Enantiospecific synthesis of the heparanase inhibitor (+)-trachyspic acid and stereoisomers from a common precursor

Article abstract of DOI:10.1039/b708594j

The total synthesis of natural (+)-trachyspic acid and its enantiomer is described starting from a common 2-deoxy-d-ribose derivative. The synthesis of the corresponding C3 epimers from the same starting material is also described. Each stereoisomer was a

Full text of DOI:10.1039/b708594j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Enantiospecific synthesis of the heparanase inhibitor (+)-trachyspic acid and
stereoisomers from a common precursor†
Steven C. Zammit,a Vito Ferro,b Edward Hammondb and Mark A. Rizzacasa*a
Received 6th June 2007, Accepted 20th June 2007
First published as an Advance Article on the web 26th July 2007
DOI: 10.1039/b708594j
The total synthesis of natural (+)-trachyspic acid and its enantiomer is described starting from a
common 2-deoxy-D-ribose derivative. The synthesis of the corresponding C3 epimers from the same
starting material is also described. Each stereoisomer was assayed for heparanase inhibition.
Its structure was determined by NMR analysis and degradation
Introduction
experiments and the relative configuration was confirmed by a
total synthesis of racemic 1.4 Compound 1 is a member of a family
Heparanase is an endo-b-glucuronidase that cleaves the heparan
of 2-alkylcitrate type natural products which includes cinatrin B5
and citrafungin A.6 We have recently reported the total synthesis of
(−)-(3R,4R,6R)-trachyspic acid (ent-1) and confirmed the absolute
configuration of natural (+)-(3S,4S,6S)-trachyspic acid (1).7 In
this article we report the full details of this work as well as the
synthesis of natural (+)-trachyspic acid (1) and the corresponding
C3 epimers from a common chiral pool precursor. Each isomer
was then tested for inhibition of heparanase.
sulfate (HS) side chains of proteoglycans that are found on
cell surfaces and as a major constituent of the extracellular
matrix (ECM) and basement membranes surrounding cells.1 The
degradation of the ECM by heparanase facilitates the spread of
metastatic tumor cells and leukocytes by allowing them to pass
into the blood stream and lodge in remote sites, where they can
form secondary tumors or cause inflammation, respectively. In
addition, heparanase is able to liberate HS-bound angiogenic
growth factors such as vascular endothelial growth factor (VEGF)
and fibroblast growth factors (FGF-1 and FGF-2) which promote
tumor growth and angiogenesis. Because of its pivotal role in these
processes, heparanase is an attractive target for the development
of antitumor, antimetastasis or anti-inflammatory drugs.2
Results and discussion
Our initial retrosynthetic analysis of (−)-trachyspic acid (ent-1)
is shown in Scheme 1. At the onset of this work, the relative
and absolute configuration of 1 was unknown. We selected ent-
1 as our initial target and envisaged that it could be synthesised
from the lactol precursor 2 by acid hydrolysis of the dioxolane
and spirocyclisation of the resultant aldehyde followed by lactol
acetylation4 and global ozonolysis of three terminal alkenes.
Trachyspic acid (1, Fig. 1) was isolated from the culture broth
of Talaromyces trachyspermu SANK 12191 and was identified
as a potent inhibitor of heparanase with an IC50 of 36 lM.3
Fig.
1 Structures of (+)-(3S,4S,6S)-trachyspic acid (1) and of
(−)-(3R,4R,6R)-trachyspic acid (ent-1).
aSchool of Chemistry, Bio21 Institute, 30 Flemington Rd, The University
of Melbourne, Victoria 3010, Australia. E-mail: masr@unimelb.edu.au;
Fax: +61 3 9347 5180; Tel: +61 3 8344 2397
bProgen Pharmaceuticals Ltd, PO Box 2403, Toowong, Queensland 4066,
Australia
† Electronic supplementary information (ESI) available: Experimental for
preparation of (+)-1, 2, 16, 20, (+)-25, 28 and ent-28, NMR comparison
tables and copies of the 1H and 13C NMR spectra of compounds 1, 23, 25,
28 and 31. See DOI: 10.1039/b708594j
Scheme 1 Initial retrosynthetic analysis of (−)-trachyspic acid (ent-1).
Base induced elimination by adapting the method of
Hatakeyama et al.4 would install the C9–C10 alkene and oxidation
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Scheme 2 Reagents and conditions: (a) NaH, PMBCl; (b) 10% HCl, MeOH; (c) NaOCl, NaClO2, TEMPO; (d) DCC, DMAP, allyl alcohol; (e) (1)
TMSCl–NEt3, LDA, THF–HMPA, −95 ◦C, (2) aq. NaOH, (3) N,Nꢀ-diisopropyl-O-tert-butylisourea; (f) (1) 10% HCl, (2) PCC; (g) (1) DDQ, (2) MsCl,
pyridine; (h) CuI, vinylMgBr, Me2S, −45 ◦C; (i) (1) O3, Me2S, (2) NaClO2, NaH2PO4, (3) N,Nꢀ-diisopropyl-O-tert-butylisourea.
followed by deprotection of the t-butyl ester would give ent-1.
Lactol 2 might be available by addition of the anion derived from
vinyl bromide 4 to the lactone 3 which could be obtained from the
2-deoxy-D-ribose derivative 5. An Ireland–Claisen rearrangement
in the presence of a b-leaving group8,9 conducted on 5 would
introduce the C3 stereocentre in a controlled fashion whilst the
bromide 4 could be secured from dimethyl malonate (6). We have
utilised the Ireland–Claisen rearrangement in the presence of a
b-leaving group for the synthesis of the 2-alkylcitrate moiety in a
number of related natural products including the cintatrins B,10 C1
confirm the structure of the minor conjugate addition diastereo-
isomer 3.
and C3 and zaragozic acid C.12
11
The synthesis of the lactone 3 is detailed in Scheme 2 and began
with protection of the known alcohol in 713 as the p-methoxybenzyl
(PMB) ether followed by acid hydrolysis to give the a-anomer and
b-anomer 8 in good yield. In practice, either anomer could be
utilised since the anomeric centre is oxidised at a later stage in
the synthesis however, we elected to continue with the b-anomer
since better yields were obtained in subsequent steps. Oxidation14
of the alcohol 8 to the acid proceeded well with only a small
amount of over-oxidation of the PMB group to the benzoyl ester.
This byproduct was easily removed in the next step. Esterification
of the crude acid mixture with allyl alcohol then gave the pure
Claisen precursor 5 after flash chromatography.
Fig. 2 X-Ray structure of ester 13 (20% ellipsoids; H atoms omitted).
The synthesis of the requisite vinyl bromide coupling partner
4 is outlined in Scheme 3. Alkylation of dimethylmalonate with
nonyl bromide gave the diester 1419 in excellent yield. Reduction to
the diol 15 was effected with LiAlH4 and monoprotection20 gave
the tert-butyldiphenylsilyl (TBDPS) ether 16. Oxidation of the
primary alcohol in 16 and Corey–Fuchs extension21 yielded alkyne
17. Bromoboration22 of 17 followed by treatment with acetic acid
Ireland–Claisen rearrangement of 5 using our previously opti-
mised conditions8,10–12 followed by hydrolysis and esterification15
gave the t-butyl ester 9 as the only detectable isomer in good
yield. The stereochemistry of 9 was confirmed by a strong NOE
interaction between H4 and the H2 protons as indicated. This
showed that the [3,3]-rearrangement had occurred exclusively
from the b-face opposite the OPMB group. We next investigated
the introduction of the C4 vinyl group via a conjugate addition
approach.16 At the onset, we wanted to access both epimers as the
relative configuration was, at the time, undetermined. Thus, the
Claisen adduct was subjected to acid hydrolysis to afford a lactol
which was oxidised to the lactone 10. Removal of the PMB group
followed by mesylation and concomitant base induced elimination
gave the a,b-unsaturated lactone 11 ready for conjugate addition.
