Design, synthesis and evaluation of bifunctional inhibitors of type II dehydroquinase.

Article abstract of DOI:10.1039/b301731a

Inhibitors of type II dehydroquinase were designed to straddle the two distinct binding sites identified for the inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid and a glycerol molecule in a crystallographic study of the Streptomyces coelicolor enzyme. A number of compounds were designed to incorporate characteristics of both ligands. These analogues were synthesized from quinic acid, and were assayed against type I (Salmonella typhi) and type II (S. coelicolor) dehydroquinases. None of the analogues showed inhibition for type I dehydroquinase. Six of the analogues were shown to have inhibition constants in the micromolar to low millimolar range against the S. coelicolor type II dehydroquinase, while two showed no inhibition. The binding modes of the analogues in the active site of the S. coelicolor enzyme were studied by molecular docking with GOLD1.2. These studies suggest a binding mode where the ring is in a similar position to (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid in the crystal structure and the side-chain occupies part of the glycerol binding-pocket.

Full text of DOI:10.1039/b301731a

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Design, synthesis and evaluation of bifunctional inhibitors of type
II dehydroquinase†
Miguel D. Toscano,a Martyn Frederickson,a David P. Evans,a John R. Coggins,b Chris Abell*a
and Concepción González-Belloc
a University Chemical Laboratory, Lensfield Road, Cambridge, UK CB2 1EW
b Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology,
University of Glasgow, Glasgow, UK G12 8QQ
c Departamento de Química Orgánica y Unidad Asociada al C.S.I.C., Facultad de Química,
Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Received 5th February 2003, Accepted 2nd May 2003
First published as an Advance Article on the web 15th May 2003
Inhibitors of type II dehydroquinase were designed to straddle the two distinct binding sites identified for the
inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid and a glycerol molecule in a crystallographic
study of the Streptomyces coelicolor enzyme. A number of compounds were designed to incorporate characteristics
of both ligands. These analogues were synthesized from quinic acid, and were assayed against type I (Salmonella
typhi) and type II (S. coelicolor) dehydroquinases. None of the analogues showed inhibition for type I
dehydroquinase. Six of the analogues were shown to have inhibition constants in the micromolar to low millimolar
range against the S. coelicolor type II dehydroquinase, while two showed no inhibition. The binding modes of the
analogues in the active site of the S. coelicolor enzyme were studied by molecular docking with GOLD1.2. These
studies suggest a binding mode where the ring is in a similar position to (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-
1-carboxylic acid in the crystal structure and the side-chain occupies part of the glycerol binding-pocket.
determined at 1.8 Å resolution, with the potent reversible
inhibitor 3 bound at the active site (PDB code: 1GU1, the
Introduction
Dehydroquinase (EC 4.2.1.10, 3-dehydroquinate dehydratase)
catalyses the conversion of 3-dehydroquinate (1) into 3-dehydro-
crystals were obtained at pH 8.5 in Tris buffer in the presence of
PEG8Kcrystallisation Na/K phosphate and sodium tartrate).10
shikimate (2) (Scheme 1).1 This is the third step in the shikimate
This complex identifies a number of key interactions involved
pathway leading to chorismate, the precursor to the aromatic
in inhibitor binding, and sheds light on aspects of the catalytic
amino-acids -phenylalanine, -tryptophan and -tyrosine, and
mechanism of the enzyme. Also present in this structure was
a molecule of glycerol bound 3.7 Å away from the inhibitor
other key metabolic intermediates (e.g. p-aminobenzoic acid).2
The pathway is present in plants, fungi, bacteria and some
(Fig. 1). The glycerol originated from the enzyme storage buffer,
parasites, and inhibitors of shikimate pathway enzymes are
but its adventitious appearance in the crystal structure suggests
candidates for antibiotic, anti-parasitic and herbicidal agents
additional binding interactions which can be exploited in
(e.g. the herbicide glyphosate inhibits the sixth enzyme, 5-enol-
inhibitor design.
pyruvyl shikimate-3-phosphate (EPSP) synthase).3 The conver-
sion of 3-dehydroquinate into 3-dehydroshikimate is also a step
on the quinate pathway in fungi.4
Scheme 1 The reaction catalysed by dehydroquinase.
There are two structurally distinct forms (type I and type II)
of dehydroquinase, which catalyse the same transformation by
distinct mechanisms.1 The type I enzyme catalyses the syn elim-
ination of water from 3-dehydroquinate via imine intermediates
that are covalently attached to a conserved lysine side chain.5
The type II enzyme catalyses an anti elimination via an enol
or enolate intermediate.6 Inhibitors have been designed which
Fig. 1 Selected view of the active-site of type II dehydroquinase
specifically inhibit type I7 and type II dehydroquinases.8
(S. coelicolor) from the crystal structure (1.8 Å resolution, PDB code
The crystal structures of both type I and type II dehydro-
quinases have been reported.9 Recently, the structure of the
Streptomyces coelicolor type II dehydroquinase has been
1GU1) showing the relative position of 3 (green) and the glycerol
molecule (purple). The distance between the two closest carbon atoms
in 3 and glycerol is 3.7 Å.
There are several examples of inhibitor design where two
inhibitors which bind to distinct regions of an enzyme active
site have been incorporated into a larger inhibitor with signifi-
† Electronic supplementary information (ESI) available: Dixon plots
and results of docking experiments. See http://www.rsc.org/suppdata/
ob/b3/b301731a/
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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cant increases in potency.11 In this paper we explore this idea by
making compounds 5–12 which straddle the binding sites of
both 3 and the glycerol molecule, and incorporate structural
features from both. The results of inhibition studies with
these compounds against type I (Salmonella typhi) and type II
dehydroquinase (S. coelicolor), and of molecular docking
studies into the active-site of S. coelicolor type II dehydro-
quinase using GOLD1.212 are described.
The strategy used for making 5–8 (Scheme 2) involved the
initial preparation of the protected trans-diaxial allylbenzoate
analogue (14). This was made in 3 steps and 47% yield, follow-
ing the methodology of Widlanski et al.13 The synthesis of 5
was achieved by a two step deprotection of 14. The lactone 14
was treated with a catalytic amount of sodium methoxide in
methanol to remove the benzoate group and form the methyl
ester 15 (87%). This was purified by extraction into water, wash-
ing with diethyl ether to remove the methyl benzoate, followed
by lyophilization. Conversion of 15 to the 3-allyl quinic acid
analogue 5 was achieved in 90% yield by ester hydrolysis, fol-
lowed by treatment with Dowex 50 (Hϩ) ion-exchange resin and
lyophilization. When deprotection of 14 was attempted in a
single step using sodium hydroxide, the product was obtained in
a mixture with benzoic acid which proved difficult to separate
even by ion-exchange chromatography.
The introduction of the 2Ј-hydroxy group into the side chain
olefin in 15 to form 6 was achieved in 98% yield by acid-cata-
lysed hydroxylation with concentrated hydrochloric acid under
reflux for 24 hours. The product was obtained as an approx-
imately 1 : 1 mixture of diastereoisomers, epimeric at C-2Ј. The
two diastereoisomers could be partially resolved by HPLC
using a preparative organic acids column, but it was not pos-
sible to separate them cleanly. The analogue 6 could also be
formed quantitatively by stirring 5 in concentrated hydrochloric
acid for 4 days at room-temperature.
The anti-Markovnikov mono-hydroxylated compound 16
was obtained in 31% yield by hydroboration of 14, using
borane–THF complex followed by work-up with sodium per-
borate and water. The modest yield was due to the difficult
purification of the product, which is partially deprotected
during the work-up. Compound 16 was purified by column
chromatography on silica gel and deprotected by treatment with
sodium methoxide in methanol to give the methyl ester 17
(98%) which was then hydrolysed to the target compound 7
(65%). The hydroxylation reaction was also attempted using
borane–dimethyl sulfoxide complex but a lower yield was
obtained. Furthermore the standard work-up with the hydro-
gen peroxide–sodium hydroxide totally deprotected the hydroxy
groups around the ring.
