph), 4.37 (d, 1 H, Jgem= 11.2 Hz,
OCH2Ph), 4.13 (m, 1 H, H1), 4.08 (m, 1 H, H6), 3.84 (m, 2 H,
H3, H4), 3.76 (s, 3 H, OCH3), 3.73 (m, 1 H, H8), 3.61 (m, 1 H,
H8¢), 2.28 (m, 2 H, H5, H7), 2.15 (m, 1 H, H2), 1.96 (m, H1,
H2¢), 1.59 (m, 1 H, H7¢). 13C NMR (100 MHz, CDCl3): d (ppm)
159.0, 138.8, 138.7, 138.6, 131.0 (ArC), 129.1-127.5 (ArC), 113.7
(ArC), 80.1, 79.8 (C3, C4), 73.6, 73.1 (OCH2Ph), 71.8 (C6), 71.7,
70.9 (OCH2Ph), 68.5 (C8), 64.1 (C1), 55.3 (OCH3), 46.5 (C5), 40.1
(C7), 38.5 (C2). HRMS (ES+): calcd. for C37H42O6Na: 605.2879,
found 605.2885.
4.25 (d, 1 H, Jgem= 10.8 Hz, OCH2Ph), 4.42, 4.39 (AB, 2 H, JAB
=
12.0 Hz, OCH2Ph), 4.01 (m, 1 H, H3), 3.83 (dd, 1 H, J = 6.8 Hz,
J = 2.7 Hz, H4), 3.76 (m, 1 H, H10), 3.72 (s, 3H, OCH3), 3.63 (q,
1 H, J5,6;6,7,6,7¢ = 5.6 Hz, H6), 3.54 (dd, 1 H, J5,10¢ = 6.8 Hz, Jgem
=
9.6 Hz, H10¢), 2.35 (m, 2 H, H7,H7¢), 2.15 (m, 1 H, H5). 13C NMR
(100 MHz, CDCl3): d (ppm) 159.1, 139.7, 138.9, 138.7 (ArC),
135.9, 135.6 (C2, C8), 131.1 (ArC), 129.5-127.2 (m, ArC), 119.1,
116.9 (C1, C9), 113.8 (ArC), 83.7 (C3), 79.5 (C4), 77.9 (C6), 74.3,
73.1, 71.4, 70.8 (OCH2Ph), 67.9 (C10), 55.4 (OCH3), 43.5 (C5),
36.4 (C7). HRMS (ES+): calcd. for C39H44O5Na: 615.3086, found
615.3075.
(3R,4R,5S,6S)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-6-O-p-
methoxybenzyl-cycloheptene (14S). Diene 13S (0.79 g,
1.33 mmol) and Grubbs’–Hoveyda 2nd generation catalyst
(CAS [301224-40-8], 16 mg, 0.026 mmol, 0.02 eq) were dissolved
in freshly distilled CH2Cl2 (4 mL) in a sealed microwave vial. The
reaction mixture was heated by microwave irradiation for 2 min
(80 ◦C) after which the formed ethylene gas was released; the
microwave irradiation was then continued for 10 min. The solvent
was removed under reduced pressure and the remaining oil was
purified by flash column chromatography (pentane–EtOAc 20 : 1)
to give the desired cycloheptene 14S as an oil (0.63 g, 84%). [a]D20
(1R,2R,3R,4S,5S)-2,3-Di-O-benzyl-4-C-benzyloxymethyl-5-O-
p-methoxybenzyl-cycloheptane-1-ol (16S). Colourless oil (1.05 g,
60%). [a]2D0 37 (c 1.0, CHCl3). Rf (pentane–EtOAc 2 : 1) = 0.46. 1H
NMR (400 MHz, CDCl3): d (ppm) 7.37-7.23 (m, 17 H, ArH), 6.87
(m, 2 H, ArH), 4.92 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.71 (d, 1 H,
Jgem= 11.2 Hz, OCH2Ph), 4.59 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph),
4.56 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.51 (d, 1 H, Jgem= 11.2 Hz,