Treatment of 11 with vinylmagnesium bromide in the presence of
CuI and Me2S17 afforded the two alkene isomers 12 and 3 with
a slight preference for the isomer 12 with what turned out to
be the incorrect relative stereochemistry. This was confirmed by
the conversion of 3 into the crystalline tri-tert-butylester 13 by
double ozonolysis, oxidation and ester formation. A single crystal
X-ray structure of 13 was determined18 (Fig. 2) which served to
Scheme 3 Reagents and conditions: (a) NaOMe, nonyl bromide, MeOH,
reflux; (b) LiAlH4, 0 ◦C; (c) NaH, TBDPSCl; (d) (1) Dess–Martin reagent,
(2) PPh3, CBr4, 0 ◦C, (3) BuLi, −78 ◦C; (e) B-Br-9-BBN, AcOH, 0 ◦C;
(f) TBAF; (g) (1) Dess–Martin reagent, (2) p-TsOH, HOCH2CH2OH,
benzene, reflux.
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Scheme 4 Reagents and conditions: (a) t-BuLi, Et2O–hexane, −78 ◦C; (b) lactone 3, THF; (c) (1) 3 M HClO4, THF, (2) Ac2O, DMAP, pyridine;
(d) lactone 13, THF; (e) O3, NaHCO3, Me2S; (f) TFA, CH2Cl2; (g) CH2N2, Et2O.
gave bromoalkene 18 which was desilylated to afford alcohol 19.
Finally, oxidation of 19 followed by acetal formation gave the
dioxolane 4.
in intermediate II. It is also not unreasonable to suggest that the
cyclisation could also take place under thermodynamic control
however, since acid treatment of 23 to afford ent-1 (see below)
did not result in any spiroisomerisation, it is most likely that the
cyclisation is under kinetic control.
Treatment of spiroketal 23 with TFA in dichloromethane then
gave (−)-ent-1 which was identical to natural 1 in all respects
except for the sign of optical rotation ([a]D −3.5 (c 0.213, MeOH);
[a]D −8.4 (c 0.35, CH2Cl2); lit.3 [a]D +3.1 (c 1.0, MeOH)). This
led to the assignment of the absolute configuration of (+)-
trachyspic acid as (3S,4S,6S) shown in Fig. 1. Synthetic ent-1
was further characterised by conversion into the trimethyl ester
(−)-ent-25 ([a]D −20.4 (c 0.13, CH2Cl2) the physical data of which
also compared well to those reported for naturally derived 25.
Unfortunately, the optical rotation for naturally derived 25 was
not reported.3
For the synthesis of the correct enantiomer (+)-1, the lactone
ent-13 would be required. We envisaged that this would be available
from the same deoxy-D-ribose derivative 7 utilised for the synthesis
of 13. As observed earlier, it is the stereochemistry at C4 (trachyspic
acid numbering) that controls the outcome of the Ireland–Claisen
rearrangement and thus the stereochemistry at C3. Therefore, if
the C4 stereogenic centre is inverted as in precursor 26 (Fig. 4),
this would allow for the introduction of the 3R stereochemistry
and provide the ester required for the production of natural (+)-
trachypsic acid (1).
With the two fragments in hand we next examined the coupling
reaction (Scheme 4). Bromide 4 was treated with t-BuLi23 in ether–
hexane solvent and the resultant anion smoothly added to the
lactone 3 to give the hemiacetal 2 as a mixture of isomers. This
compound was then subjected to acid hydrolysis and acetylation4
to give a complex mixture of 5,5-spiroketal diastereoisomers 20
characterised by the presence of four acetate methyl signals and
one spiro carbon signal in its 13C NMR spectrum. Unfortunately,
all attempts at oxidative cleavage of the three alkenes in the
presence of base did not afford the desired product.
At this point we envisaged that the lactone 13 may serve as an
alternative coupling partner. Although there are several carbonyl
groups present in 13, we reasoned the lactone carbonyl would
be the most reactive. Treatment of lactone 13 with the anion
derived from 4 afforded the lactols 22 in reasonable yield along
with some starting lactone 13 (48% based on recovered 13). Acid
induced cyclisation and acetylation of 22 followed by ozonolysis
in the presence of NaHCO34,7 installed the desired a,b-unsaturated
system and afforded the spiroketal isomers 23 and 24 in a ratio of
∼9 : 1 which were separable by flash chromatography.
The stereochemical outcome of the cyclisation can be ratio-
nalised by considering the two possible modes of attack on the
oxonium ion intermediate as shown in Fig. 3.4 Cyclisation from
the least hindered a-face as in intermediate I affords the major
isomer 23. On the other hand, cyclisation from the b-face is not
preferred as there is a larger steric interaction present as indicated
Fig. 4 Proposed synthesis of ent-13.
The conversion of the above theory into practice is outlined
in Scheme 5. 2-Deoxy-D-ribose derivative 7 was subjected to a
modified Mitsunobu inversion24 and subsequent methanolysis,
benzylation and trityl group hydrolysis afforded 27 along with
the corresponding b-anomer. Oxidation of 27 was best achieved
using a two step protocol rather than the one pot procedure
utilised previously. Esterification then gave allyl ester 26 which was
subjected to Ireland–Claisen rearrangement and esterification to
Fig. 3 Proposed spirocyclisation of intermediates I and II.
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Scheme 5 Reagents and conditions: (a) (1) Ph3P, diisopropyl azodicarboxylate (DIAD), p-NO2C6H4CO2H, (2) K2CO3, MeOH; (b) NaH, PMBCl;
(c) 10% HCl–MeOH; (d) (1) Dess–Martin reagent, (2) Ag2O, KOH; (e) DCC, DMAP, allyl alcohol; (f) (1) TMSCl–NEt3, LDA, THF–HMPA, −95 ◦C,
(2) aq. NaOH, (3) N,Nꢀ-diisopropyl-O-tert-butylisourea; (g) t-BuLi, Et2O–hexane, −78 ◦C; (h) lactone ent-13, THF; (i) (1) 3 M HClO4, THF, (2) Ac2O,
DMAP, pyridine; (j) O3, NaHCO3, Me2S; (k) TFA, CH2Cl2; (l) CH2N2, Et2O.
(+)-3-epi-trachyspic acid (28) ([a]2D2 +2.9 (c 0.10, MeOH)) and in
a similar manner, ent-29 was converted into (−)-3-epi-trachyspic
acid (ent-28) ([a]2D4 −3.4 (c 0.25, MeOH)).
give ent-9. This was then carried through the same sequence as
for 9 (see Scheme 2) to eventually give ent-13 in a comparable
overall yield. Addition of the anion derived from 4 to ent-13 gave
a mixture of lactols which underwent acid induced cyclisation and
ozonolysis to give the spiroketals ent-23 and ent-24. Deprotection
of ent-23 with TFA afforded (+)-trachyspic acid (1) which now had
chiroptical properties that matched the natural product ([a]D +3.1
(c 0.30, MeOH); [a]D +7.1 (c 0.30, CH2Cl2); lit.3 [a]D +3.1 (c 1.0,
MeOH)). In addition, this was further characterised by conversion
into the trimethyl ester (+)-25 ([a]D +17.3 (c 0.10, CH2Cl2)).