Results
Synthesis
All of the target compounds were accessible from quinic acid
(13). The first series 5–8 involved replacement of the C-3
hydroxy with an allyl group, which was subsequently elaborated
to introduce hydroxy groups. The second series 11–12 involved
attachment and modification of an allyl group attached to the
C-3 hydroxy group of quinic acid (13). A similar approach
using compounds epimeric at C-3 was used to make 9–10.
Scheme 2 Reagents and conditions: (i) ref. 13; (ii) MeOH, NaOMe, RT; (iii) 1. NaOH, H2O, RT, 2. Dowex 50 (Hϩ); (iv) HCl, H2O, ∆; (v) 1. BH3–
THF, THF, 0 ЊC, 2. NaBO3, H2O, 0 ЊC; (vi) OsO4 (cat), NMO, acetone–H2O (1 : 1), RT.
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The first step towards the dihydroxylated analogue 8 required
catalytic osmylation of 14. This was achieved using N-methyl-
morpholine N-oxide to reoxidise the catalyst14 to form 18 as a
1 : 1 mixture of diastereoisomers (epimeric at C-2Ј) in 22%
yield. Attempts to separate the diastereoisomers by HPLC on
an organic acids column and by acetylation and benzoylation
of the free hydroxy groups were unsuccessful. The deprotection
of 18 was carried out by initial treatment with sodium
methoxide in methanol, to give the methyl ester 19 (83%). This
was then hydrolysed with sodium hydroxide, followed by ion
exchange to yield the acid 8 quantitatively. Osmylation of 14
using trimethylamine N-oxide as a reoxidant and dichloro-
methane as the only solvent15 led to no reaction. Catalytic
osmylation on the methyl ester 15 using N-methylmorpholine
N-oxide was not successful.
Compounds 9 and 10 were synthesized from the protected
alcohol 20 prepared using a previously reported protocol
(Scheme 3).16 Ketone 21 was obtained via oxidation of 20 with
pyridinium dichromate under dry conditions (77%), and was
subsequently reduced to give alcohol 22.17 Allylation of 22
was achieved by treatment with allyl methyl carbonate and a
catalytic amount of Pd2(dba)2/dppb to give 23 in 69% yield.18
This step was particularly difficult because a competitive
diallylation process was observed, which could be overcome by
adding the solution of the carbonate slowly to the reaction
mixture. The desired acid 9 was obtained by a two-step depro-
tection sequence. First, treatment with aqueous trifluoroacetic
acid cleaved the bismethoxyacetal protecting group giving 24
in 94% yield, which upon basic hydrolysis followed by ion
exchange gave the acid 9 (97%). Treatment of the allyloxy ether
23 with osmium tetraoxide (catalytic) resulted in hydroxylation
with concomitant lactonization to afford 25 in 78% yield.
Deprotection of 25 was achieved in the same manner as for 23,
to give the acid 10 as a 1.2 : 1 mixture of diastereoisomers in
95% yield.
Compounds 11 and 12 were synthesized from the protected
alcohol 20 (Scheme 4). The allyl side-chain was introduced using
palladium coupling with allyl methyl carbonate in quantitative
yield to give 26. Treatment of 3-allyloxy derivative 26 with
aqueous trifluoroacetic acid led to deprotection of the bis-
methoxyacetal together with partial allyl migration to afford
a chromatographically separable mixture of 3-allyloxy and
4-allyloxy derivatives 27 (72%) and 28 (25%). Finally, hydrolysis
of the methyl ester 27 under basic conditions followed by ion
exchange gave acid 11 in 89% yield. Hydroxylation of 26 with
catalytic osmium tetraoxide gave 29 in 76% yield, which was
deprotected using a combination of TFA, lithium hydroxide
and Amberlite IR-120 to give 12 as a 1.2 : 1 mixture of
diastereoisomers.
Scheme
4
Reagents and conditions: (i) CH2᎐᎐CHCH2OCO2Me,
Pd2(dba)2, dppb, THF, ∆; (ii) TFA–H2O (20 : 1); (iii) 1. LiOH, H2O, RT,
2. Amberlite IR-120; (iv) OsO4(cat), NMO, dioxane–H2O, RT; (v) 1.
TFA–H2O (20 : 1), RT, 2. LiOH, H2O, RT, 3. Amberlite IR-120.
Assay results
Inhibition studies against type I dehydroquinase (S. typhi) and
type II dehydroquinase (S. coelicolor) were performed with
compounds 5, 7, 9 and 11, and compounds 6, 8, 10 and 12 as
the prepared diastereomeric mixtures. The UV spectrophoto-
metric assay6c was used to measure the initial rate of product
formation, detecting the enone-carboxylate chromophore at
Scheme 3 Reagents and conditions: (i) ref. 16; (ii) PDC, 4 Å MS, DCM,
RT; (iii) ref. 17; (iv) CH2᎐᎐CHCH2OCO2Me, Pd2(dba)2, dppb, THF, ∆;
(v) TFA–H2O (20 : 1), RT; (vi) 1. LiOH, H2O, RT, 2. Amberlite IR-120;
(vii) OsO4 cat, NMO, dioxane–H2O, RT; (viii) 1. TFA–H2O (20 : 1), RT,
2. LiOH, H2O, RT, 3. Amberlite IR-120.
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Table 1 Enzyme assay results (Ki values in µM)
Compound
Type I dehydroquinase S. typhia
Type II dehydroquinase S. coelicolora
3
3000 1000b
4500 500b
>20000
>20000
>20000
>20000
>20000
>20000
>20000
30 10b
600 200b
420 50
4
5
6 (R ϩ S)
>20000
7
180 20
8 (R ϩ S)
9
3000 500
1200 150
530 50
>20000
3500 400
10 (R ϩ S)
11
12 (R ϩ S)
>20000
a KM values of 16 µM (type I dehydroquinase, S. typhi) and 250 µM (type II dehydroquinase, S. coelicolor) were obtained at the assay conditions.
b Ref. 8.
234 nm in 3-dehydroshikimate (2). The Ki values (Table 1) were
obtained from Dixon plots (1/v vs. [I]).19
the crystal structure. All the ligands have the ring in a similar
position as 3 and the side-chain in the glycerol binding-pocket.
For ligands 5–10 the ring adopts a chair conformation. How-
ever, in the dockings of ligands 11 and 12 the ring is flipped
into a boat-conformation. This moves the side-chain onto an
equatorial position so that it extends into the glycerol binding-
pocket, and avoids steric clashes with protein side chains below
the ring. From the ligand positions in the docking results no
H-bond interactions are recognised between the hydroxy
groups in the ligand side-chains and the enzyme.
The most striking observation is that none of the compounds
5–12 showed any measurable inhibition against the type I
dehydroquinase. This result was not too surprising as substrate
analogues where the carbonyl oxygen in 3-dehydroquinate (1)
was replaced by either CH2 or NOH had previously been shown
not to inhibit type I dehydroquinase.8
The best inhibitor of type II dehydroquinase was 7 which
had a Ki of 180 µM. This compared with a KM for substrate of
250 µM under the same assay conditions. The related allyl
compound 5 lacking the terminal hydroxy group had an
increased Ki of 420 µM. A similar pattern was observed for the
dihydroxylated ether 10 (Ki 530 µM) and corresponding allyl
compound 9 (Ki 1200 µM). The diols 8 and 12 showed modest
inhibition (3.0 and 3.5 mM, respectively), while no inhibition
was observed for the secondary alcohol 6 and the C-3 epimeric
allyl ether 11.
Molecular modelling
The structures (ligands) of 4, 5, 7, 9, 11 and each stereo-
isomer of 6, 8, 10 and 12 were docked in the active-site of
the structure of type II dehydroquinase from S. coelicolor
(receptor) (Fig. 1), using the program GOLD (version 1.2).12
The structures of the ligands were prepared using the pro-
gram SYBYL6.520 and energy minimised using the Tripos
force field. The receptor was also prepared using SYBYL6.5.