OCH2Ph), 4.49 (s, 2 H, OCH2Ph), 4.40 (d, 1 H, Jgem= 11.2 Hz,
OCH2Ph), 3.90 (m, 2 H, H3, H5), 3.81 (s, 3 H, OCH3), 3.75 (m, 1
H, H1), 3.70 (m, 2 H, H8, H8¢), 3.45 (m, 1 H, H2), 2.68 (bs, 1 H,
OH), 2.42 (dq, 1 H, J4,5 = 2.4 Hz, J3,4;4,8;4,8¢ = 6.8 Hz, H4), 2.03 (m,
1 H, H6), 1.83 (m, 1 H, H6¢), 1.75 (m, 1 H, H7), 1.58 (m, 1 H, H7¢).
13C NMR (100 MHz, CDCl3): d (ppm) 159.2, 138.6, 138.5, 138.4,
131.0 (ArC), 129.2-127.6 (m, ArC), 113.8 (ArC), 89.4 (C2) 79.8
(C3/C5), 75.0 (OCH2Ph), 74.5 (C3/C5) 73.4, 73.3 (OCH2Ph),
71.3 (C1), 70.8 (OCH2Ph), 68.3 (C8), 55.4 (OCH3), 46.0 (C4), 28.8
1
17 (c 1.0, CHCl3). Rf (pentane–EtOAc 10 : 1) = 0.37. H NMR
(400 MHz, CDCl3): d (ppm) 7.36-7.23 (m, 17 H, ArH), 6.86 (m,
2 H, ArH), 5.75 (m, 1 H, H2), 5.65 (m, 1 H, H1), 4.82 (d, 1 H,
Jgem= 11.0 Hz, OCH2Ph), 4.69 (s, 2 H, OCH2Ph), 4.56 (d, 1 H,
Jgem= 11.0 Hz, OCH2Ph), 4.47 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph),
4.47 (s, 2 H, OCH2Ph), 4.42 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph),
438 | Org. Biomol. Chem., 2010, 8, 433–441
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13
=
(C6), 26.8 (C7). HRMS (ES+): calcd. for C37H42O6Na: 605.2879,
found 605.2874.
C NMR (100 MHz, CDCl3): d (ppm) 165.9 (C O), 138.6, 138.4,
138.2 (ArC), 133.0 (ArC), 130.4 (ArC), 129.7-127.5 (m, ArC), 83.5
(C2), 78.2 (C3), 74.7 (OCH2Ph), 74.3 (C1), 74.3, 73.3 (OCH2Ph),
68.5 (C8), 60.9 (C5), 48.4 (C4), 28.1, 24.6 (C6, C7). HRMS (ES+):
calcd. for C36H37N3O5Na: 614.2631, found 614.2636.
(1R,2R,3R,4S,5S)-1-O-Benzoyl-2,3-di-O-benzyl-4-C-benzyl-
oxymethyl-cycloheptane-5-ol (18S). To a stirred solution of al-
cohol 16S (1.05 g, 1.80 mmol) in dry pyridine (10 mL) under
an atmosphere of nitrogen was added benzoyl chloride (0.42 mL,
3.62 mmol, 2.0 eq) and a catalytic quantity of DMAP (0.02 g,
0.19 mmol, 0.1 eq). The reaction mixture was stirred at rt overnight
before the reaction was quenched by addition of water (25 mL) in
small portions. After stirring for 10 min the mixture was diluted
with EtOAc (100 mL) and the organic phase washed with a 1 M
hydrochloric acid (3 ¥ 50 mL), saturated aq. NaHCO3 (50 mL),
brine (2 ¥ 50 mL), dried over MgSO4, filtered and concentrated.