Since the C4 isomers 12 and ent-12 were also produced in
the conjugate addition, we elected to carry each one though to
provide (+)-3-epi-trachyspic acid (28) and its enantiomer (−)-
ent-28 for biological testing (Scheme 6). Ozonolysis of 12 proved
troublesome with the formation of a dimethyl acetal byproduct.
However, this could be removed by chromatography and the tri-
t-butyl ester 29 and its enantiomer ent-29 could be produced in
moderate yield. Coupling of the anion derived from 4 with 29
afforded the lactols 30 which spirocyclised under acidic conditions.
Ozonolysis then afforded triester 31 as the major compound with
a trace of the spiroisomer. In this case, cyclisation occurs from the
b-face of the oxonium ion (cf. Fig. 3) almost exclusively to give the
3R,4S,6S stereochemistry, epimeric at C3 compared to the natural
(+)-trachyspic acid (1) configuration. Deprotection then afforded
Synthetic (+)-trachyspic acid (1) and (−)-ent-1 as well as (+)-
3-epi-trachyspic acid (28) and (−)-ent-28 were all tested for
inhibition of human platelet heparanase and the results are
shown in Table 1. The assays were performed using a Microcon
ultrafiltration assay25 which uses [3H]-labelled HS as substrate and
an ultrafiltration device to separate longer native HS from the
smaller, cleaved products of heparanase catalysis which are then
quantified by scintillation counting. Synthetic (+)-trachyspic acid
(1) had a comparable IC50 value to that obtained for the natural
product by an alternative heparanase assay.3 Interestingly, all other
stereoisomers had comparable activities with (−)-ent-28 being the
most active. Thus, it appears the stereochemistry is not critical for
heparanase inhibition.
Table 1 Inhibition of heparanase activity
Compound
IC50/lM
(+)-1
33.5 6.5
26.5 5.4
31.7 5.6
24.5 5.4
(−)-ent-1
(+)-28
(−)-ent-28
Scheme 6 Reagents and conditions: (a) (1) O3, Me2S, (2) NaClO2, NaH2PO4, (3) N,Nꢀ-diisopropyl-O-tert-butylisourea; (b) t-BuLi, Et2O–hexane, −78 ◦C;
(c) lactone 29, THF; (d) (1) 3 M HClO4, THF, (2) Ac2O, DMAP, pyridine; (e) O3, NaHCO3, Me2S; (f) TFA, CH2Cl2.
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the organic phase was extracted with Et2O. The combined organic
extracts were washed with water then brine, and dried over MgSO4.
The solvent was removed under reduced pressure and the crude
residue was dissolved in MeOH (1000 cm3) and treated with 10%
HCl (1 cm3). The reaction was stirred at rt overnight and quenched
with saturated aqueous NaHCO3. The MeOH was removed under
reduced pressure and the aqueous phase was extracted with Et2O,
washed with water then brine, and dried. The crude product was
purified by column chromatography with 20% EtOAc–petrol as
eluent to afford the b-anomer 8 (9.22 g, 36% for 2 steps) as a
yellow oil: [a]1D4 −38.4 (c 1.07, CH2Cl2); vmax (film) 3447, 2937,
2836, 1613, 1514, 1037 cm−1; dH (400 MHz) 2.08 (br s, 1H), 2.16–
2.27 (m, 2H), 3.39 (s, 3H), 3.57 (dd, J = 12.0, 3.6 Hz, 1H), 3.73
(dd, J = 12.0, 2.6 Hz, 1H), 3.80 (s, 3H), 4.26 (m, 2H), 4.42 (s,
2H), 5.12 (dd, J = 5.6, 2.6 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 7.24
(d, J = 8.6 Hz, 2H); dC (100 MHz, CDCl3) 40.2, 55.2, 55.4, 64.0,
71.4, 78.6, 85.7, 105.7, 113.8, 129.3, 129.8, 159.3; HRMS (ESI):
calculated for C14H20O5Na [M + Na]+ 291.1208, found 291.1205.
Further elution with 40% EtOAc–petrol afforded the a-anomer
(11.7 g, 44%) as a yellow gum: [a]1D4 +139.5 (c 1.04, CH2Cl2); vmax
3461, 2911, 2837, 1613, 1514, 1037 cm−1; dH (400 MHz) 1.73 (br s,
1H), 1.98 (dd, J = 13.9, 1.6 Hz, 1H), 2.18 (ddd, J = 13.9, 8.0,
4.8 Hz, 1H), 3.37 (s, 3H), 3.52–3.74 (m, 2H), 3.77 (s, 3H), 3.95
(m, 1H), 4.09 (dd, J = 8.0, 4.4 Hz, 1H), 4.45 (ABq, J = 12.0 Hz,
2H), 5.02 (d, J = 4.8 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 7.24 (d,
J = 8.8 Hz, 2H); dC (100 MHz) 39.3, 55.1, 55.2, 62.7, 71.5, 77.6,
83.0, 105.2, 113.8, 129.4, 130.0, 159.2; HRMS (ESI): calculated
for C14H20O5Na [M + Na]+ 291.1208, found 291.1204.
Conclusions
The synthesis of both natural (+)-trachyspic acid (1) and its
enantiomer (−)-ent-1 as well as both enantiomers of 3-epi-
trachyspic acid (28 and ent-28) has been achieved from a
common chiral precursor 7 derived from 2-deoxy-D-ribose. The
key steps in the sequences used are a stereoselective Ireland–
Claisen rearrangement in the presence of a b-leaving group,
a selective carbanion addition to a lactone tri-t-butylester and
a spirocyclisation–ozonolysis–elimination sequence to construct
the spirolactone system in a stereoselective manner. The activity
against heparanase of all four stereoisomers is similar indicating
that the stereochemistry of the triacid is not critical for activity
against this target.
Experimental
General
Optical rotations were recorded in a 10 cm microcell. High
resolution mass spectra (HRMS) were run using electrospray
ionisation (ESI). Proton nuclear magnetic resonance (1H NMR,
300, 400 and 500 MHz) and proton decoupled carbon nuclear
magnetic resonance spectra (13C NMR, 75.5, 100 and 125 MHz)
were recorded for deuteriochloroform solutions with residual chlo-
roform as internal standard unless otherwise stated. Analytical
thin layer chromatography (TLC) was conducted on aluminium
backed 2 mm thick silica gel GF254. Compounds were visualised
with solutions of 20% w/w phosphomolybdic acid in ethanol, 20%
w/w potassium permanganate in water or under UV (365 nm).