The structures of 3, the glycerol molecule, and all of the
water molecules (except a key structural water molecule that is
present in all the crystal structures, shown in Fig. 1) were
removed from the crystal structure. No energy minimisation
was performed on the receptor since the crystal structure
used has the enzyme in the desired conformation, with bound
ligands. All the ligands were docked as carboxylate anions
and 25 independent GOLD runs were performed for each
ligand.
Ligand 3 was initially docked as a control, and the result
compared to the crystal structure of the enzyme inhibitor com-
plex. All 25 GOLD runs gave results within 0.5 Å of each other,
and the position of the docked ligand in the active-site
coincided with the position in the crystal structure to within
0.3 Å RMSD. The reduced analogue 4 also docked consistently
at the same site with an RMSD of 1.0 Å.
Fig.
2
Results from the docking experiments with GOLD1.2:
positions of the ligands in the active-site of type II dehydroquinase
(S. coelicolor), against a surface (coloured by electrostatic potential)21
calculated for the protein residues shown in Fig. 1, omitting Y28 for
clarity. Oxygen atoms are shown in orange. (a) 3 (green) and glycerol
(purple), (b) 7 (blue), (c) 8R (light blue) and (d) 10S (magenta).
The docking of ligands 5–9 also gave highly consistent
results. All 25 GOLD runs for each ligand showed the 6-mem-
bered ring in a similar position to 3, with only small variations
in the position of the side-chains. Compounds 10, 11 and 12
also docked in this position in the large majority of the 25
experiments, but other binding modes were also found where
the ring position was substantially changed. These were not
considered further in this study.
Discussion and conclusions
The observation of a glycerol molecule adventitiously bound at
a distal site in the structure of the complex between S. coelicolor
type II dehydroquinase and inhibitor 3 inspired the design of
bifunctional inhibitors which would straddle the two sites
and incorporate features of both 3 and glycerol. The target
compounds 5–12 were all synthesized from quinic acid (13).
Compounds with secondary hydroxy groups in their side chain
were prepared and tested as diastereomeric mixtures.
Fig. 2 shows the docking results of ligands 7, 8R and 10S,
compared with the position of 3 and the glycerol molecule in
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None of the compounds 5–12 inhibited type I dehydro-
quinase. This was expected as the type I enzyme mechanism
involves initial attack on the C-3 carbonyl of 3-dehydroquinate
(1) by an active site lysine.5a Substrate analogues substituted at
C-3 would be expected to suffer unfavourable steric clashes with
this side chain.
Five compounds showed competitive inhibition against
type II dehydroquinase, although all were less potent than the
original inhibitor 3. The most potent inhibitor 7 has a Ki below
the KM for substrate, and is more potent than 4, the reduced
analogue of 3 with sp3 hybridisation at C-3. In analysing the
inhibition data several trends were discerned:
(i) Introduction of a hydroxy group in the side chain three
atoms remote from C-3 of quinic acid 13 is favorable. This is
most clearly seen in the comparison of 7 vs. 5.
(ii) Extending the side chain to four atoms appears to be less
favourable, e.g. 5 vs. 9 and 7 vs 10.
(iii) Introduction of a hydroxy group in the side chain two
atoms remote from C-3 is unfavourable. This is clearly seen in
the comparison of 6 with 5 and 7, and also 8 vs. 7.
(iv) There is a preference for the side chain to be on the
β-face, i.e. the (R)- stereochemistry at C-3 is less favourable
when comparing 10 vs. 12 and 9 vs. 11.
These trends consistently account for the relative affinities of
the compounds 5–12. They are further supported by consider-
ation of the structures of the ligands docked into the active site
using GOLD, shown in Fig. 2.
commercial silica gel 60 0.25 mm plates using either UV absorp-
tion, iodine staining, ceric molybdate solution or potassium
manganate() spray for visualisation. RF values are quoted
with respect to the solvent system used to develop the
plate. Column chromatography was carried out using 230–
400 mesh silica gel 60. Melting points are uncorrected. NMR
spectra were recorded in deuterated solvents. In the spectra of
diastereomeric mixtures, all resolvable peaks are given. Infrared
spectra were recorded as NaCl plates, Nujol mulls or KBr discs.
[α]D values are given in 10Ϫ1 deg cm2 gϪ1. Ultraviolet–visible
spectra were recorded using black-walled quartz cuvettes.
Carboxylic acids were analysed or purified by HPLC on a pre-
parative (300 mm × 16 mm) Bio-Rad Aminex Ion Exclusion
HPX-87H Organic Acids column. The eluant used for these
columns was 0.05 M aqueous formic acid, at a flow rate of
1.0 ml minϪ1. All procedures involving the use of ion-exchange
resins were carried out at room temperature and used Milli-Q
deionised water. Dowex 50W-X8 (H) (cation exchanger) and
Amberlite IR-120 (H) (cation exchanger) were washed alter-
nately with water, 10% HCl, water, 10% sodium hydroxide,
water, 10% HCl and finally with water before use. Purified
S. typhi type I dehydroquinase and S. coelicolor type II
dehydroquinase were stored in aliquots as concentrated solu-
tions in glycerol–water at Ϫ20 ЊC and 4 ЊC, respectively.
Dehydroquinase assay
The dehydroquinase enzymes were assayed by monitoring
the increase in absorbance at 234 nm in the UV spectrum due
to the absorbance of the enone-carboxylate chromophore of
3-dehydroshikimate (2 ε = 1.2 × 104 MϪ1cmϪ1). The assays were
performed at 25 ЊC in potassium phosphate (0.05 M, pH 7)
buffer for type I dehydroquinase and Tris-HCl (0.05 M, pH 7)
buffer for type II dehydroquinase. The assay mixture was pre-
pared in situ and the assay was initiated by addition of the
enzyme solution to the mixture. Solutions of 3-dehydroquinate
(1) were calibrated by equilibration with type I dehydroquinase
and measurement from the change in UV absorbance at 234
nm. In these assays the concentration of inhibitor (I) was varied
for a constant concentration of substrate and enzyme. The
assays were repeated for a number of different concentrations
of substrate and the Ki values were obtained through a Dixon
plot (1/v vs. [I]).
Fig. 2a shows the position of 3 and the glycerol molecule in the
active-site. Fig. 2b overlays of the docked structure of 7, where
the side-chain reaches into the glycerol binding-pocket and the
terminal hydroxy overlaps the C-1 position of the glycerol mole-
cule. Docking of 5 shows the ligand in a similar position to 7.
The docking of the dihydroxy ligand 8R is shown on Fig. 2c.
This suggests a position where the secondary hydroxy group at
C-2Ј abuts Leu17. This result is independent of whether or not
the adjacent water molecule is removed. Ligands 6R, 6S and 8S
dock in a similar position to 8R. The terminal primary hydroxy
group on C-3Ј of 8 reaches into the glycerol pocket and may
account for its affinity relative to 6. The extended diol 10S is
shown in Fig. 2d. The diol 10 has the longest side chain and it
docks so as to overlap both C-1 and C-2 of glycerol. This might
have been expected to increase the affinity compared to 7, but
must be offset against the increased entropic cost of immobil-
izing the longer side chain. Finally, the docking results for lig-
ands 11 and 12 (both with (R)-stereochemistry at C-3) suggest
they bind in a similar position to 10S. However, to accom-
modate the side-chain in the glycerol pocket the six-membered
ring is flipped into a boat conformation. This conformational
switch, which would be forced by steric crowding below C-3 (see
Fig. 1) preventing the side chain extending downwards, is likely
to disrupt hydrogen bonding to the hydroxys at C-4 and C-5,
contributing to their reduced affinity.