The resulting product 17S (1.26 g) was dissolved in CH2Cl2
(23 mL) and H2O (0.8 mL), and vigorously stirred with DDQ
(0.47 g, 2.05 mmol, 1.1 eq) for 1 h. A saturated aq. solution of
NaHCO3 (100 mL) and CH2Cl2 (100 mL) were then added to
the reaction mixture and the layers separated before the organic
layer was washed with brine (3 ¥ 100 mL), dried over MgSO4,
filtered and concentrated. The crude product was purified by flash
column chromatography (pentane–EtOAc 10 : 1→5 : 1) to give the
desired product 18S (0.87 g, 85% (two steps)) as a colourless oil.
[a]2D0 68 (c 1.0, CHCl3). Rf (pentane–EtOAc 4 : 1) = 0.34. 1H NMR
(400 MHz, CDCl3): d (ppm) 8.01 (m, 2 H, o-Bz), 7.58-7.54 (m,
1 H, ArH), 7.44-7.40 (m, 3 H, ArH), 7.37-7.25 (m, 12 H, ArH),
(2S,3R,4R,5R)-5-Azido-2,3-di-O-benzyl-4-C-benzyloxymethyl-
cycloheptanone (21). Sodium (0.04 g, 1.65 mmol, 1.4 eq) was
dissolved in dry MeOH (10 mL) and the resulting solution added
to 19 (0.69 g, 1.17 mmol) under an atmosphere of nitrogen. The
reaction mixture was stirred overnight before being diluted with
EtOAc (70 mL) and the organic phase washed with water (3 ¥
50 mL), dried over MgSO4, filtered and concentrated to give
crude 20. Dess–Martin periodinane (0.86 g, 2.03 mmol, 1.6 eq)
was added to 20 (0.61 g) dissolved in CH2Cl2 (10 mL). The
reaction mixture was stirred at rt for 30 min and then diluted
with Et2O (50 mL) after which stirring was continued for 30 min.
The resulting mixture was washed with a saturated aq. solution of
Na2S2O3 (3 ¥ 50 mL), brine (2 ¥ 50 mL), dried over MgSO4, filtered
and concentrated. The crude product was purified by flash column
chromatography (pentane–EtOAc 20 : 1) to give the product 21
(0.46 g, 80% (two steps)) as colourless crystals. The crystals were
◦
recrystallised from CH2Cl2 and heptane. Mp 74.6-76.5 C, [a]D20
-0.3 (c 1.0, CHCl3). Rf (pentane–EtOAc 9 : 1) = 0.36. H NMR
1
(400 MHz, CDCl3): d (ppm) 7.30 - 7.16 (m, 15 H, ArH), 4.56
(d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.45 (d, 1 H, Jgem= 11.6 Hz,
OCH2Ph), 4.42 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph), 4.41 (d, 1 H,
Jgem= 11.6 Hz, OCH2Ph), 4.38 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph),
4.33 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.11 (d, 1 H, J2,3 = 6.4 Hz,
H2), 3.88 (t, 1 H, J2,3;3,4 = 6.4 Hz, H3), 3.80 (dt, 1 H, J5,6¢ = 2.0 Hz,
J4,5;5,6 = 9.2 Hz, H5), 3.66 (dd, 1 H, J4,8 = 3.6 Hz, Jgem = 9.2 Hz,
H8), 3.55 (dd, 1 H, J4,8¢ = 5.6 Hz, Jgem = 9.2 Hz, H8¢), 2.53 (m,
2 H, H7, H7¢), 2.18 (m, 1 H, H6), 2.00 (m, 2 H, H6¢, H4). 13C
7.18-7.16 (m, 2 H, ArH), 5.51 (m, 1 H, H1), 4.80 (d, 1 H, Jgem
=
11.2 Hz, OCH2Ph), 4.71 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.69
(d, 1 H, Jgem= 10.8 Hz, OCH2Ph), 4.55 (d, 1 H, Jgem= 12.0 Hz,
OCH2Ph), 4.49 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph), 4.48 (d, 1 H,
Jgem= 10.8 Hz, OCH2Ph), 4.36 (m, 1 H, H5), 4.05 (t, 1 H, J1,2;2,3
=
4.8 Hz, H2), 3.99 (dd, 1 H, J4,8 = 6.4 Hz, Jgem = 8.8 Hz, H8), 3.89
(dd, 1 H, J2,3 = 4.8 Hz, J3,4 = 9.2 Hz, H3), 3.66 (dd, 1 H, J4,8
=
=
3.6 Hz, Jgem = 8.8 Hz, H8¢), 3.48 (bs, 1 H, OH), 2.32 (m, 1 H,
NMR (100 MHz, CDCl3): d (ppm) 209.3 (C O), 138.3, 137.9,
H4), 2.24 (m, 1 H, H7), 1.97 (m, 3 H, H6, H6¢, H7¢). 13C NMR
137.0 (ArC), 128.7-127.8 (ArC), 87.9 (C2), 76.4 (C3), 73.2, 72.9,
72.9 (OCH2Ph), 68.9 (C8), 61.2 (C5), 48.1 (C4), 37.6 (C7), 26.8
(C6). HRMS (ES+): calcd. for C29H31N3O4Na: 508.2212, found
508.2212.