Anhydrous tetrahydrofuran (THF) and diethyl ether were distilled
from sodium benzophenone ketyl and sodium metal under a
nitrogen atmosphere. Petrol refers to the fraction boiling at 40–
Allyl ester 5
A
solution of alcohol
8
(2.83 g, 10.5 mmol) and
tetramethylpiperidine-N-oxyl (TEMPO, 115 mg, 0.738 mmol) in
acetonitrile (52 cm3) and pH 7 buffer (41 cm3) was warmed
to 35 ◦C and solutions of 80% NaClO2 (2.39 g, 21.0 mmol)
in water (10.5 cm3) and 8% NaOCl (196 lL, 0.211 mmol) in
water (5.2 cm3) were then added simultaneously to the reacti◦on
mixture over 15 min. The reaction mixture was stirred at 35 C
for 24 h and then cooled to rt. Water (75 cm3) was added and the
pH was adjusted to 8 using 1 M aqueous NaOH. The mixture
was cooled to 0 ◦C and a solution of Na2SO3 (3.15 g) in water
(49 cm3) was added. The solution was warmed to rt and stirred for
30 min. Et2O was added and the organic phase was separated and
concentrated under reduced pressure to recover starting alcohol 8
(0.62 g). The aqueous layer was acidified with 10% HCl and the
aqueous phase was extracted with Et2O and the combined organic
extracts were washed with water then brine, and dried. The solvent
was removed under reduced pressure and the crude residue was
dissolved in dry CH2Cl2 (45 cm3), cooled to 0 ◦C and DMAP
(90 mg, 0.737 mmol), allyl alcohol (551 lL, 8.11 mmol) and 1,3-
dicyclohexylcarbodiimide (DCC, 1.60 g, 7.74 mmol) were added.
The mixture was stirred for 1 h at 0 ◦C then warmed to rt and
stirred for another 1 h. The white suspension was filtered through
Celite and the filtrate was concentrated under reduced pressure.
The crude residue was purified by column chromatography with
10% EtOAc–petrol as eluent to give ester 5 (1.98 g, 75% for 2 steps)
as a pale yellow oil: [a]1D3 −58.8 (c 1.05, CH2Cl2); vmax (thin film)
2934, 2836, 1756, 1613, 1250 cm−1; dH (400 MHz) 2.06 (m, 1H),
2.21 (m, 1H), 3.36 (s, 3H), 3.77 (s, 3H), 4.46 (ABq, J = 11.6 Hz,
◦
60 C. All other commercial reagents were used as received. All
air and moisture sensitive reactions were performed in glassware
that was either flame dried under an atmosphere of dry argon or
oven dried at 150 ◦C.
Methyl furanoside 8
A solution of commercial 2-deoxy-D-ribose (14.8 g, 0.110 mol) in
anhydrous MeOH (235 cm3) was cooled to 0 ◦C and treated with
concentrated H2SO4 (5 cm3). The solution was stirred at 0 ◦C for
1 h and then neutralised with solid NH4HCO3. The suspension
was filtered and the filtrate was concentrated. The crude residue
was dissolved in N,N-dimethylformamide (DMF, 140 cm3) and
pyridine (19.2 cm3), 4-(dimethylamino)pyridine (DMAP, 1.34 g,
0.011 mol) and trityl chloride (61.3 g, 0.220 mol) were added. The
solution was stirred at rt for 16 h and saturated aqueous NaHCO3
was added and the aqueous phase was extracted with Et2O. The
combined organic extracts were washed with saturated aqueous
CuSO4, water then brine, dried and concentrated. Purification
by flash chromatography with 10–40% EtOAc–petrol as eluent
gave alcohol 7 (37.4 g, 87%) as a yellow gum. A solution of 7
(37.4 g, 0.0993 mol) in DMF (250 cm3) was added via cannula to
a suspension of 60% NaH dispersion in oil (4.77 g, 0.119 mol) in
DMF (200 cm3). To the suspension was added p-methoxybenzyl
chloride (PMBCl, 16.2 cm3, 0.119 mol) and the reaction mixture
was stirred at rt for 16 h. The reaction was quenched with water and
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1H), 4.52 (ABq, J = 11.6 Hz, 1H), 4.53 (m, 1H), 4.55 (m, 2H),
4.64 (d, J = 6.0 Hz, 1H), 5.13 (dd, J = 5.2, 1.2 Hz, 1H), 5.24 (d,
J = 10.6 Hz, 1H), 5.33 (dd, J = 17.2, 1.2 Hz, 1H), 5.91 (ddd, J =
17.2, 10.6, 5.6 Hz, 1H), 6.84 (d, J = 8.6 Hz, 2H), 7.24 (d, J =
8.6 Hz, 2H); dC (100 MHz) 39.7, 55.2, 55.3, 65.9, 71.7, 80.6, 81.9,
106.2, 113.8, 118.8, 129.4, 129.6, 131.6, 159.3, 171.3; HRMS (ESI):
calculated for C17H22O6Na [M + Na]+ 345.1314, found 345.1313.
solvent was removed under reduced pressure. The remaining dark
coloured residue was purified by column chromatography with
20% EtOAc–petrol as eluent to give the lactone 10 (820 mg, 84%
for 2 steps) as a pale yellow oil: [a]2D0 +3.8 (c 1.09, CH2Cl2); vmax
(thin film) 2934, 1756, 1613, 1514, 1037 cm−1; dH (400 MHz) 1.45
(s, 9H), 2.53 (dd, J = 14.8, 7.2 Hz, 1H), 2.69 (d, J = 6.8 Hz, 2H),
2.84 (dd, J = 14.8, 7.2 Hz, 1H), 3.80 (s, 3H), 4.16 (t, J = 7.2 Hz,
1H), 4.46 (ABq, J = 11.6 Hz, 1H), 4.52 (ABq, J = 11.6 Hz, 1H),
5.15 (m, 2H), 5.73 (ddt, J = 16.8, 10.0, 7.2 Hz, 1H), 6.86 (d, J =
8.6 Hz, 2H), 7.20 (d, J = 8.6 Hz, 2H); dC (100 MHz) 27.9, 34.9,
39.3, 55.2, 72.3, 77.9, 83.1, 88.4, 113.8, 120.4, 128.7, 129.4, 130.7,
159.5, 167.3, 173.4; HRMS (ESI): calculated for C20H26O6Na [M +
Na]+ 385.1627, found 385.1617.
tert-Butyl ester 9
A solution of nBuLi in hexanes (3.4 cm3, 2.3 M, 7.71 mmol) was
added dropwise to a solution of iPr2NH (0.98 cm3, 7.01 mmol) in
dry THF (17 cm3) at 0 ◦C under argon. The resultant lithium diiso-
propylamide (LDA) solution was stirred at 0 ◦C for 5 min, cooled
to −78 ◦C and added dropwise via cannula to a solution of the allyl
ester 5 (1.13 g, 3.51 mmol), hexamethylphosphoramide (HMPA,
4.6 cm3) and the supernatant from a centrifuged mixture of freshly
distilled trimethylsilyl chloride (TMSCl, 2.5 cm3, 19.3 mmol) and
NEt3 (2.5 cm3, 17.5 mmol) in dry THF (26 cm3) at −100 ◦C (liquid
N2/MeOH bath). The reaction mixture was stirred at −100 ◦C for
10 min and then allowed to warm to rt and left to stir for 2 h. The
a,b-Unsaturated lactone 11
To a solution of the lactone 10 (820 mg, 2.26 mmol) in CH2Cl2
(100 cm3) and water (5.6 cm3) was added 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ, 1.28 g, 5.66 mmol). The biphasic
suspension was stirred at rt for 16 h and the crude reaction mixture
was filtered through Celite and the filtrate was concentrated. The
crude product was purified by column chromatography with 30%
EtOAc–petrol as eluent to give an alcohol (507 mg, 92%) as a pale
pink oil: [a]1D8 +13.5 (c 1.01, CH2Cl2); vmax (thin film) 3466, 2983,
1789, 1736, 1267, 1161 cm−1; dH (400 MHz) 1.47 (s, 9H), 2.47 (dd,
J = 14.4, 7.0 Hz, 1H), 2.65 (dd, J = 18.2, 4.4 Hz, 1H), 2.74 (dd, J =
14.4, 7.0 Hz, 1H), 2.84 (dd, J = 18.2, 7.4 Hz, 1H), 3.34 (br s, 1H),
4.47 (dd, J = 7.4, 4.4 Hz, 1H), 5.16 (s, 1H), 5.19 (d, J = 8.8 Hz, 1H),
5.72 (ddt, J = 17.2, 10.0, 7.0 Hz, 1H); dC (100 MHz) 27.9, 36.9,
39.5, 72.1, 83.8, 90.2, 120.5, 130.2, 167.8, 174.3; HRMS (ESI):
calculated for C12H18O5Na [M + Na]+ 265.1052, found 265.1047.