GOLD docking
All ligands and the receptor were prepared using SYBYL6.5
and used as MOL2 files. The ligands were designed as carb-
oxylate anions and their structure was energy minimised using
the Tripos force-field prior to docking. Each ligand was docked
using GOLD1.2 in 25 independent genetic algorithm (GA)
runs, and for each of these a maximum number of 100000
GA operations was performed on a single population of
50 individuals. Operator weights for crossover, mutation and
migration in the entry box were used as default parameters (95,
95 and 10, respectively), as well as the hydrogen bonding (4.0 Å)
and van der Waals (2.5 Å) parameters. The position of the
active-site was introduced and the radius was set to 10 Å, with
the automatic active-site detection on. The “flip ring corners”
flag was switched on, while all the other flags were off.
In conclusion, the strategy of making inhibitors combining
structural features of both 3 and glycerol has resulted in the
formation of new inhibitors. The docking experiments suggest
that most of the compounds bind in a similar manner to 3, with
their side chains extending into the glycerol binding site. The
modest affinity of the inhibitors is probably a consequence of
the entropic cost of immobilising a flexible side chain, and the
sensitivity of binding to sp2 hybridisation at C-2 and C-3 of the
six membered ring. Both of these issues are being addressed in
the design of the next generation of inhibitors.
Methyl (1S,3R,4R,5S )-5-allyl-1,3,4-trihydroxycyclohexane-
1-carboxylate (15)
The allyl carbolactone 1413 (140 mg, 0.47 mmol) was dissolved
in methanol (10 ml) and stirred in the presence of sodium
methoxide (5 mg) for 24 hours at room temperature. The
solvent was removed at reduced pressure, the crude product
was taken in water (10 ml) and washed with diethyl ether
(3 × 20 ml). The ester 15 was lyophilised and collected as an
amorphous yellow solid (94 mg, 87%). δH (400 MHz, D2O): 5.65
Experimental
General
All organic solvents were freshly distilled prior to use and
Milli-Q deionised water was used for all biochemical work.
Analytical thin layer chromatography was carried out on
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(1 H, m), 4.95 (2 H, m), 3.62 (3 H, s), 3.60 (1 H, ddd, J = 12.0,
9.4 and 5.1 Hz), 3.05 (1 H, t, J = 9.4 Hz), 2.35 (1 H, dddd,
J = 14.2, 6.1, 3.2 and 1.5 Hz), 2.00–1.80 (2 H, m), 1.75–1.60
(3 H, m), 1.45 (1 H, t, J = 14.2 Hz); δC (100 MHz, DEPT, D2O):
175.2 (C), 134.3 (CH), 115.1 (CH2), 75.3 (CH), 72.6 (C), 68.7
(CH), 51.2 (OCH3), 37.8 (CH2), 35.3 (CH2), 34.9 (CH), 33.2
(CH2); HRMS calcd for C11H18O5Na: MNaϩ, 253.1052. Found:
MNaϩ, 253.1072.
5.20 (1 H, dd, J = 5.0 and 2.3 Hz), 4.89 (1 H, t, J = 5.0 Hz), 3.63
(2 H, m), 3.52 (1 H, s), 2.58–2.45 (2 H, m), 2.33 (1 H, dd,
J = 13.3 and 9.1 Hz), 2.23 (1 H, m), 1.86 (1 H, m), 1.75–1.60
(3 H, m), 1.50 (1 H, m); δC (100 MHz, CDCl3): 181.2, 167.5,
135.8, 131.8, 131.5, 130.8, 77.8, 74.2, 72.9, 64.4, 39.5, 39.3, 39.2,
33.0, 32.9; HRMS calcd for C17H20O6Na: MNaϩ, 343.1143.
Found: MNaϩ, 343.1158.
Methyl (1S,3R,4R,5S )-5-(3Ј-hydroxypropyl)-1,3,4-trihydroxy-
cyclohexane-1-carboxylate (17)
(1S,3R,4R,5S )-5-Allyl-1,3,4-trihydroxycyclohexane-
1-carboxylic acid (5)
The lactone 16 (87 mg, 0.27 mmol) was dissolved in methanol
(5 ml) and stirred in the presence of sodium methoxide (5 mg)
for 2 hours at room temperature. The solvent was removed at
reduced pressure, the crude product was taken in water (10 ml)
and washed with diethyl ether (3 × 10 ml). The product was
lyophilised to give ester 17 as a colourless glass (66 mg, 98%).
υmax (Nujol)/cmϪ1 3330b (OH) and 1730b (OH); δH (400 MHz,
D2O): 3.66 (3 H, s), 3.60 (1 H, m), 3.50 (2 H, t, J = 6.5 Hz), 3.08
(1 H, td, J = 9.0 and 7.2 Hz), 1.99 (1 H, ddd, J = 13.2, 4.0 and
3.1 Hz), 1.85Ϫ1.45 (6 H, m), 1.36 (1 H, m), 1.15 (1 H, m);
δC (100 MHz, DEPT, D2O): 178.7 (C), 79.6 (CH), 76.0 (C), 73.0
(CH), 63.3 (CH2), 54.6 (OMe), 42.3 (CH2), 39.7 (CH2), 38.3
(CH), 29.7 (CH2), 28.4 (CH2).
The methyl ester 15 (94 mg, 0.41 mmol) was dissolved in water
(10 ml) and taken to pH∼12 with 10% NaOH, and stirred at
room temperature overnight. The solution was acidified with
Dowex 50 (Hϩ) ion-exchange resin, filtered and lyophilised to
give the allyl acid 5 as an amorphous white solid (80 mg, 90%).
HPLC retention time (organic acids column): 37 minutes;
υmax (Nujol)/cmϪ1 3368b (OH) and 1712s (CO); δH (500 MHz,
D2O): 5.80 (1 H, m), 5.05 (2 H, m), 3.70 (1 H, ddd, J = 14.0,
9.3 and 4.9 Hz), 3.17 (1 H, dd, J = 10.1 and 9.3 Hz), 2.45
(1 H, dddd, J = 14.0, 6.3, 3.2 and 1.6 Hz), 2.00 (2 H, m), 1.80
(3 H, m), 1.58 (1 H, dd, J = 14.0 and 12.7 Hz); δC (100 MHz,
DEPT, D2O): 179.3 (C), 136.2 (CH), 116.8 (CH2), 77.2 (CH),
74.4 (C), 70.7 (CH), 39.8 (CH2), 37.2 (CH2), 36.9 (CH), 34.0
(CH2); HRMS calcd for C10H16O5: Mϩ Ϫ H, 215.0920. Found:
Mϩ Ϫ H, 215.0939.
(1S,3R,4R,5S )-5-(3Ј-Hydroxypropyl)-1,3,4-trihydroxycyclo-
hexane-1-carboxylic acid (7)
The methyl ester 17 (10 mg, 0.040 mmol) was dissolved in water
(2 ml), the pH adjusted to 12 with sodium hydroxide (10%) and
the solution stirred at room temperature for 2 hours. The solu-
tion was acidified with Amberlite IR-120 (Hϩ) ion-exchange
resin, filtered and lyophilised to give the 3Ј-hydroxy acid 7 as a
colourless glass (6 mg, 65%); HPLC retention time (organic
acids column): 28 minutes; υmax (Nujol)/cmϪ1 3393b (OH) and
1711s (CO); δH (400 MHz, D2O): 3.66 (1 H, ddd, J = 11.8, 9.5
and 4.6 Hz), 3.54 (2 H, t, J = 6.5 Hz), 3.12 (1 H, t, J = 9.5 Hz),
2.02 (1 H, ddd, J = 13.3, 4.6 and 3.1 Hz), 1.83–1.52 (6 H, m),
1.41 (1 H, ddd, J = 17.1, 11.2 and 6.5 Hz), 1.20 (1 H, m); δC (100
MHz, DEPT, D2O): 179.4 (C), 77.6 (CH), 74.2 (C), 70.4 (CH),
61.7 (CH2), 39.7 (CH2), 37.0 (CH2), 36.6 (CH), 27.8 (CH2), 26.5
(CH2); HRMS calcd for C10H18O6Na: MNaϩ, 257.1001. Found:
MNaϩ, 257.1001.