=
(100 MHz, CDCl3): d (ppm) 165.8 (C O), 138.5, 138.0, 137.7
(ArC), 133.0 (ArC), 130.4 (ArC), 129.7-127.5 (m, ArC), 83.0 (C2),
78.4 (C3), 73.9, 73.6 (OCH2Ph), 73.5 (C1), 73.1 (OCH2Ph), 71.9
(C8), 70.0 (C5), 47.1 (C4), 30.2 (C6), 22.2 (C7). HRMS (ES+):
calcd. for C36H38O6Na: 589.2566, found 589.2565.
(1R,2S,3R,4R,5R)-2,3-Di-O-benzyl-4-C-benzyloxymethyl-8-
azabicyclo[3.2.1]octane-1-ol (22). PPh3 (0.09 g, 0.32 mmol, 1.6
eq) was added to a stirred solution of ketone 21 (0.10 g, 0.21 mmol)
in MeOH (5 mL) and H2O (0.25 mL). The reaction mixture was
stirred at 40 ◦C for 150 min. during which N2 evolution was
observed. The reaction mixture was concentrated and the crude
product purified by flash column chromatography (EtOAc) to give
the desired product 22 (0.064 g, 68%) as a colourless oil. [a]2D0 26
(c 1.0, CHCl3) Rf (EtOAc) = 0.16. 1H NMR (400 MHz, CDCl3):
d (ppm) 7.40-7.21 (m, 15 H, ArH), 5.10 (d, 1 H, Jgem= 11.2 Hz,
OCH2Ph), 4.86 (d, 1 H, Jgem= 10.8 Hz, OCH2Ph), 4.78 (d, 1 H,
Jgem = 11.2 Hz, OCH2Ph), 4.51 (d, 1 H, Jgem= 10.8 Hz, OCH2Ph),
4.49 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph), 4.41 (d, 1 H, Jgem= 12.0 Hz,
OCH2Ph), 3.62 (dd, 1 H, J4,8 = 3.2 Hz, Jgem= 9.6 Hz, H8), 3.56
(m, 2 H, H5, H2/H3), 3.34 (m, 2 H, H8¢, H2/H3), 2.22 (m, 1 H,
H7), 1.98 (m, 2 H, H4, H6), 1.72 (m, 1 H, H6¢), 1.53 (m, 1 H, H7¢).
13C NMR (100 MHz, CDCl3): d (ppm) 139.3, 138.7, 138.3 (ArC),
128.7-127.5 (m, ArC), 92.9 (C1) 88.4, 79.0 (C2, C3) 75.1, 74.6,
73.2 (OCH2Ph), 68.6 (C8), 53.5 (C5), 49.7 (C4), 31.0 (C7), 23.5
(C6). HRMS (ES+): calcd. for C29H33NO4Na: 482.2307, found
482.2306.
