Design and synthesis of aromatic inhibitors of anthranilate synthase

Article abstract of DOI:10.1039/b503802b

Aromatic analogues of chorismate were synthesised as potential inhibitors of anthranilate synthase. Molecular modelling using GOLD2.1 showed that these analogues docked into the active site of Serratia marcescens anthranilate synthase in the same conformation as chorismate. Most compounds were found to be micromolar inhibitors of S. marcescens anthranilate synthase. The most potent analogue, 3-(1-carboxy-ethoxy)-4-hydroxybenzoate (K₁ 3 μM). included a lactyl ether side chain. This appears to be a good replacement for the enol-pyruvyl side chain of chorismate. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b503802b

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A R T I C L E
Design and synthesis of aromatic inhibitors of anthranilate synthase
Richard J. Payne,a Miguel D. Toscano,a Esther M. M. Bulloch,a Andrew D. Abellb and
Chris Abell*a
a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
UK CB2 1EW. E-mail: ca26@cam.ac.uk; Fax: +44 1223 336362; Tel: +44 1223 336405
b Department of Chemistry, University of Canterbury, Christchurch, New Zealand
Received 15th March 2005, Accepted 15th April 2005
First published as an Advance Article on the web 6th May 2005
Aromatic analogues of chorismate were synthesised as potential inhibitors of anthranilate synthase. Molecular
modelling using GOLD2.1 showed that these analogues docked into the active site of Serratia marcescens
anthranilate synthase in the same conformation as chorismate. Most compounds were found to be micromolar
inhibitors of S. marcescens anthranilate synthase. The most potent analogue, 3-(1-carboxy-ethoxy)-4-
hydroxybenzoate (KI 3 lM), included a lactyl ether side chain. This appears to be a good replacement for the
enol-pyruvyl side chain of chorismate.
TrpG–TrpE heterodimer or TrpG2–TrpE2 heterotetramer. TrpG
Introduction
belongs to the family of “triad” glutamine amidotransferases
that hydrolyse the amido side chain of glutamine. In the case
of anthranilate synthase, the nascent ammonia is believed to be
transferred through an intramolecular channel to the synthase
active site of TrpE.8 The TrpE subunit catalyses the production
of anthranilate in two steps (Scheme 1).
The product of the shikimate pathway, chorismate (1), is
a branchpoint for five biosynthetic pathways leading to the
formation of the major aromatic metabolites, including the
aromatic amino acids phenylalanine, tyrosine and tryptophan.
The folate coenzymes, benzoid and naphthenoid quinones and
siderophores are also produced through these pathways (Fig. 1).1
Three of the branchpoint enzymes; anthranilate synthase, 4-
amino-4-deoxychorismate synthase and isochorismate synthase,
appear to share many mechanistic features and are believed to
have diverged from a common ancestor.2–6 Chorismate-utilising
enzymes only occur in plants, bacteria, fungi and apicomplexan
parasites. Inhibitors of these enzymes may therefore have
herbicidal, antibiotic, fungicidal or antiparasitic activity.
Scheme 1 The two reactions catalysed by the TrpE subunit of
anthranilate synthase.
The first reaction catalysed by TrpE is the nucleophilic attack
at C-2 of chorismate (1) with ammonia (produced by the TrpG
subunit) to give an intermediate, 2-amino-2-deoxyisochorismate
(ADIC) (2).9 The second reaction is the elimination of pyruvate
from ADIC to produce anthranilate (3). It has been shown that
the TrpE subunit alone is capable of producing anthranilate from
chorismate when ammonia is supplied.10
Previous inhibition studies on anthranilate synthase have
focused on substrate analogues based on cyclohexadiene (as
observed in chorismate, 1) or cyclohexene rings with varied
functionality.11,12 The inhibition constants against the Serratia
marcescens enzyme ranged from sub-micromolar tomillimolar.12
The most potent of these inhibitors are shown in Fig. 2. We have
designed and synthesised a series of substrate analogues built
around an aromatic core. The use of this simplified core allowed
us to explore the functionality around the ring. The flat aromatic
ring is a reasonable mimic of the almost planar cyclohexadiene
core of chorismate.
Fig. 1 Chorismate-utilising enzymes.