A solution of the alcohol (507 mg, 2.09 mmol) in pyridine (20 cm3)
was cooled to 0 ◦C. The solution was treated with MsCl (486 lL,
6.28 mmol) at 0 ◦C for 2 h and then warmed to rt and stirred
overnight. Water and Et2O were added and the aqueous phase
was extracted further with Et2O. The combined organic extracts
were washed with saturated aqueous CuSO4, water, then brine,
dried and concentrated under reduced pressure. The crude residue
was purified by column chromatography, eluting with 15–20%
EtOAc–petrol to afford the a,b-unsaturated lactone 11 (423 mg,
90%) as a pale yellow low melting crystalline solid: [a]1D3 +161.8 (c
1.01, CH2Cl2); vmax (thin film) 2983, 1772, 1372, 1258, 1133 cm−1;
dH (400 MHz) 1.45 (s, 9H), 2.64 (dd, J = 14.4, 6.8 Hz, 1H), 2.84
(dd, J = 14.4, 7.6 Hz, 1H), 5.17 (s, 1H), 5.20 (d, J = 4.8 Hz, 1H),
5.67 (m, 1H), 6.13 (d, J = 5.8 Hz, 1H), 7.39 (d, J = 5.8 Hz, 1H);
dC (100 MHz) 27.8, 39.8, 84.0, 89.5, 120.9, 122.2, 129.4, 154.6,
165.9, 171.5; HRMS (ESI): calculated for C12H16O4Na [M + Na]+
247.0946, found 247.0944.
◦
solution was cooled to 0 C and aqueous 1 M NaOH (25 cm3)
was added followed by Et2O and water. The organic phase was
separated and discarded and the aqueous phase was then acidified
with 10% HCl at 0 ◦C and extracted with Et2O. The combined
extracts were washed with water then brine, and dried over MgSO4.
The solvent was removed under reduced pressure and the residue
was dissolved in dry CH2Cl2 (50 cm3) and treated with N,Nꢀ-
diisopropyl-O-tert-butylisourea (3.23 g, 16.1 mmol) for 16 h at rt.
The white suspension was filtered through Celite and the filtrate
was concentrated under reduced pressure. The crude residue was
purified by column chromatography with 10% EtOAc–petrol as
eluent to give the tert-butyl ester 9 (1.02 g, 77% for 2 steps) as a
colourless oil: [a]D14 −55.1 (c 0.98, CH2Cl2); vmax (thin film) 2979,
2837, 1733, 1614, 1515, 1249, 1036 cm−1; dH (400 MHz) 1.43 (s,
9H), 2.11 (ddd, J = 13.1, 6.4, 1.6 Hz, 1H), 2.28 (m, 1H), 2.46
(dd, J = 14.2, 7.2 Hz, 1H), 2.81 (dd, J = 14.2, 7.2 Hz, 1H), 3.37
(s, 3H), 3.80 (s, 3H), 4.13 (t, J = 6.8 Hz, 1H), 4.44 (ABq, J =
11.6 Hz, 1H), 4.52 (ABq, J = 11.6 Hz, 1H), 5.10 (s, 1H), 5.13 (d,
J = 7.2 Hz, 1H), 5.21 (dd, J = 5.6, 1.6 Hz, 1H), 5.86 (ddt, J =
17.2, 10.0, 7.2 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 7.22 (d, J =
8.6 Hz, 2H); dC (100 MHz) 28.0, 38.3, 41.9, 55.2, 55.3, 71.9, 81.5,
82.7, 88.6, 104.8, 113.6, 118.3, 129.2, 129.8, 133.1, 159.1, 169.8;
HRMS (ESI): calculated for C21H30O6Na [M + Na]+ 401.1940,
found 401.1936.
Lactone 10
To a solution of the ester 9 (1.02 g, 2.70 mmol) in THF (50 cm3)
was added 10% aqueous HCl (35 cm3) and the reaction was stirred
for 40 h at rt and diluted with water and Et2O. The aqueous phase
was extracted with Et2O, washed with water then brine, and dried
over MgSO4. The solvent was removed under reduced pressure and
the resulting lactol mixture was dissolved in dry CH2Cl2 (60 cm3)
Dienes 12 and 3
To a flame dried flask containing CuI (395 mg, 2.07 mmol)
was added dry THF (10 cm3) under argon. The pale brown
suspension was cooled to −78 ◦C and a solution of vinylMgBr
in THF (4.6 cm3, 0.9 M, 4.15 mmol) was added. The resulting
yellow suspension was stirred for 10 min at −78 ◦C and Me2S
(102 lL, 1.38 mmol) was then added and the suspension was
stirred at −78 ◦C for 90 min. A solution of the lactone 11 (155 mg,
0.691 mmol) in dry THF (4.5 cm3) was then added via cannula.
˚
and 4 A sieves (1.54 g) were added. The suspension was cooled to
0 ◦C and pyridinium chlorochromate (PCC, 880 mg, 4.04 mmol)
was added. The reaction was stirred for 2 h at 0 ◦C, warmed
to rt and stirred overnight. The brown suspension was filtered
through Fluorosil, washed with large quantities of Et2O and the
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The reaction mixture was immediately warmed to −45 ◦C and
stirred for 1 h then quenched with saturated aqueous NH4Cl and
diluted with Et2O. The aqueous phase was extracted with Et2O
and the combined organic extracts were washed with water then
brine, dried and concentrated. The crude product was purified
by column chromatography with 80% CH2Cl2–petrol as eluent to
afford lactone 12 (80 mg, 46%) as a colourless oil: [a]2D3 −24.8
(c 1.03, CH2Cl2); vmax (thin film) 2981, 1796, 1733, 1370, 1252,
1152 cm−1; dH (400 MHz) 1.45 (s, 9H), 2.51 (dd, J = 14.8, 8.0 Hz,
1H), 2.58 (dd, J = 17.2, 8.8 Hz, 1H), 2.67 (dd, J = 17.2, 11.2 Hz,
1H), 2.86 (dd, J = 14.8, 6.4 Hz, 1H), 3.12 (dd, J = 17.8, 7.6 Hz,
1H), 5.16–5.23 (m, 4H), 5.68 (ddd, J = 17.2, 10.4, 7.6 Hz, 1H),
5.79 (m, 1H); dC (100 MHz) 28.0, 33.2, 38.7, 47.2, 83.6, 87.8,
119.3, 120.4, 131.1, 132.5, 168.1, 174.9; HRMS (ESI): calculated
for C14H20O4Na [M + Na]+ 275.1259, found 275.1257.