(1S,3R,4R,5S )-5-(2Ј-Hydroxypropyl)-1,3,4-trihydroxycyclo-
hexane-1-carboxylic acid (6)
The acid 5 (11 mg, 0.052 mmol) was stirred with concentrated
hydrochloric acid (2 ml) under reflux for 24 hours. The acidic
solution was diluted in water and the solvent and some of the
acid removed at reduced pressure. The residue was redissolved
in water (5 ml) and lyophilised to give a 1 : 1 mixture of
diastereoisomers of the secondary hydroxy acid 6 as an
amorphous white solid (12 mg, 98%). HPLC retention time
(organic acids column): 36 and 37 minutes; υmax (Nujol)/cmϪ1
3381b (OH) and 1719s (CO); δH (500 MHz, D2O): 4.27 (1 H, tq,
J = 9.1 and 6.2 Hz), 3.81 (1 H, ddd, J = 11.0, 9.6 and 4.8 Hz),
3.30 (1 H, dd, J = 10.3 and 9.6 Hz), 2.20 (1 H, ddd, J = 12.0,
6.2 and 6.0 Hz), 1.98 (2 H, m), 1.85 (1 H, dd, J = 13.5 and
3.3 Hz), 1.75 (1 H, dd, J = 13.7 and 11.0 Hz), 1.69 (1 H, t,
J = 13.5 Hz), 1.34 (1 H, td, J = 12.0 and 9.1 Hz), 1.25 (3 H, d,
J = 6.2 Hz); δC (100 MHz, DEPT, D2O): 179.7 (C), 86.3 (CH),
78.0 (CH), 77.3 (C), 70.4 (CH), 42.0 (CH2), 40.2 (CH), 39.3
(CH2), 37.5 (CH2), 22.4 (CH3); only ten peaks observed in 13C
spectrum of the mixture. HRMS calcd for C10H15O5: M( Ϫ Hϩ
Ϫ H2O), 215.0920. Found: M( Ϫ Hϩ Ϫ H2O), 215.0937.
(1S,3R,4R,5S )-5-(2Ј,3Ј-Dihydroxypropyl)-4-benzoyl-1-hydroxy-
cyclohexane-1,3-carbolactone (18)
To a solution of the carbolactone 14 (167 mg, 0.55 mmol) in
acetone (2 ml) was added N-methylmorpholine oxide (76.7 mg,
0.65 mmol), water (2 ml) and osmium tetraoxide (1.1 mg, 55 µl
of 2.5% wt. solution in tert-butanol, 5.5 µM). The mixture was
degassed with nitrogen and stirred for 20 hours under argon,
with additional osmium tetraoxide (1.1 mg, 55 µl of 2.5% solu-
tion in tert-butanol, 5.5 µM) added after 7 hours. A saturated
solution of sodium bisulfite (10 ml) was added to destroy excess
of osmium tetraoxide. The acetone was removed at reduced
pressure and the product was extracted with ethyl acetate (2 ×
20 ml). The extract was washed with water (4 × 20 ml). The
solution was dried with MgSO4 and evaporated at reduced
pressure. The product was purified by column chromatography
on silica gel eluting with ethyl acetate to give the diol (1 : 1
mixture of diastereomers) 18 as a colourless oil (40 mg, 22%).
RF 0.25 (ethyl acetate); δH (400 MHz, CDCl3): 7.95 (2 H, d,
J = 8.2 Hz), 7.55 (1 H, t, J = 8.2 Hz), 7.40 (2 H, t, J = 8.2 Hz),
5.10 (1 H, m), 4.82 (1 H, t, J = 4.5 Hz), 4.05–3.35 (5 H, m), 2.60–
2.20 (4 H, m), 1.94 (1 H, dd, J = 10.2 and 9.0 Hz), 1.65 (1 H, m),
1.45 (1 H, m); δC (100 MHz, CDCl3): 179.9, 179.8, 166.1, 166.0,
134.1, 134.0, 130.1 (2×), 129.7, 129.6, 129.0, 128.9, 76.1, 76. 0,
72.5, 72.2, 71.0 (2×), 70.5, 70.1, 67.0, 66.8, 38.4, 38.3, 37.8, 37.5,
37.2, 35.9, 34.2, 33.5; HRMS calcd for C17H20O7Na: MNaϩ,
359.1109. Found: MNaϩ, 359.1158.
(1S,3S,4R,5S )-5-(3Ј-Hydroxypropyl)-4-benzoyl-1-hydroxy-
cyclohexane-1,3-carbolactone (16)
A solution of borane–THF complex (1.89 ml ca. 1.0 M in THF,
1.89 mmol, 2 equiv.) was added to a solution of the allyl
carbolactone 14 (286 mg, 0.94 mmol) in THF (5 ml), at 0 ЊC in
an ice-bath. The solution was stirred for 1 hour, and a solution
of sodium perborate (286 mg, 1.89 mmol) in water (5 ml) was
added and the ice-bath removed. The solution was stirred for a
further 2 hour at room temperature. The THF was removed at
reduced pressure, the product was dissolved in ethyl acetate
(20 ml) and washed with water (3 × 20 ml). The organic layer
was dried with anhydrous Na2SO4 and the solvent removed at
reduced pressure. The crude product was purified by column
chromatography on silica gel, eluting with ethyl acetate–petrol-
eum ether 40–60 (3 : 1). The carbolactone 16 (94 mg, 31%) was
obtained as a colourless oil. RF 0.22 [EtOAc–petroleum ether
40–60 (3 : 1)]; υmax (NaCl)/cmϪ1 3452b (OH) and 1790s (CO),
1723s (CO) and 1601s (Ar); δH (400 MHz, CDCl3): 8.10 (2 H, d,
J = 8.0 Hz), 7.60 (1 H, t, J = 8.0 Hz), 7.46 (2 H, t, J = 8.0 Hz),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 7 5 – 2 0 8 3
2080

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Methyl (1S,3R,4R,5S )-5-(2Ј,3Ј-dihydroxypropyl)-
1,3,4-trihydroxycyclohexane-1-carboxylate (19)
The resultant green suspension was heated under reflux for
20 hours. The solvent was removed under reduced pressure and
the crude mixture was purified by flash chromatography eluting
with diethyl ether–hexane (75%) to afford the allyl ether 23 as a
colourless oil (170 mg, 69%). [α]2D0 ϩ136 (c 1.40 in CHCl3); υmax
(NaCl)/cmϪ1 3700b (OH) and 1733s (CO); δH (250 MHz,
CDCl3): 5.91–5.76 (1 H, m), 5.24–5.06 (2 H, m), 3.92–3.75 (4 H,
m), 3.67 (3 H, s), 3.40 (1 H, t, J = 9.6 Hz), 3.22 (3 H, s), 3.18
(3 H, s), 2.32 (1 H, ddd, J = 13.9, 4.6 and 2.8 Hz), 2.14 (1 H,
ddd, J = 13.9, 4.2 and 2.8 Hz), 1.85–1.64 (2 H, m), 1.27 (3 H, s),
1.23 (3 H, s); δC (63 MHz,DEPT, CDCl3): 172.5 (C), 134.0
(CH), 117.0 (CH3), 99.5 (C), 99.5 (C), 79.0 (C), 76.2 (CH), 66.5
(CH), 65.7 (CH3), 64.7 (CH), 52.3 (OCH3), 47.8 (2 × OCH3),
37.3 (CH2), 34.9 (CH2), 17.6 (2 × CH3); MS (CI) m/z (%)
329 [(MHϩ) Ϫ HOCH3]; HRMS calcd for C16H25O7: MHϩ,
329.1600. Found: MHϩ, 329.1601.
The dihydroxy lactone 18 (40 mg, 0.12 mmol) was dissolved in
methanol (10 ml) and stirred in the presence of sodium methox-
ide (1 mg) for 6 hours at room temperature. The solvent was
removed at reduced pressure, the crude product was taken in
water (10 ml) and washed with diethyl ether (3 × 20 ml). The
solution was lyophilised to give the ester (1 : 1 mixture of
diastereomers) 19 as a brown solid (27 mg, 83%). δH (400 MHz,
D2O): 3.68 (3 H, s), 3.67 (2 H, m) 3.48 (1 H, ddd, J = 17.9, 12.1
and 4.0 Hz), 3.36 (1 H, ddd, J = 17.9, 12.1 and 7.2 Hz), 3.08
(1 H, td, J = 9.0, 6.3 Hz), 2.02 (1 H, m), 1.90–1.73 (4 H, m), 1.60
(1 H, t, J = 12.0 Hz), 1.22 (1 H, m); δC (100 MHz, DEPT, D2O):
179.5 (2 × C), 81.1 (CH), 80.4 (CH), 76.9 (CH2), 76.8 (CH2),
73.1 (CH), 72.9 (CH), 71.4 (2 × CH), 68.6 (CH2), 67.7 (CH2),
55.0 (2 × OCH3), 42.0 (2 × CH2), 40.8 (CH2), 39.8 (CH2), 37.3
(CH2), 37.2 (CH), 36.9 (CH2), 36.2 (CH).