(1R,2R,3R,4R,5R)-5-Azido-1-O-benzoyl-2,3-di-O-benzyl-4-C-
benzyloxymethyl-cycloheptane (19). To a cooled (0 ◦C) and
stirred solution of alcohol 18S (0.87 g, 1.54 mmol) in dry THF
(10 mL) under an atmosphere of nitrogen were consecutively
added PPh3 (0.45 g, 1.70 mmol, 1.1 eq) and DIAD (0.35 mL,
1.69 mmol, 1.1 eq) in a drop wise fashion. After stirring for 10 min,
DPPA (0.43 mL, 1.99 mmol, 1.3 eq) was added, and the reaction
mixture allowed to reach rt and then stirred for 2 h. The reaction
mixture was concentrated and the resulting residue purified by
flash column chromatography (pentane–EtOAc 50 : 1→20 : 1) to
give the desired product 19 (0.69 g, 76%) as a clear oil. [a]2D0 52 (c
1.0, CHCl3). Rf (pentane–EtOAc 9 : 1) = 0.53. 1H NMR (400 MHz,
CDCl3): d (ppm) 8.05 (m, 2 H, o-Bz), 7.60 (m, 1 H, ArH), 7.46
(m, 2 H, ArH), 7.42-7.21 (m, 15 H, ArH), 5.44 (m, 1H, H1), 4.82
(d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.80 (d, 1 H, Jgem= 10.8 Hz,
OCH2Ph), 4.74 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.57 (d, 1 H,
Jgem= 11.6 Hz, OCH2Ph), 4.55 (d, 1 H, Jgem= 10.8 Hz, OCH2Ph),
4.51 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 3.97 (m, 3 H, H8, H2,
H5), 3.79 (m, 2 H, H8¢, H3), 2.08 (m, 5 H, H4, H6, H6¢, H7, H7¢).
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(1R,2S,3R,4R,5R)-4-(hydroxymethyl)-8-azabicyclo[3.2.1]oct-
ane-1,2,3-triol (5). Protected noeurostegine (22) (0.060 g,
0.12 mmol) was dissolved in 96% EtOH (3 mL) and flushed
with N2. Pearlmann’s catalyst (37 mg) was added and the reaction
mixture was stirred overnight under an atmosphere of H2 (balloon)
before the reaction mixture was then transferred directly to a
flash column (silica), where the product was eluted with EtOAc–
isopropanol–H2O 1 : 1 : 1 to give the desired product 5 (21 mg,
91%) as a clear oil. [a]D20 26 (c 0.5, H2O). Rf (5% aq. ammonium
hydroxide in EtOH (99.9%)) = 0.13. 1H NMR (400 MHz, D2O): d
(ppm) 3.87 (dd, 1 H, J = 4.0 Hz, J = 11.6 Hz, H3), 3.63 (m, 1 H,
H5), 3.56 (m, 2 H, H2, H8), 3.32 (m, 1 H, H8¢), 2.04 (m, 2 H, H6,
as described for the formation of 15S and 16S and gave compound
15R and 16R (2.10 g, 90%) as a 2 : 1 mixture of regioisomers which
were easily separated.
15R Colourless oil (0.77 g, 33%). [a]2D0 -28 (c 1.0, CHCl3). Rf
1
(pentane–EtOAc 2 : 1) = 0.28. H NMR (400 MHz, CDCl3): d
(ppm) 7.35-7.20 (m, 17 H, ArH), 6.84 (m, 2 H, ArH), 4.66-4.40
(m, 8 H, OCH2Ph), 3.98 (m, 2 H), 3.83 (m, 1 H), 3.80 (s, 3 H,
OCH3), 3.74 (m, 1 H), 3.59 (m, 1 H), 3.50 (m, 1 H), 2.44 (bs, 1
H), 2.36 (m, 1 H), 2.10 (m, 5 H). 13C NMR (100 MHz, CDCl3):
d (ppm) 159.3, 139.0, 138.7, 138.7, 130.4 (ArC), 129.7-127.5 (m,
ArC), 113.9 (ArC), 79.2, 78.2, 76.1, 73.0, 72.6, 71.6, 70.9, 70.5,
67.0, 55.4, 48.1, 38.6, 36.9. HRMS (ES+): calcd. for C37H42O6Na:
605.2879, found 605.2881.