Anthranilate synthase catalyses the first committed step in
the biosynthesis of tryptophan, in which chorismate is first
aminated and then the enol-pyruvyl side chain cleaved to form
the aromatic product anthranilate (3).7 Anthranilate synthase
is a multifunctional enzyme composed of a small TrpG and
large TrpE subunit encoded by the trpG and trpE genes,
respectively. In most bacteria the enzyme consists of either a
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 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1
2 2 7 1
©

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Fig. 2 Inhibition constants for inhibitors 4–6 against anthranilate
synthase from S. marcescens.12
Results and discussion
Inhibitor design and modelling
Crystal structures of anthranilate synthase from Sulfolobus
solfataricus (1QDL),9 Salmonella typhimurium (1I1Q)13 and
Serratia marcescens (1I7Q and 1I7S)8 have been reported. Of
the two structures reported for S. marcescens, one contains a
tryptophan bound to an allosteric binding site, whilst the other
has benzoate and pyruvate ligands bound in the TrpE active
site. The latter structure was used in this study.8,14 The TrpE
active site of S. marcescens anthranilate synthase was prepared
for modelling studies using SYBYL6.5.15 This involved first
removing the benzoate and pyruvate ligands. Chorismate was
then docked into the vacant active site using GOLD2.1 (Genetic-
Optimisation Ligand Docking).16 Chorismate docks with the
C-1 carboxylate forming an electrostatic interaction with an
enzyme bound magnesium ion, while the C-4 hydroxyl is in
the vicinity of a glutamate residue (Glu309) (Fig. 3A). The enol-
pyruvyl side chain at C-3 sits in a binding pocket where it can
form hydrogen bonds to Arg469 and Tyr449. The RMSD of the
common heavy atoms in the chorismate docking compared with
Fig. 3 (A) Chorismate (1) and (B) aromatic analogue (R-21) docked
into the active site of S. marcescens anthranilate synthase (1I7Q).8
˚
the benzoate ligand in the original crystal structure is 0.62 A.
explore the enol-pyruvyl binding pocket of anthranilate synthase
and to identify groups that could be used to replace the enol-
pyruvyl side chain of chorismate, which is difficult to incorporate
synthetically.
The first series of inhibitors were the 4-methoxybenzoate
analogues (7–9), a simple replacement for hydroxyl that intro-
duces possible steric interactions. The second series were the 4-
aminobenzoate analogues (10–12). In this series, two different C-
3 substituents were used; one retaining the ether linkage (10, 11)
of chorismate and the other with a secondary amine linkage (12).
In considering the inhibitor design the proposal was to use a
flat aromatic scaffold with a C-1 carboxylate, which was assumed
to bind to the magnesium ion in the active site of the enzyme.
A variety of substituents were incorporated at C-3 and C-4.
The substituents at C-4 included OMe (7–9), NH2 (10–12) and
OH (13–21) (Fig. 4). These were intended to probe the role of
the C-4 hydroxyl in chorismate, which is central to the enzyme
reaction and was therefore expected to be relatively intolerant
to change. The different side chains at C-3 were designed to
Fig. 4
2 2 7 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1

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The final series of compounds proposed as targets were the
4-hydroxybenzoate analogues (13–21). These analogues most
closely mimic chorismate, but with the secondary hydroxyl
at C-4 replaced by a more acidic phenolic hydroxyl. Again,
two types of side chains were used at C-3. One contained an
ether linkage as found in chorismate (21). The other series of
analogues incorporated an additional methylene unit before
the ether linkage on the C-3 side chain (13–20). Inspection
of the active site of anthranilate synthase indicated that there
was unoccupied space where the enol-pyruvyl side chain of
chorismate binds, which could be explored by extending the side
chain. The second step of the reaction catalysed by anthranilate
synthase involves elimination of the enol-pyruvyl side chain. It
was envisaged that extension of the side chain may mimic this
departure.
Molecular docking was used to predict the binding of the
target compounds in the active site of S. marcescens anthranilate
synthase. The ligands were first built in SYBYL6.5 and energy
minimised using the Tripos force field.15 The aromatic analogues
(7–21) were docked into the active site using GOLD2.1.16
The docking results suggested that these ligands would bind
in a similar orientation to chorismate (the docking result of
the R enantiomer of 21 is shown in Fig. 3B). A number of
general observations were made about the dockings. The C-
1 carboxylate of the analogues docked in such a way that it
would interact with the metal ion, while the para-substituents
were situated close to Glu309, suggesting a hydrogen-bonding
interaction. The para-methoxy group in ligands (7–9) cannot
donate a hydrogen bond to Glu309, however this does not affect
the orientation of the ring in the docking. The C-3 side chains
docked in the same binding pocket as the enol-pyruvyl side chain
of chorismate (1), where they could potentially form hydrogen
bonding interactions with Arg469 and Tyr449. The docking
of the ligands with larger C-3 side chains (13–20) resulted in
conformations where the aromatic ring was displaced slightly.