Methyl (1S,3R,4R,5R)-3-allyloxy-1,4,5-trihydroxycyclohexane-
1-carboxylate (24)
(1S,3R,4R,5S )-5-(2Ј,3Ј-Dihydroxypropyl)-1,3,4-trihydroxy-
cyclohexane-1-carboxylic acid (8)
A solution of acetal 23 (117 mg, 0.33 mmol) in 2 ml of a solu-
tion of 20 : 1 (v/v) of trifluoroacetic acid–water was stirred for
30 min and then the solvent was concentrated in vacuo. The
crude mixture was purified by flash chromatography eluting
with ethyl acetate–dichloromethane–methanol (7 : 2 : 1) to
afford 24 as a colourless oil (78 mg, 94%). [α]2D0 Ϫ0.2 (c 2.6 in
H2O); υmax (NaCl)/cmϪ1 3399b (OH) and 1731s (CO); δH (300
MHz, D2O): 6.01–5.98 (1 H, m), 5.46–5.32 (2 H, m), 4.08
(2 H, d, J = 5.9 Hz), 3.88 (3 H, s), 3.85 (1 H, m), 3.41 (1 H, t,
J = 9.1 Hz), 2.42 (2 H, dd, J = 2.8 and 11.9 Hz), 1.94 (2 H, dd,
J = 12.3 and 13.5 Hz); δC (125 MHz, DEPT, D2O): 174.0 (C),
133.2 (CH), 118.2 (CH2), 79.1 (C), 78.7 (CH), 68.6 (2 × CH),
65.8 (CH2), 52.9 (CH3), 37.1 (2 × CH2); MS (CI) m/z (%) 247
(MHϩ); HRMS calcd for C11H19O6: MHϩ, 247.1174. Found:
MHϩ, 247.1182.
The ester 19 (27 mg, 0.10 mmol) was dissolved in water (10 ml),
the pH of the solution adjusted to 12 with sodium hydroxide
(10%) and stirred at room temperature overnight. The solution
was acidified with Dowex 50 (Hϩ) ion-exchange resin, filtered
and lyophilised to give the two diastereisomers of the dihydroxy
acid 8 (1 : 1 mixture) as a white solid (25 mg, quantitative).
HPLC retention time (organic acids column): 22 and 24 min-
utes; υmax (Nujol)/cmϪ1 3401b (OH) and 1718s (CO); δH (400
MHz, D2O): 3.73 (1 H, ddd, J = 14.0, 6.5 and 3.4 Hz), 3.64 (1 H,
ddd, J = 14.1, 9.2 and 4.5 Hz), 3.53 (1 H, dd, J = 13.0 and
3.4 Hz), 3.36 (1 H, dd, J = 13.0 and 6.5 Hz), 3.10 (1 H, t,
J = 9.2 Hz), 1.98 (1 H, ddd, J = 14.0, 4.5 and 3.1 Hz), 1.90–1.65
(4 H, m), 1.60 (1 H, t, J = 12.9 Hz), 1.20 (1 H, m); δC (100 MHz,
DEPT, D2O): 179.7 (C), 179.6 (C), 78.0 (CH), 77.8 (CH), 74.3
(CH2), 74.2 (CH2), 70.5 (CH), 70.4 (CH), 70.3 (CH), 68.6 (CH),
65.9 (CH2), 64.9 (CH2), 39.6 (2 × CH2), 38.3 (CH2), 37.3 (CH2),
34.7 (CH), 34.6 (CH2), 34.3 (CH2), 33.7 (CH); HRMS calcd for
C10H17O7: M Ϫ H, 249.0974. Found: M Ϫ H, 249.0968.
(1S,3R,4R,5R)-3-Allyloxy-1,4,5-trihydroxycyclohexane-1-carb-
oxylic acid (9)
A solution of the methyl ester 24 (70 mg, 0.28 mmol) in aque-
ous lithium hydroxide (3 ml, 0.2M) was stirred for 1 hour. The
reaction mixture was diluted with water and was washed with
diethyl ether (3 × 5 ml). The aqueous layer was treated with
Amberlite IR-120 until the pH was 6.0. The resin was filtered
and washed with water. The filtrate was freeze-dried to afford
the acid 9 as a colourless oil (67 mg, 97%). [α]2D0 Ϫ0.5 (c 2.2 in
H2O); υmax (NaCl)/cmϪ1 3399b (OH) and 1683s (CO); δH (300
MHz, D2O): 6.11–6.00 (1H, m), 5.46–5.30 (2H,m), 4.05 (2 H, d,
J = 5.9 Hz), 3.85–3.76 (2 H, m), 3.41 (1 H, t, J = 9.2 Hz),
2.39 (2 H, dd, J = 4.1 and 12.3 Hz), 1.88 (2 H, t, J = 12.8 Hz);
δC (125 MHz, DEPT, D2O): 177.3 (C), 133.8 (CH), 117.8 (CH2),
79.9 (C), 79.0 (CH), 69.0 (2 × CH), 65.6 (CH2), 37.6 (2 × CH2);
MS (CI) m/z (%) 233 (MHϩ); HRMS calcd for C10H17O6: MHϩ,
233.1025. Found: MHϩ, 233.1018.
Methyl (1S,3S,4S,6S,9R)-9-hydroxy-3,4-dimethoxy-3,4-di-
methyl-7-oxo-2,5-dioxabicyclo[4.4.0]decane-9-carboxylate (21)
To a stirred suspension of the alcohol 2016 (2.69 g, 8.41 mmol)
and activated molecular sieves 4 Å (3.4 g) in dry dichloro-
methane (80 ml) was added pyridinium dichromate (4.74 g,
12.61 mmol). The resultant suspension was stirred at room
temperature for 2 hours and then diluted with diethyl ether
(60 ml) and filtered over Celite. The solution was evaporated
and the crude reaction was purified by flash chromatography,
eluting with ethyl acetate, and then recrystallised from diethyl
ether to afford the ketone 21 as white needles (2.06 g, 77%). Mp
194–195 ЊC (diethyl ether); δH (250 MHz, CDCl3): 4.19 (1 H, dd,
J = 10.3 and 0.9 Hz), 4.02 (1 H, td, J = 11.0 and 4.2 Hz), 3.60
(3 H, s), 3.02 (3 H, s), 2.99 (3 H, s), 2.69 (1 H, d, J = 14.3 Hz),
2.28 (1 H, dd, J = 14.3 and 2.8 Hz), 2.12 (1 H, t, J = 12.4 Hz),
1.87 (1 H, m), 1.15 (3 H, s), 1.06 (3 H, s); δC (63 MHz, DEPT,
CDCl3): 199.5 (C), 174.1 (C), 100.5 (C), 99.6 (C), 77.2 (CH),
74.0 (C), 67.0 (CH), 53.6 (OCH3), 49.0 (CH2), 48.3 (OCH3),
48.0 (OCH3), 37.8 (CH2), 17.6 (CH3), 17.5 (CH3). MS (CI) m/z
(%) 287 [MHϩ Ϫ CH3OH]; HRMS calcd. For C13H19O7: MHϩ,
287.1130. Found MHϩ, 287.1127.
Methyl (1S,3S,4S,6S,7S,9R)-7-(2Ј,3Ј-dihydroxy)propyloxy-
9-hydroxy-3,4-dimethoxy-3,4-dimethyl-2,5-dioxabicyclo[4.4.0]-
decane-9-carboxylate (25)
To a stirred solution of allyl ether 23 (87 mg, 0.24 mmol) and
N-methylmorpholine oxide (34 mg, 0.29 mmol) in 50% aqueous
dioxane (4 ml) was added a freshly made aqueous solution of
sodium tetraoxide (0.3 ml, 0.12 M). After stirring for 1 hour
ethyl acetate was added and then saturated sodium bisulfite.