Further elution gave the C4 epimer 3 (56 mg, 32%) as a pale
yellow oil: [a]2D2 +18.1 (c 0.87, CH2Cl2); vmax (thin film) 2981, 1791,
1732, 1370, 1132 cm−1; dH (400 MHz) 1.47 (s, 9H), 2.42 (dd, J =
14.4, 6.0 Hz, 1H), 2.45 (dd, J = 17.6, 6.0 Hz, 1H), 2.58 (dd, J =
14.4, 8.0 Hz, 1H), 2.73 (dd, J = 18.0, 8.4 Hz, 1H), 3.23 (dd, J =
14.4, 8.4 Hz, 1H), 5.14–5.29 (m, 4H), 5.72–5.86 (m, 2H); dC (100
MHz) 27.9, 33.7, 37.9, 46.4, 83.2, 87.9, 119.1, 120.0, 130.9, 133.2,
169.5, 174.5; HRMS (ESI): calculated for C14H20O4Na [M + Na]+
275.1259, found 275.1260.
Alkyne 17
Dess–Martin periodinane (12.51 g, 29.5 mmol) was added to a
cooled solution of alcohol 16 (8.29 g, 18.8 mmol) in CH2Cl2
(200 cm3) at 0 ◦C. The solution was warmed to rt, stirred for
30 min and quenched with saturated aqueous NaHCO3 and 1.5 M
Na2S2O3. The biphasic solution was stirred for 15 min, until two
clear layers formed and the aqueous phase was extracted with
Et2O. The combined organic extracts were washed with water then
brine, dried and concentrated. A ◦solution of the crude aldehyde in
CH2Cl2 (150 cm3) was cooled to 0 C and PPh3 (19.72 g, 75.2 mmol)
and CBr4 (12.47 g, 37.6 mmol) were added. The orange solution
was stirred at 0 ◦C for 1 h and the solvent was removed under
reduced pressure. The crude residue was triturated with petrol,
filtered and the filtrate was concentrated to afford a yellow oil
(8.73 g, 14.6 mmol) which was dissolved in dry THF (200 cm3),
cooled to −78 C and a solution of BuLi in hexanes (2.1 M,
14.0 cm3, 29.4 mmol) was added dropwise. Water was added and
the aqueous phase was extracted with petrol and the combined
organic extracts were washed with water then brine, dried and
concentrated. Purification by flash chromatography with 2.5%
EtOAc–petrol as eluent gave the alkyne 17 (5.40 g, 66%) as a
colourless oil: vmax 2928, 2857, 1428, 1112 cm−1; dH (400 MHz)
0.89 (t, J = 6.8 Hz, 3H), 1.07 (s, 9H), 1.28 (br s, 14H), 1.66 (m,
2H), 2.04 (d, J = 2.4 Hz, 1H), 2.56 (m, 1H), 3.61 (dd, J = 10.0,
7.6 Hz, 1H), 3.75 (dd, J = 10.0, 5.6 Hz, 1H), 7.40 (m, 5H), 7.69
(m, 5H); dC (100 MHz) 14.1, 19.3, 22.7, 26.8, 26.8, 29.3, 29.4,
29.5, 29.6, 30.9, 31.9, 34.5, 66.1, 69.8, 85.6, 127.6, 129.6, 133.6,
135.6, 135.6; HRMS (ESI): calculated for C29H42OSiH [M + H]+
435.3083, found 435.3084.
◦
n
Lactone triester 13
Ozone gas was bubbled through a solution of the diene 3 (30 mg,
0.119 mmol) in CH2Cl2 (3.0 cm3) and MeOH (180 lL) at −78 ◦C
until a pale blue colour persisted. Me2S (87 lL, 1.19 mmol)
was added and the solution was allowed to warm to rt and
stirring was continued for a further 4 h. Water and Et2O were
added and the aqueous phase was extracted with Et2O. The
combined extracts were washed with water then brine, and dried
over MgSO4 and concentrated under reduced pressure. The crude
dialdehyde was dissolved in tert-butanol (3.0 cm3) and treated
with 2-methyl-2-butene (500 lL). A solution of NaH2PO4·H2O
(131 mg, 0.951 mmol) and 80% NaClO2 (215 mg, 1.90 mmol)
in water (1.6 cm3) was then added and the reaction mixture
was stirred at rt for 16 h. Water and Et2O were added and the
organic layer was separated and discarded. The aqueous phase
was acidified with 10% HCl and extracted with Et2O and the
combined extracts were washed with water then brine, and dried.
The solvent was removed under reduced pressure and the residue
was dissolved in dry CH2Cl2 (9.5 cm3) and treated with N,Nꢀ-
diisopropyl-O-tert-butylisourea (476 mg, 2.38 mmol) for 16 h at
rt. The suspension was filtered through Celite and the solvent was
removed under reduced pressure. The crude residue was purified
by column chromatography with 10% EtOAc–petrol as eluent to
give the triester 13 (30 mg, 63%) as a colourless crystalline solid;
[a]1D4 −15.5 (c 0.99, CH2Cl2); vmax (thin film) 2980, 1803, 1732,
1369, 1148 cm−1; dH (400 MHz) 1.44 (s, 9H), 1.48 (s, 9H), 1.49 (s,
9H), 2.75 (dd, J = 17.6, 4.0 Hz, 1H), 2.83 (dd, J = 17.6, 8.6 Hz,
1H), 2.87 (ABq, J = 17.2 Hz, 1H), 3.08 (ABq, J = 17.2 Hz, 1H),
3.39 (dd, J = 8.6, 4.0 Hz, 1H); dC (100 MHz) 27.7, 27.9, 28.0,
32.1, 39.0, 47.6, 82.0, 83.2, 83.6, 83.7, 167.6, 168.3, 169.0, 174.0;
HRMS (ESI): calculated for C20H32O8Na [M + Na]+ 423.1995,
found 423.1990.
Bromoalkene 18
A solution of alkyne 17 (5.40 g, 12.4 mmol) in CH2Cl2 (100 cm3)
was cooled to 0 ◦C and B-bromo-9-borabicyclo[3.3.1]nonane (B-
Br-9-BBN, 1.0 M in THF, 24.8 cm3, 24.8 mmol) was added. The
reaction was stirred at 0 ◦C for 3 h and glacial acetic acid (11.4 cm3,
198 mmol) was then added and the solution was stirred for 1 h.
Water was added and the aqueous phase was extracted with petrol.
The combined organic extracts were washed with water then brine,
dried and concentrated and the crude product was purified by flash
chromatography with petrol as eluent to give vinyl bromide 18
(5.51 g, 86%) as a colourless oil: vmax 2928, 2856, 1428, 1113 cm−1;
dH (400 MHz) 0.89 (t, J = 6.8 Hz, 3H), 1.05 (s, 9H), 1.25 (br s,
14H), 1.32 (m, 2H), 2.41 (quint., J = 6.0 Hz, 1H), 3.55 (dd, J =
10.0, 5.2 Hz, 1H), 3.66 (dd, J = 10.0, 8.0 Hz, 1H), 5.54 (s, 1H),
5.68 (s, 1H), 7.40 (m, 5H), 7.68 (m, 5H); dC (100 MHz) 14.1,
19.3, 22.7, 26.7, 26.8, 29.1, 29.3, 29.4, 29.5, 29.5, 31.9, 52.3, 65.3,
118.4, 127.6, 127.6, 129.6, 133.6, 133.8, 135.6, 135.7, 137.2; HRMS
(ESI): calculated for C29H43BrOSiNa [M + Na]+ 537.2164, found
537.2169.