H6¢), 1.92 (m, 1 H, H4), 1.78 (m, 1 H, H7), 1.61 (m, 1 H, H7¢). 13
C
NMR (100 MHz, D2O): d (ppm) 92.0 (C1) 78.2, 70.8, 59.8, 53.2,
49.0, 28.0, 22.0. HRMS (ES+): calcd. for C8H15NO4Na: 212.0899,
found 212.0890.
16R Colourless oil (1.33 g, 57%). [a]2D0 -2 (c 1.0, CHCl3). Rf
1
(pentane–EtOAc 2 : 1) = 0.60. H NMR (400 MHz, CDCl3): d
(ppm) 7.38-7.25 (m, 17 H, ArH), 6.89 (m, 2 H, ArH), 4.92 (d,
1 H, Jgem= 11.6 Hz, OCH2Ph), 4.83 (d, 1 H, Jgem= 11.6 Hz,
OCH2Ph), 4.63 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.59 (d, 1 H,
Jgem= 11.6 Hz, OCH2Ph), 4.54 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph),
4.46 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph), 4.39 (d, 1 H, Jgem= 12.0 Hz,
OCH2Ph), 4.37 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 3.82 (s, 3 H,
(3R,4R,5S,6R)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-6-O-p-
methoxybenzyl-1,8-nonadiene (13R). 13R was prepared accord-
ing to the procedure described for 13S and obtained (2.21 g, 97%)
as a clear oil. [a]2D0 4 (c 1.0, CHCl3). Rf (pentane–EtOAc 16 : 1) =
1
0.38. H NMR (400 MHz, CDCl3): d (ppm) 7.37-7.26 (m, 15
OCH3), 3.72 (m, 5 H, H1, H2, H3, H5, H8), 3.54 (dd, 1 H, J4,8¢
=
H, ArH), 7.23-7.20 (m, 2 H, ArH), 6.88-6.84 (m, 2 H, ArH),
5.86 (m, 2 H, H2, H8), 5.33 (m, 2 H, H1/H9), 5.06 (m, 2 H,
H1/H9), 4.94 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph), 4.63 (d, 1 H,
Jgem= 12.0 Hz, OCH2Ph), 4.59 (d, 1 H, Jgem= 11.6 Hz, OCH2Ph),
4.50 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph), 4.46 (d, 1 H, Jgem= 12.0 Hz,
OCH2Ph), 4.40 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph), 4.40 (s, 2 H,
OCH2Ph), 4.18 (t, 1 H, J2,3;3,4 = 7.2 Hz, H3), 3.84 (m, 2 H, H4,
H10), 3.82 (s, 3H, OCH3), 3.64 (m, 2 H, H6, H10¢), 2.43 (m, 2
H, H5, H7), 2.27 (m, 1 H, H7¢). 13C NMR (100 MHz, CDCl3):
d (ppm) 159.2, 139.4, 138.9, 138.8 (ArC), 136.1, 136.0 (C2, C8),
131.0 (ArC), 129.5-127.3 (ArC), 119.1, 116.6 (C1, C9) 113.8 (ArC),
83.4 (C3), 79.6 (C4), 78.3 (C6), 73.9, 73.0, 71.0, 70.7 (OCH2Ph),
67.8 (C10), 55.4 (OCH3), 42.2 (C5), 35.7 (C7). HRMS (ES+):
calcd. for C39H44O5Na: 615.3086, found 615.3080.
4.8 Hz, Jgem = 8.8 Hz, H8¢), 3.36 (bs, 1 H, OH), 2.23 (m, 1 H,
H4), 2.07 (m, 1 H, H6), 1.87 (m, 2 H, H6¢,H7), 1.66 (m, 1 H,
H7¢). 13C NMR (100 MHz, CDCl3): d (ppm) 159.2, 138.7, 138.6,
138.6, 130.9 (ArC), 129.4-127.6 (m, ArC), 113.9 (ArC), 86.0 78.9,
75.1, 74.4, 73.1 (OCH2Ph), 72.9, 70.3 (OCH2Ph), 70.0 (C8), 55.4
(OCH3), 48.8 (C4), 26.7(C6), 24.6 (C7). HRMS (ES+): calcd. for
C37H42O6Na: 605.2879, found 605.2876.