This movement of the ring would affect both the interaction of
the C-4 substituent with Glu309 and the C-1 carboxylate with
the magnesium ion.
Scheme 2
Synthesis
The 4-methoxybenzoate series. Treatment of the aldehyde
iso-vanillin (22) with hydrogen peroxide, perchloric acid and
catalytic vanadium pentoxide in methanol, using a procedure
developed by Gopinath et al.17 produced the desired methyl
ester 23 in one step. Treatment with methylbromoacetate in the
presence of sodium iodide in acetone led to alkylation at C-3
and formation of 24 in quantitative yield. Finally, hydrolysis of
the methyl esters by treatment with potassium hydroxide gave
the diacid 7. In a similar manner, 23 was alkylated with methyl-
2-bromopropionate and methyl-2-(bromomethyl)acrylate to af-
ford 25 and 26 respectively. Hydrolysis of the methyl esters of
25 and 26 with aqueous potassium hydroxide gave the desired
diacids 8 and 9 in high yields (Scheme 2).
Scheme 3
Synthesis of the 4-amino analogue 12 began from methyl
3,4-diaminobenzoate. The meta-amine of 30 is the most nu-
cleophilic, so a weak base was used to alkylate at this posi-
tion. Reaction of methyl-2-bromo-propionate with 30 in the
presence sodium iodide and anhydrous potassium carbonate
gave the desired diester 31 in moderate yield (Scheme 4).
Finally, deprotection with aqueous lithium hydroxide yielded
the dicarboxylate 12.
The 4-aminobenzoate series. Methyl-3-hydroxy-4-amino
benzoate (27) was used as the starting material in the synthesis
of 10 and 11. The choice of base proved to be crucial for the
selectivity of the alkylation reaction. The phenolate anion is a
stronger nucleophile than the deactivated para-amino group, so
a strong base was required to deprotonate the 3-hydroxyl and
achieve selectivity at this position. Treatment of 27 with potas-
sium tert-butoxide and sodium iodide followed by dropwise
addition of methyl-2-bromopropionate gave only substitution at
the 3-hydroxyl position to produce 28 in 87% yield (Scheme 3).
The diester 28 was hydrolysed with aqueous lithium hydroxide
to afford the dicarboxylate 10 as the lithium salt. Treatment of
27 with potassium tert-butoxide and sodium iodide followed by
addition of methyl-2-(bromomethyl)acrylate, gave the desired
phenol ether 29 in 70% yield. Finally, hydrolysis of the methyl
ester with lithium hydroxide gave the dicarboxylate 11.
The 4-hydroxybenzoate series. Compounds (13–20) were
synthesised in five steps from 4-hydroxy-3-methyl benzoate (32).
Formation of the methyl ester by treatment with thionyl chloride
and methanol proceeded in 76% yield (Scheme 5). Subsequent
acetylation with acetyl chloride in pyridine formed the diester 34
in 76% yield. Radical bromination of the methyl group at C-3 of
34 using N-bromosuccinimide and AIBN proceeded smoothly
in 79% yield to form the desired bromomethyl compound 35.
This was the key intermediate in the synthesis of the desired
analogues.
Ethers were formed by treatment of 35 with silver oxide in
diethyl ether using a variety of alcohols. The reactions proceed-
ing in moderate yields (35–50%). A number of commercially
available alcohols were selected to produce the small library
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1
2 2 7 3

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Scheme 4
Scheme 6
Assay Results
The aromatic chorismate analogues synthesised (7–21) were
tested against S. marcescens anthranilate synthase using a
previously reported fluorescence assay.18 All the compounds
were found to be competitive reversible inhibitors. The inhibition
results are shown in Table 1.