The reaction mixture was stirred for 20 min. The organic layer
was separated and the aqueous layer was extracted with ethyl
acetate (3 × 10 ml). The combined organic layers were dried
(anhydrous Na2SO4) and concentrated in vacuo. The crude
residue was purified by flash chromatography eluting with
95% ethyl acetate–methanol to afford a 1.2 : 1 mixture of
diastereoisomers of the lactone 25 (68 mg, 78%) as white
Methyl (1S,3S,4S,6S,7S,9R)-7-allyloxy-9-hydroxy-
3,4-dimethoxy-3,4-dimethyl-2,5-dioxabicyclo[4.4.0]dec-
9-anecarboxylate (23)
To a stirred solution of Pd2(dba)2 (16 mg, 17 µmol) and dppb
(29 mg, 68 µmol) in dry tetrahydrofuran (3 ml) under argon was
added the alcohol 22 (217 mg, 0.68 mmol) and then a solution
of allyl methyl carbonate in tetrahydrofuran (1.6 ml, 0.5 M).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 7 5 – 2 0 8 3
2081

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needles. υmax (NaCl)/cmϪ1 3425b (OH) and 1750s (CO); δH (250
MHz, CDCl3): 4.58 (m, 1H), 3.94–3.66 (6 H, m), 3.48 (1 H, t,
J = 9.6 Hz), 3.27 (3 H, s), 3.23 (3 H, s), 2.64 (br s), 2.45–2.16
(2 H, m), 2.08–1.77 (2 H, m), 1.30 (3 H, s), 1.28 (3 H, s); δC (63
MHz, DEPT, CDCl3): 170.2 (C), 99.7 (C), 99.6 (C), 77.4 (C),
77.3 (C), 76.0 (CH), 66.4 (CH), 64.6 (CH), 61.4 (CH2), 61.3
(CH2), 59.2 (CH2), 59.1 (CH2), 48.0 (OCH3), 47.9 (OCH3), 39.4
(CH2), 34.7 (CH2), 17.7 (CH3), 17.6 (CH3); MS (CI) m/z (%)
331 [(MHϩ) Ϫ HOCH3]; HRMS calcd for C15H23O8: MHϩ,
331.1393. Found: MHϩ, 331.1393.
5.35 (1 H, dq, J = 10.3 and 1.1 Hz), 4.28–4.08 (4 H, m), 3.86
(3 H, s), 3.77 (1 H, dd, J = 8.2 and 3.5 Hz), 2.24–2.06 (4 H, m);
δC (75 MHz, DEPT, CD3OD): 172.9 (C), 131.3 (CH), 115.5
(CH2), 73.2 (CH), 72.2 (C), 70.0 (CH), 67.9 (CH2), 64.2 (CH),
50.3 (OCH3), 36.6 (CH2), 31.0 (CH2); MS (CI) m/z (%) 247
(MHϩ); HRMS calcd for C11H19O6: MHϩ, 247.1182. Found:
MHϩ, 247.1185.
Data for methyl (1S,3S,4R,5R)-4-allyloxy-1,3,5-trihydroxy-
cyclohexane-1-carboxylate (28). [α]2D0 Ϫ6 (c 2.95 in (CH3)2CO);
υmax (NaCl)/cmϪ1 3419b (OH) and 1731s (CO); δH (300 MHz,
(CD3)3CO): 5.79 (1 H, m), 5.13 (1 H, dq, J = 17.3 and 1.8 Hz),
4.97 (1 H, dq, J = 10.4 and 1.7 Hz), 3.94–3.70 (5 H, m), 3.65
(1 H, d, J = 5.4 Hz), 3.56 (3 H, s), 3.40 (1 H, d, J = 7.3 Hz),
2.10–2.00 (2 H, m), 1.95–1.79 (2 H, m); δC (75 MHz,
DEPT, (CD3)3CO): 172.1 (C), 134.8 (CH), 115.2 (CH2),
79.8 (C), 74.1 (CH), 67.9 (CH), 67.2 (CH), 64.8 (CH2), 51.2
(OCH3), 36.5 (CH2), 35.1 (CH2); MS (CI) m/z (%) 247 (MHϩ);
HRMS calcd for C11H19O6: MHϩ, 247.1182. Found: MHϩ,
247.1184.
(1S,3R,4R,5R)-1,4,5-Trihydroxy-3-(2Ј,3Ј-dihydroxy)propyloxy-
cyclohexane-1-carboxylic acid (10)
A solution of acetal 25 (46 mg, 0.13 mmol) in 2 ml of a solution
of trifluoroacetic acid–water (20 : 1 (v/v)) was stirred for 30 min
and then concentrated in vacuo. The crude residue was redissol-
ved in an aqueous lithium hydroxide (1.7 ml, 0.2 M) and stirred
for 1 hour. The aqueous layer was washed with diethyl ether
(3 × 5 ml) and then was treated with Amberlite IR-120 until
the pH was 6.0. The resin was filtered and washed with water.
The filtrate was freeze-dried to afford a 1.2 : 1 mixture of
diastereoisomers of the acid 10 as a colourless oil (34 mg,
95%). υmax (Nujol)/cmϪ1 3390b (OH) and 1682s (CO); δH (300
MHz, D2O): 3.70 (1 H, br s), 3.55–3.20 (5 H, m), 3.13 (1 H, t,
J = 9.2 Hz), 2.10 (2 H, d, J = 11.5 Hz), 1.60 (2 H, t, J = 12.5 Hz);
δC (75 MHz, DEPT, D2O): 166.1 (C), 165.6 (C), 82.0 (C ϩ CH),
73.6 (CH), 72.0 (2 × CH), 68.0 (CH2), 65.6 (CH2), 40.5 (CH2),
40.3 (CH2); MS (CI) m/z (%) 249 [(MHϩ) Ϫ H2O]; HRMS calcd
for C10H17O7: MHϩ, 249.0974. Found: MHϩ, 249.0970.
(1S,3S,4R,5R)-3-Allyloxy-1,4,5-trihydroxycyclohexane-
1-carboxylic acid (11)
A solution of the ester 27 (34 mg, 0.14 mmol) in aqueous
lithium hydroxide (1.7 ml, 0.2 M) was stirred at room temper-
ature for 2 hours. The resultant solution was diluted with water
and treated with Amberlite IR-120 until the pH was 6.0. The
resin was filtered and washed with water. The filtrate was con-
centrated to afford the acid 11 (29 mg, 89%) as a colourless oil.
[α]2D0 Ϫ25 (c 1.7 in H2O); υmax (NaCl)/cmϪ1 3418b (OH) and
1713s (CO); δH (300 MHz, D2O): 6.02 (1 H, m), 5.40 (1 H, d,
J = 17.4 Hz), 5.31 (1 H, d, J = 10.2 Hz), 4.26–4.07 (3 H, m), 3.71
(1 H, dd, J = 11.2 and 2.6 Hz), 2.24–1.97 (4 H m); δC (75 MHz,
DEPT, D2O): 176.1 (C), 131.2 (CH), 115.4 (CH2), 73.7 (CH),
73.0 (C), 70.9 (CH), 67.9 (CH2), 64.2 (CH), 37.2 (CH2), 31.2
(CH2); MS (CI) m/z (%) 215 [(MHϩ) Ϫ H2O]; HRMS calcd for
C10H15O5: MHϩ, 215.0916. Found: MHϩ, 215.0919.