(2R,3R,4R,5R)-5-O-Benzoyl-3,4-di-O-benzyl-2-C-benzyloxy-
methyl-cycloheptanone (23). Benzoyl protection of 16R was con-
ducted as described for the formation of 17S. DDQ deprotection
of the crude product was carried out as described for the formation
of 18S. The resulting alcohol was oxidised directly using Dess–
Martin periodinane as described for the formation of 21. This
gave the desired ketone 23 (1.17 g, 90% (three steps)) as an oil.
[a]2D0 10 (c 1.0, CHCl3). Rf (pentane–EtOAc 5 : 1) = 0.46. 1H NMR
(400 MHz, CDCl3): d (ppm) 7.99 (m, 2 H, o-Bz), 7.56 (m, 1 H,
ArH), 7.44-7.40 (m, 2 H, ArH), 7.37-7.24 (m, 13 H, ArH), 7.19-
7.17 (m, 2 H, ArH), 5.48 (m 1 H, H5), 4.78 (d, 1 H, Jgem= 11.6 Hz,
OCH2Ph), 4.75 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.73 (d, 1 H,
Jgem= 11.6 Hz, OCH2Ph), 4.55 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph),
4.48 (s, 2 H, OCH2Ph), 4.07-3.97 (m, 3 H, H3, H4, H8), 3.75 (dd,
1 H, J2,8¢ = 4.8 Hz, Jgem = 8.8 Hz, H8), 3.08 (m, 1 H, H2), 2.77
(m, 1 H, H7), 2.67 (m, 1 H, H7¢), 2.34 (m, 2 H, H6, H6¢). 13C
(3R,4R,5S,6R)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-6-O-p-
methoxybenzyl-cycloheptene (14R). 14R as prepared according
to the procedure described for 14S and obtained (1.90 g, 91%) as
an oil. [a]2D0 -24 (c 1.0, CHCl3). Rf (pentane–EtOAc 10 : 1) = 0.45.
1H NMR (400 MHz, CDCl3): d (ppm) 7.37-7.26 (m, 15 H, ArH),
7.23-7.20 (m, 2 H, ArH), 6.88-6.84 (m, 2 H, ArH), 5.85 (m, 1 H,
H2), 5.76 (m, 1 H, H1), 4.93 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph),
4.70 (s, 2 H, OCH2Ph), 4.56 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph),
4.52 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.44 (d, 1 H, Jgem= 12.0 Hz,
OCH2Ph), 4.41 (d, 1 H, Jgem= 12.0 Hz, OCH2Ph), 4.37 (d, 1 H,
Jgem= 11.2 Hz, OCH2Ph), 4.36 (m, 1 H, H3), 3.80 (s, 3 H, OCH3)
3.78-3.74 (m, 3 H, H4, H8, H8¢), 3.70 (dt, 1 H, J6,7 = 2.4 Hz,
J5,6;6,7¢ = 8.0 Hz, H6), 2.44 (m, 2 H, H7, H7¢), 2.18 (m, 1 H, H5).
13C NMR (100 MHz, CDCl3): d (ppm) 159.2, 139.1, 139.0, 138.8
(ArC), 133.1 (C2), 130.8 (ArC), 129.5-127.5 (m, ArC), 126.7 (C1),
113.9 (ArC), 81.1 (C3), 78.9 (C4), 75.5 (C6), 74.7, 73.0, 72.7, 70.7
(OCH2Ph), 68.6 (C8), 55.4 (OCH3), 50.9 (C5), 31.3 (C7). HRMS
(ES+): calcd. for C37H40O5Na: 587.2773, found 587.2775.