Several general conclusions can be made from the inhibition
data. Extension of the C-3 side chain (13–20) had a detrimental
effect on binding of these analogues to the enzyme. Analogues
containing an extra methylene linkage before the ether side
chain at C-3 were relatively weak inhibitors of the enzyme,
with inhibition constants ranging from 160–1600 lM. The
introduction of methyl substituents in the side chain further
reduced the affinity of these analogues and, in general, the
longer the side chain the lower the potency of the inhibitor.
The inclusion of a double bond in the side chain of analogue 18
recovered some binding affinity, possibly due to its similarity to
the enol-pyruvyl side chain of chorismate.
Compounds 8, 10, 12 and 21, which all contain a lactyl side
chain, were the most potent inhibitors of anthranilate synthase
with KI’s of 25, 43, 50 and 3 lM, respectively. It is unclear if
this is because the lactyl side chain is a good mimic of the enol-
pyruvyl side chain of chorismate. Alternatively, there may be
additional bonding interactions associated with the stabilisation
of the transition state that leads to the loss of the side chain and
aromatisation of ADIC to form anthranilate.
Scheme 5
of triesters 36–43 with side chains of differing length and
functionality. Methyl ester and acetate hydrolysis was achieved
in one step in generally high yields by treatment with aqueous
potassium hydroxide to afford the diacids 13–20.
Table 1 Inhibition constants of the aromatic inhibitors against S.
marcescens anthranilate synthase
Synthesis of compound 21 was achieved from methyl-
3,4-dihydroxybenzoate (44). Selective alkylation of the meta-
hydroxyl of methyl-3,4-dihydroxybenzoate proved to be more
problematic than for previous analogues. Treatment of methyl-
3,4-dihydroxybenzoate (44) with methyl-2-bromopropionate
and sodium hydride in DMF at 25 ◦C for five days, gave the
dialkylated product (16%) and an inseparable mixture of both
mono-alkylated products (27%). The mixture of mono-alkylated
products was further treated with benzoyl chloride in pyridine in
order to protect the free alcohol (Scheme 6). Column chromatog-
raphy of the crude product allowed separation of a small amount
of the desired C-3 alkylated product. The regiochemistry of 45
was confirmed by NOE analysis. Irradiation of the doublet signal
arising from the methyl group of the lactyl side chain caused
enhancement of the doublet for the C-2 ring proton and vice
versa. Hydrolysis of the methyl and benzoyl esters with aqueous
potassium hydroxide and acidic work-up gave the desired diacid
21 as well as benzoic acid, which could be separated by HPLC.
Inhibitor
KI/lMa
7
650 140
8
25
3
9
120 10
43 10
120 20
50 10
360 60
560 120
480 100
480 90
530 60
160 30
1600 400
780 140
10
11
12
13
14
15
16
17
18
19
20
21
3
0.3
a KM = 3.7 0.5 lM, kcat = 5.6 s−1 at 25 ◦C.
2 2 7 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1

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Somewhat surprisingly, anthranilate synthase appears to be
relatively insensitive to the functionality at C-4. Analogues 9 and
11, both containing an acrylate side chain and a methoxy and
amino moiety at C-4, respectively, exhibited identical inhibition
constants (120 lM).
The most potent of the aromatic chorismate analogues was
21 (Fig. 5), which exhibited a KI of 3 lM. This compound is
five times less potent than 5. Of all the compounds tested it
is the closest structural analogue of chorismate, with the lactyl
group replacing the enol-pyruvyl side chain and an aromatic
ring replacing the cyclohexadiene ring in chorismate.
trometer using NaCl plates or on a Perkin Elmer Spectrum One
FTIR spectrometer using attenuated transmittance reflectance
(ATR). High resolution mass spectrometry was carried out using
a Micromass Quadrapole-Time of Flight (Q–Tof) spectrometer.
HPLC purification was carried out on a Gilson HPLC system
fitted with a Gilson 118 UV/Vis detector, with detection at
254 nm. Where noted, compounds were analysed using a Waters
Xterra MS C18 5 lm 4.6 × 50 mm column and purified
using a Waters Xterra MS C18 5 lm 19 × 50 mm column.
Columns were eluted with a gradient of between 5 : 95 and
95 : 5 acetonitrile : water, both containing 0.1% TFA. Liquid-
chromatography mass-spectrometry (LCMS) was carried out
using an Alliance HT Waters 2795 Separations Module coupled
to a Waters Micromass ZQ Quadrapole Mass Analyzer. Samples
were detected using a photomultiplier detection system. Samples
were run on a gradient from 10 mM ammonium acetate
containing 0.1% formic acid to 95% acetonitrile over a period
of 8 min.