Methyl (1S,3S,4S,6S,7R,9R)-7-allyloxy-9-hydroxy-
3,4-dimethoxy-3,4-dimethyl-2,5-dioxabicyclo[4.4.0]decane-
9-carboxylate (26)
To a stirred solution of Pd2(dba)2 (14 mg, 15.8 µmol) and dppb
(27 mg, 63 µmol) in dry tetrahydrofuran (2 ml) under argon was
added the alcohol 20 (200 mg, 0.63 mmol) and then a solution
of allyl methyl carbonate (85 µl, 0.75 mmol) in dry tetrahydro-
furan (1.5 ml). The resultant green suspension was heated at
60 ЊC for 2 h. The solvent was removed under reduced pressure
and the crude mixture was purified by flash chromatography
eluting with 75% diethyl ether–hexane to afford the allyl ether
26 as a colourless oil (226 mg, 99%). [α]2D0 ϩ125 (c 1.9 in CHCl3);
υmax (NaCl)/cmϪ1 3479b (OH) and 1737s (CO); δH (300 MHz,
CDCl3): 5.80 (1 H, m), 5.11 (2 H, m), 4.22 (2 H, m), 4.07–3.86
(2 H, m), 3.64 (3 H, s), 3.45 (1 H, ddd, J = 18.2, 10.2 and
2.5 Hz), 3.14 (3 H, s), 3.13 (3 H, s), 2.25 (2 H, m), 1.90–1.74
(2 H, m), 1.19 (3 H, s, CH3), 1.16 (3 H, s, CH3); δC (75 MHz,
DEPT, CDCl3): 173.4 (C), 134.3 (CH), 116.8 (CH2), 99.6 (C),
99.1 (C), 83.9 (C), 75.8 (CH), 73.2 (CH), 72.1 (CH2), 62.5 (CH),
52.3 (OCH3), 47.6 (OCH3), 47.5 (OCH3), 39.0 (CH2), 36.6
(CH2), 17.5 (CH3), 17.4 (CH3); MS (CI) m/z (%) 329 [(MHϩ) Ϫ
HOCH3]; HRMS calcd for C16H25O7: MHϩ, 329.1600. Found:
MHϩ, 329.1591.
Methyl (1S,3S,4S,6S,7R,9R)-9-hydroxy-7-(2Ј,3Ј-dihydroxy)-
propyloxy-3,4-dimethoxy-3,4-dimethyl-2,5-dioxabicyclo[4.4.0]-
decane-9-carboxylate (29)
To a stirred solution of allyl ether 26 (629 mg, 1.75 mmol) and
N-methylmorpholine oxide (246 mg, 2.10 mmol) in 50% aque-
ous dioxane (20 ml) was added a freshly made aqueous solution
of sodium tetraoxide (2.2 ml, 0.12 M). After stirring for 1 hour,
ethyl acetate was added followed by saturated sodium bisulfite.
The reaction mixture was stirred for 20 min. The organic layer
was separated and the aqueous layer was extracted with ethyl
acetate (3 × 20 ml). The combined organic layers were dried
(anhydrous Na2SO4) and concentrated in vacuo. The crude resi-
due was purified by flash chromatography eluting with 10%
methanol–ethyl acetate, and crystallised from ethyl ether–
hexane to afford a 1.2 : 1 mixture of diastereoisomers of the
glycerol 29 (500 mg, 76%) as white needles. υmax (Nujol)/cmϪ1
3313b (OH) and 1735s (CO); δH (250 MHz, CDCl3): 4.33–4.22
(1 H, m), 3.89–3.53 (5 H, m), 3.73 (3 H, s), 3.24 (3 H, s), 3.23
(3 H, s), 2.22–1.83 (4H, m), 1.30 (3 H, s), 1.25 (3 H, s); δC (63
MHz, DEPT, CDCl3): 174.1 (C), 173.9 (C), 99.9 (C), 99.8 (C),
77.4 (CH), 77.3 (CH), 75.4 (C), 75.3 (C), 74.0 (CH2), 73.4 (CH),
73.0 (CH), 72.8 (CH2), 70.7 (CH), 69.9 (CH), 52.6 (OCH3), 47.9
(OCH3), 47.9 (OCH3), 38.8 (CH2), 38.7 (CH2), 36.5 (CH2), 36.4
(CH2), 17.5 (CH3), 17.4 (CH3); MS (CI) m/z (%) 363 [(MHϩ) Ϫ
HOCH3]; HRMS calcd for C16H27O9: MHϩ, 363.1655. Found:
MHϩ, 363.1650.
Methyl (1S,3S,4R,5R)-3-allyloxy-1,4,5-trihydroxycyclohexane-
1-carboxylate (27) and methyl (1S,3S,4R,5R)-4-allyloxy-
1,3,5-trihydroxycyclohexane-1-carboxylate (28)
A solution of the acetal 26 (174 mg, 0.48 mmol) in a mixture
20 : 1 (v/v) of trifluoroacetic acid–water was stirred at room
temperature for 15 min. The solvent was removed under
reduced pressure and the crude mixture was purified by flash
chromatography eluting with 50% acetone–dichloromethane to
afford the 3-allyloxy ester 27 (85 mg, 72%) and the 4-allyloxy
ester 28 (30 mg, 25%), both as colourless oils.
(1S,3S,4R,5R)-1,4,5-Trihydroxy-3-(2Ј,3Ј-dihydroxy)propyloxy-
cyclohexane-1-carboxylic acid (12)
Data for methyl (1S,3S,4R,5R)-3-allyloxy-1,4,5-trihydroxy-
cyclohexane-1-carboxylate (27). [α]2D0 Ϫ16 (c 2.05 in (CH3)2CO);
υmax (NaCl)/cmϪ1 3423b (OH) and 1731s (CO); δH (300 MHz,
CD3OD): 6.05 (1 H, m), 5.43 (1 H, dq, J = 17.2 and 1.5 Hz),
A solution of acetal 29 (95 mg, 0.24 mmol) in a solution
of trifluoroacetic acid–water (2 ml, 20 : 1 (v/v)) was stirred for
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 7 5 – 2 0 8 3
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22237–22242; (c) A. P. Leech, R. James, J. R. Coggins and
30 min and then the solvent was removed in vacuo. The crude
residue was dissolved in an aqueous lithium hydroxide (3 ml,
0.5 M) and stirred for 1 hour. The aqueous layer was washed
with diethyl ether (3 × 5 ml) and then was treated with Amber-
lite IR-120 until the pH was 6.0. The resin was filtered and
washed with water. The filtrate was freeze-dried to afford a
1.2 : 1 mixture of diastereoisomers of the acid 12 (60 mg, 94%)
as an amorphous solid. υmax (Nujol)/cmϪ1 3430b (OH) and
1725s (CO); δH (300 MHz, D2O): 4.64 (1 H, dt, J = 10.1 and
4.5 Hz), 4.53 (1 H, q, J = 3.4 Hz), 4.49–4.41 (1 H, m), 4.29–3.97
(5 H, m), 2.49–2.03 (4 H, m); δC (75 MHz, DEPT, D2O): 181.3
(C), 81.6 (CH), 81.3 (CH), 78.4 (C), 77.3 (CH), 73.9 (CH2), 73.7
(CH2), 73.5 (CH), 73.4 (CH), 69.5 (CH), 65.5 (CH2), 43.3
(CH2), 43.2 (CH2), 36.7 (CH2), 36.5 (CH2); MS (CI) m/z (%) 249
[(MHϩ) Ϫ H2O]; HRMS calcd for C10H17O7: MHϩ, 249.0974.
Found: MHϩ, 249.0967.
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Acknowledgements
Financial support from the Xunta de Galicia under project
PGIDIT02RAG20901PR and Fundação para a Ciência e a
Tecnologia is gratefully acknowledged.
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