=
=
NMR (100 MHz, CDCl3): d (ppm) 209.5 (C O), 165.7 (C O,
ester) 138.2, 138.1, 137.8 (ArC), 133.2 (ArC) 129.9 (ArC), 129.8-
127.7 (m, ArC), 82.4 (C3/C4) 77.7 (C3/C4), 74.2 (OCH2Ph), 73.4
(OCH2Ph), 72.9 (C5), 69.6 (C8) 55.8 (C2), 39.0 (C7), 24.3 (C6).
HRMS (ES+): calcd. for C36H36O6Na: 587.2410, found 587.2405.
(1R,2R,3R,4S,5S)-1-O-Benzoyl-2,3-di-O-benzyl-4-C-benzyl-
oxymethyl-cycloheptane-5-ol (18S). Li(OtBu)3AlH (2.90 g,
11.42 mmol, 5.0 eq) was added to a stirred solution of ketone
23 (1.29 g, 2.29 mmol) in dry Et2O (40 mL) at -78 ◦C. After 1 h
the solution was allowed to warm to rt and stirred overnight before
diluted with Et2O (100 mL). The organic phase was then washed
with 1 M HCl until acidic (2 ¥ 50 mL) and the aqueous phase
extracted with EtOAc (2 ¥ 50 mL). The combined organic phases
(1R/S,3R,4R,5S,6R)-3,4-Di-O-benzyl-5-C-benzyloxymethyl-
6-O-p-methoxybenzyl-cycloheptane-1-ol (15R) and (1R,2R,3R,
4S,5R)-2,3-di-O-benzyl-4-C-benzyloxymethyl-5-O-p-methoxy-
benzyl-cycloheptane-1-ol (16R). Hydroboration was conducted
440 | Org. Biomol. Chem., 2010, 8, 433–441
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were washed with a saturated aq. solution of NaHCO3 (2 ¥ 50 mL),
brine (50 mL), dried over MgSO4, filtered and concentrated.
The crude product was purified by flash column chromatography
(pentane–EtOAc 10 : 1→8 : 1→5 : 1) to give the product 18 (1.10 g,
85%) as a 1 : 4 mixture of R- and S-diastereomers. 1H NMR data
for 18S (colourless oil (0.86 g, 66%)) was in accordance with data
given above.
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18R: Colourless oil (0.23 g, 18%) 1H NMR (400 MHz, CDCl3):
d (ppm) 8.00 (m, 2 H, o-Bz), 7.57-7.53 (m, 1 H, ArH), 7.43-7.17
(m, 17 H, ArH), 5.35 (m, 1 H, H1), 4.73 (d, 1 H, Jgem= 11.2 Hz,
OCH2Ph), 4.72 (d, 1 H, Jgem= 11.2 Hz, OCH2Ph), 4.65 (d, 1 H,
Jgem= 11.2 Hz, OCH2Ph), 4.50 (s, 2 H, OCH2Ph), 4.39 (d, 1 H,
Jgem= 11.2 Hz, OCH2Ph), 4.04-3.91 (m, 3 H, H2, H5, H8), 3.65
(m, 3 H, H3, H8¢, OH), 2.32 (m, 1 H, H4), 2.05 (m, 1 H, H7)
1.96-1.86 (m, 3 H, H6, H6¢, H7¢). 13C NMR (100 MHz, CDCl3): d
=
(ppm) 166.0 (C O), 138.2, 138.1, 137.9 (ArC), 133.0 (ArC), 130.5
(ArC), 129.8 (ArC), 128.6-127.7 (m, ArC), 84.3, 78.6, 76.0, 74.5,
74.2, 73.6, 71.4, 70.6, 48.5, 30.5, 23.8. HRMS (ES+): calcd. for
C36H38O6Na: 589.2566, found 589.2568.
Acknowledgements
We are grateful for support from The Faculty of Natural Sciences
and OChem Graduate School, Aarhus University and for the
Carlsberg Scholarship to TSR.
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of calysegine B2; see ref. 18 and 19.
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The Royal Society of Chemistry 2010
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