Cloning and purification of anthranilate synthase
The vector ptttSmEDC containing S. marcescens trpEGDC
was kindly supplied by Glen Spraggon (Genomics Institute of
the Novartis Research Foundation, San Diego, California) and
Charles Yanofsky (Stanford University, Stanford, California).
The trpEG gene fragment was amplified with a BamHI restric-
tion enzyme site at the 5ꢀ-end and a NcoI site at the 3ꢀ-end by PCR
using the pttSmEDC vector as a template. The primers were 5ꢀ-
CGC GGA TCC ATG AAC ACC AAA CCA CAA TTG ACA
C-3ꢀ (forward) and 5ꢀ-GCA TGC CAT GGT TAC TTC GCC
AGC GCC CAG-3ꢀ (reverse). The trpEG fragment was inserted
between the BamHI and NcoI sites of the expression vector
mini-pRSETA. Anthranilate synthase is over-expressed from the
resulting trpEG/mini-pRSETA vector with a hexahistidine tag
(MRGSHHHHHHGLVPRGS) attached to the N-terminus of
the TrpE subunit.
Fig. 5 Least squares fitting and Lineweaver–Burk plots for the
reversible competitive inhibition of anthranilate synthase by 21.
These studies indicate that the use of an aromatic template in
place of a more saturated ring system does not lead to a marked
decrease in inhibition against anthranilate synthase when com-
pared to those synthesised previously.12 Compound 21 is the
second most potent inhibitor of anthranilate synthase reported
to date and serves as a lead for future studies in this area.
Conclusions
Cells of the Escherichia coli strain C41(DE3) were trans-
formed with trpEG/mini-pRSETA and used to inoculate a 10 ml
LB (50 lg ml◦−1 ampicillin) preculture. Precultures were grown
for 16 h at 30 C with 200 rpm shaking. A 1 L 2xYT (50 lg ml−1
ampicillin) culture was inoculated with the preculture and grown
at 37 ◦C with 250 rpm shaking. Once an optical density (600 nm)
of 0.6–0.8 was reached, induction of over-expression of the His6-
tagged anthranilate synthase was carried out by the addition of
IPTG (1 mM). Growth was continued for approximately 4 h.
Cells were harvested by centrifugation at 11 000 × g for 15 min
and cell pellets were stored at −80 ◦C.
New aromatic inhibitors of anthranilate synthase were designed
and synthesised. Molecular modelling suggests that these in-
hibitors bind in a similar manner to the substrate. The inhibition
results suggest that the lactyl side chain at C-3 is a good
replacement for the enol-pyruvyl side chain of chorismate.
Interestingly, altering the substitution at C-4 does not appear
to affect the inhibition of anthranilate synthase substantially.
This will be explored further and will be the focus of future
work in this area.
Experimental
The following steps were all carried out on ice or at 4 ◦C. Cell
pellets were defrosted and resuspended in Buffer N1 (50 mM
sodium phosphate buffer pH 8.0, 300 mM NaCl, 10 mM
imidazole, 1 mM b-mercaptoethanol). Cells were lysed by
sonication and cell debris was removed by centrifugation at
39 000 × g for 30 min. The cell-free lysate was applied to a
5 ml Ni–NTA (nitriloacetic acid) column that had been pre-
equilibriated with Buffer N1. The column was washed with
24 column volumes of Buffer N2 (Buffer N1 with 50 mM
imidazole). His6-tagged anthranilate synthase was eluted from
the column with 4–5 column volumes of Buffer N3 (Buffer N1
with 250 mM imidazole). The eluted protein was dialysed into
storage buffer (50 mM Tris HCl pH 7.5, 1 mM DTT and 0.1 mM
EDTA), separated into aliquots (100–500 ll) and flash frozen in
liquid nitrogen. Aliquots were stored at −80 ◦C.
General
All organic solvents were freshly distilled prior to use. Milli-Q
deionised water was used for all biochemical work. Analytical
thin layer chromatography was carried out on commercial silica
gel 60 0.25 mm plates using either UV absorption or potassium
permanganate stain for visualisation (3 g potassium perman-
ganate, 20 g potassium carbonate, 5 ml of 5% sodium hydroxide,
300 ml water). 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. Petroleum ether
refers to the fraction distilled between 40–60 ◦C. Unless specified
in the experimental, 1H NMR spectra were recorded on a Bruker
AM-400 spectrometer in deuterated solvents, as indicated.