Acetal 4
Tetrabutylammonium fluoride trihydrate (TBAF·3H2O, 8.43 g,
26.7 mmol) was added to a solution of the silyl ether 18 (5.51 g,
10.7 mmol) in THF (150 cm3) and the solution was stirred at rt for
16 h. Water was added and the aqueous phase was extracted with
Et2O and the combined organic extracts were washed with water
then brine, dried and concentrated to afford the alcohol 19 (2.96 g,
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100%) as a yellow oil: vmax 3339, 2926, 2855, 1466, 1050 cm−1; dH
(400 MHz) 0.87 (t, J = 6.8 Hz, 3H), 1.25 (br s, 14H), 1.38 (m, 2H),
1.61 (br s, 1H), 2.41 (quint., J = 4.4 Hz, 1H), 3.51 (dd, J = 11.2,
5.2 Hz, 1H), 3.59 (dd, J = 11.2, 8.8 Hz, 1H), 5.59 (d, J = 1.0 Hz,
1H), 5.74 (d, J = 1.0 Hz, 1H); dC (100 MHz) 14.1, 22.6, 26.7, 29.1,
29.3, 29.4, 29.4, 29.5, 31.8, 52.5, 64.2, 119.7, 136.6; HRMS (ESI):
calculated for C13H25BrONa [M + Na]+ 299.0986, found 299.0982.
A solution of the alcohol 19 (2.96 g, 10.7 mmol) in CH2Cl2
(200 cm3) was cooled to 0 ◦C and Dess–Martin periodinane (5.90 g,
13.9 mmol) was added. The solution was warmed to rt and stirred
for 30 min, then quenched with saturated aqueous NaHCO3 and
1.5 M Na2S2O3. The biphasic solution was stirred for 15 min until
two clear layers formed and the aqueous phase was extracted with
Et2O. The combined organic extracts were washed with water then
brine, dried and concentrated. The crude residue was dissolved in
dry benzene (150 cm3) and p-TsOH (204 mg, 1.07 mmol) and
ethylene glycol (6.0 cm3, 107 mmol) were added and the solution
was heated under reflux on a Dean Stark apparatus for 16 h and
then cooled to rt. Saturated aqueous NaHCO3 was added and the
aqueous phase was extracted with EtOAc. The combined organic
extracts were washed with water then brine, dried and concentrated
and the crude product was purified by flash chromatography with
2.5% EtOAc–petrol as eluent to afford acetal 4 (2.96 g, 87%) as
a colourless oil: vmax 2925, 2855, 1466, 1120 cm−1; dH (400 MHz)
0.87 (t, J = 7.2 Hz, 3H), 1.25 (br s, 14H), 1.49–1.62 (m, 2H), 2.34
(m, 1H), 3.86–3.99 (m, 4H), 4.85 (d, J = 6.4 Hz, 1H), 5.58 (d,
J = 1.2 Hz, 1H), 5.72 (d, J = 1.2 Hz, 1H); dC (100 MHz) 14.1,
22.6, 26.5, 28.1, 29.3, 29.4, 29.5, 31.9, 53.8, 64.9, 65.0, 104.9, 119.5,
119.6, 133.4; HRMS (ESI): calculated for C15H27BrO2H [M + H]+
319.1266, found 319.1266.
HRMS (ESI): calculated for C35H60O10Na [M + Na]+ 663.4084,
found 663.4083.
(−)-Trachyspic acid tri-tert-butyl ester 23
A solution of the lactol mixture 22 (18 mg, 0.0281 mmol) in
◦
THF (1.0 cm3) was cooled to 0 C and treated with 3 M HClO4
(0.5 cm3). The solution was stirred at 0 ◦C for 1 h, quenched with
saturated aqueous NaHCO3 and the aqueous phase was extracted
with Et2O, washed with water then brine and dried. The solvent
was removed under reduced pressure and the residue was dissolved
in pyridine (1.0 cm3) and DMAP (0.34 mg, 0.00281 mmol) and
acetic anhydride (27 lL, 0.281 mmol) were added. The solution
was stirred at rt overnight and then diluted with water and Et2O
and the aqueous phase was extracted with Et2O. The organic
extracts were washed with saturated aqueous CuSO4, water then
brine, dried and concentrated. The crude residue was dissolved in
CH2Cl2 (2.0 cm3) and MeOH (200 lL) and ozone gas was bubbled
through the solution at −78 ◦C until a pale blue colour persisted.
Me2S (21 lL, 0.281 mmol) and NaHCO3 (21 mg, 0.281 mmol)
were added and the solution was warmed to rt and stirred for 16 h.
Water and Et2O were added and the aqueous phase was extracted
with Et2O. The combined organic extracts were washed with water
then brine, dried and concentrated. The crude residue was purified
by column chromatography with 5–10% EtOAc–petrol as eluent
to afford the minor spiroisomer 24 (1 mg, 6% for 3 steps) as a
thin film: [a]2D4 −25.5 (c 0.03, CH2Cl2); vmax (thin film) 2927, 1732,
1367, 1151 cm−1; dH (500 MHz) 0.88 (t, J = 8.0 Hz, 3H), 1.25 (br s,
14H), 1.42 (s, 9H), 1.47 (s, 9H), 1.52 (s, 9H), 2.08 (t, J = 8.0 Hz,
2H), 2.51 (dd, J = 13.5, 5.5 Hz, 1H), 2.67 (dd, J = 13.5, 9.0 Hz,
1H), 3.03 (d, J = 16.5 Hz, 1H), 3.17 (d, J = 17 Hz, 1H), 3.68 (dd,
J = 9.0, 5.5 Hz, 1H), 7.86 (s, 1H); HRMS (ESI): calculated for
C32H52O9Na [M + Na]+ 603.3509, found 603.3507.
Lactols 22
t
A solution of BuLi in hexanes (392 lL, 1.4 M, 0.549 mmol)
Further elution provided spiroisomer 23 (9 mg, 55% for 3 steps)
as a colourless oil: [a]1D7 −14.4 (c 0.30, CH2Cl2); vmax (thin film)
2928, 1733, 1368, 1151 cm−1; dH (400 MHz) 0.87 (t, J = 8.0 Hz,
3H), 1.25 (br s, 14H), 1.42 (s, 9H), 1.48 (s, 9H), 1.50 (s, 9H), 2.06
(t, J = 8.0 Hz, 2H), 2.24 (dd, J = 13.2, 7.2 Hz, 1H), 2.63 (t,
J = 12.8 Hz, 1H), 2.87 (s, 2H), 3.53 (dd, J = 12.4, 6.8 Hz, 1H),
7.88 (s, 1H); dC (100 MHz) 14.1, 21.1, 22.7, 27.7, 27.9, 28.0, 28.0,
29.3, 29.3, 29.5, 31.8, 38.1, 39.5, 50.1, 81.1, 82.2, 82.3, 87.2, 108.4,
118.0, 168.0, 168.4, 169.6, 172.3, 198.1; HRMS (ESI): calculated
for C32H52O9Na [M + Na]+ 603.3509, found 603.3508.
was added dropwise to a solution of bromoalkene 4 (100 mg,
0.313 mmol) in dry Et2O (1.0 cm3) and dry hexane (0.5 cm3) at
−78 ◦C under argon. The solution was stirred at −78 ◦C for
5 min and a solution of the triester 13 (44 mg, 0.110 mmol)
in Et2O (1.0 cm3) and hexane (0.5 cm3) was added dropwise
via cannula to◦ the anion solution. The reaction mixture was
stirred at −78 C for 4 h and diluted with water and Et2O. The
aqueous phase was extracted with Et2O and the combined organic
extracts were washed with water then brine and dried. The solvent
was removed under reduced pressure and purification by column
chromatography with 10% EtOAc–petrol gave recovered triester
13 (20 mg). Further elution afforded the lactols 22 (24 mg, 41%,
62% based on recovered 13) as a pale yellow oil: vmax (thin film)
3497, 2928, 2856, 1735, 1369, 1153 cm−1; dH (400 MHz) 0.86 (m,
3H), 1.23 (m, 14H), 1.43 (s, 9H), 1.44 (s, 9H), 1.47 (s, 9H), 1.63 (m,
2H), 2.50 (dd, J = 18.0, 2.8 Hz, 0.5H), 2.65 (dd, J = 18.0, 2.8 Hz,
0.5H), 2.80 (dd, J = 16.8, 7.6 Hz, 1H), 2.93 (d, J = 17.2 Hz, 1H),
3.05 (m, 1H), 3.39 (dd, J = 18.0, 11.2 Hz, 0.5H), 3.50 (dd, J =
18.0, 11.6 Hz, 0.5H), 3.72–3.94 (m, 5H), 4.78 (d, J = 5.2 Hz, 0.5H),
4.85 (d, J = 5.2 Hz, 0.5H), 5.81 (d, J = 4.4 Hz, 1H), 6.18 (d, J =
8.0 Hz, 1H); dC (100 MHz, CDCl3) 14.1, 22.7, 26.9, 27.0, 27.0,
27.8, 27.9, 28.0, 29.1, 29.2, 29.3, 29.4, 29.5, 29.5, 29.6, 29.7, 31.9,
32.2, 35.8, 35.9, 42.3, 42.6, 42.6, 43.5, 48.6, 48.7, 50.2, 61.3, 64.7,
64.8, 65.0, 66.2, 75.1, 75.1, 81.4, 81.4, 83.1, 105.8, 106.3, 117.2,
125.0, 125.8, 136.0, 147.2, 147.4, 170.0, 170.2, 172.6, 199.3, 199.5;
(−)-Trachyspic acid (ent-1)
A solution of the tri-tert-butylester 23 (10 mg, 0.0172 mmol)
in dry CH2Cl2 (1.5 cm3) was cooled to 0 ◦C and treated with
trifluoroacetic acid (TFA, 250 lL). The solution was stirred at
0
◦C for 1 h, warmed to rt and stirred for an additional 2 h.
Toluene (2.0 cm3) was added and the solvent was removed under
reduced pressure to afford ent-1 (7.0 mg, 99%) as a thin film: [a]D21
−3.5 (c 0.213, MeOH); [a]2D3 −8.4 (c 0.350, CH2Cl2); vmax (thin film)
3425, 2927, 2856, 1724, 1611, 1370, 1139 cm−1; dH (400 MHz, d6-
DMSO) 0.84 (t, J = 6.6 Hz, 3H), 1.23 (br s, 12H), 1.39 (m, 2H),
2.02 (t, J = 7.8 Hz, 2H), 2.36 (m, 2H), 2.67 (d, J = 16.8 Hz, 1H),
2.85 (d, J = 16.8 Hz, 1H), 3.56 (dd, J = 11.8, 7.8 Hz, 1H), 8.45
(s, 1H); dC (100 MHz, d6-DMSO) 14.0, 20.5, 22.1, 27.5, 28.6, 28.7,
28.9, 31.3, 37.6, 38.7, 48.4, 86.5, 108.1, 116.7, 170.1, 170.6, 171.3,
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174.5, 198.2; HRMS (ESI): calculated for C20H28O9H [M + H]+
413.1812, found 413.1809.
Jonathan White for the X-ray crystallographic analysis and Dr
Hideyuki Shiozawa (Sankyo Co., Ltd.) for copies of the NMR
spectra of natural (+)-trachyspic acid (1) and trachyspic acid
trimethyl ester (25).
(−)-Trachyspic acid trimethyl ester (−)-25
A solution of (−)-trachyspic acid (ent-1) (7.0 mg, 15.8 lmol)
in MeOH (1.0 cm3) and Et2O (250 lL) was cooled to 0 ◦C
and treated with exces◦s CH2N2. The solvent was removed under
reduced pressure at 0 C and the crude product was purified by
flash chromatography with 20% EtOAc–petrol as eluent to give
trimethyl ester (−)-25 (3.0 mg, 42%) as a thin film: [a]2D3 −20.4 (c
0.13, CH2Cl2); vmax 2926, 2854, 1742, 1612, 1367, 1135 cm−1; dH
(400 MHz, d6-DMSO) 0.84 (t, J = 6.8 Hz, 3H), 1.23 (br s, 12H),
1.38 (m, 2H), 2.02 (t, J = 7.6 Hz, 2H), 2.39 (dd, J = 13.2, 12.7 Hz,
1H), 2.47 (dd, J = 13.2, 7.4 Hz, 1H), 2.87 (d, J = 16.8 Hz, 1H), 2.93
(d, J = 16.8 Hz, 1H), 3.55 (s, 3H), 3.64 (s, 3H), 3.70 (s, 3H), 3.80
(dd, J = 12.7, 7.4 Hz, 1H), 8.47 (s, 1H); dC (100 MHz, d6-DMSO)
14.0, 20.5, 22.2, 27.5, 28.7, 28.8, 29.0, 31.4, 37.3, 38.5, 47.7, 51.9,
52.6, 53.0, 86.5, 107.7, 117.0, 169.1, 169.5, 169.8, 174.7, 197.7;
HRMS (ESI): calculated for C23H34O9Na [M + Na]+ 477.2101,
found 477.2093.
Notes and references
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Reactions were set up in a volume of 100 lL containing 40 mM
acetate buffer (pH 5.0), bovine serum albumin (BSA, 0.1 mg cm−3),
heparanase (90 ng), 5 lM [3H]-HS and various concentrations of
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assays were then initiated by adding [3H]-HS and immediately
20 lL was taken, quenched with 80 lL of 10 mM phosphate
(pH 7.0) and the 100 lL transferred to a Microcon YM-10
concentrator which was centrifuged at approximately 14 000 g for
5 min. The solution that passed through the membrane (filtrate)
was retained. This sample was considered the time = 0 sample.
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for 4 h and then the filtration step was repeated for three aliquots
of 20 lL from each assay. The time = 0 filtrate and the three time =
4 h filtrate samples were counted for 3H using Optiphase HiSafe 2
scintillant (Perkin Elmer) in a Microbeta 1450 counter (Wallac).
The difference between the time = 0 and the averaged time =
4 h samples gave the amount of heparanase activity. Relative
inhibition assays were run with a heparanase standard assay which
was identical in composition except no inhibitor was present,
and the amount of heparanase inhibition in the other assays was
determined by comparison with this standard. IC50 values were
determined for each compound.
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Acknowledgements
This work was funded by an Australian Research Council Dis-
covery Grant (DP0556064). We also thank Associate Professor
2834 | Org. Biomol. Chem., 2007, 5, 2826–2834
This journal is
The Royal Society of Chemistry 2007
©