Otherwise, 1H NMR spectra were recorded on a Bruker Avance
500 spectrometer. Unless specified in the experimental, all 13C
NMR spectra were recorded on a Bruker AM-400 spectrometer
operating at 100 MHz. Otherwise, 13C NMR spectra were
carried out on a Bruker Avance 500 spectrometer operating at
125 MHz. All chemical shifts are quoted in parts per million
Anthranilate synthase assay
Kinetic parameters for S. marcescens anthranilate synthase were
determined using the kinetic fluorescence assay developed by
Bauerle et al.18 Fluorescence was detected at 390 nm after initial
excitation at 313 nm at 25 ◦C. Reactions were initiated by the ad-
dition of anthranilate synthase (20 ll of a 0.01 mg ml−1 solution)
to the following assay mixture (total volume 200 ll): 100 mM
1
(ppm) d. Coupling constants for H NMR spectroscopy are
assigned where possible and are given in Hz. Infrared spectra
were recorded on an ATI Mattson Genesis FTIR infrared spec-
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1
2 2 7 5

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potassium phosphate buffer (pH = 7.0); 10 mM magnesium
chloride solution; 20 mM glutamine solution. For the inhibitor
studies, various concentrations of inhibitor (10 lM–1 mM) were
added to the assay mixture using chorismate concentrations
varying from 1–50 lM. Initial rates were determined by measur-
ing the increase in fluorescence over the first minute.
126.8, 148.6, 155.3, 168.3, 170.8; HRMS calcd for C12H14O6Na:
MNa+, 277.0688. Found: MNa+, 277.0698.
3-Carboxymethoxy-4-methoxy-benzoic acid 7
Diester 24 (0.15 g, 0.59 mmol) was dissolved in THF–water
(1 : 1 v/v,8 ml). Potassium hydroxide (0.03 g, 2.35 mmol) was
added and the reaction stirred at 40 ◦C for 2 h. The solvent was
removed in vacuo and the resulting residue dissolved in water.
The aqueous fraction was washed with ethyl acetate (10 ml)
before acidifying to pH 1 with 1 M HCl. The aqueous fraction
was extracted with ethyl acetate (2 × 20 ml), dried (MgSO4) and
solvent removed in vacuo to afford the desired diacid 7 as a white
solid (0.10 g, 75%).
Enzyme Kinetics Pro19 or Grafit20 software were used to
construct Michaelis–Menton plots of the kinetic data and carry
out a least squares fitting for the inhibition of anthranilate syn-
thase. The software was also utilised to calculate the inhibition
constants KI and associated standard errors, assuming reversible
competitive inhibition.
Gold docking
mmax. (ATR): 3432, 3090, (br, acid OH stretch) 2931, 2570 (Ar
−1
1
All ligands and the receptor were prepared using SYBYL6.5 and
used as MOL2 files.15 The ligands were prepared as carboxylate
anions and their structures were energy minimised using the
Tripos force-field. Gasteiger–Huckel charges were calculated
prior to docking. Each ligand was docked using GOLD2.1 in
25 independent genetic algorithm (GA) runs. For each of these,
a maximum number of 100 000 GA operations was performed
on a single population of 50 individuals.16 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
=
=
C–H stretch), 1732, 1690 (C O), 1606, 1581 (C C, ar) cm ; H
NMR (d6-acetone) d 3.90 (3H, s, OCH3), 4.77 (2H, s, CH2), 7.07
(1H, d, J 8.5 Hz, ArH, H-5), 7.53 (1H, d, J 2.0 Hz, ArH, H-2),
7.68 (1H, dd, J 8.5, 2.0 Hz, ArH, H-6); 13C NMR (d6-acetone) d
57.9, 68.0, 114.0, 117.6, 125.3, 127.1, 149.8, 156.3, 168.9, 171.8;
HRMS calcd for C10H10O6Na: MNa+, 249.0375. Found: MNa+,
249.0364.
4-Methoxy-3-(1-methoxycarbonylethoxy)-benzoic acid methyl
ester 25
˚
˚
hydrogen bonding (4.0 A) and van der Waals (2.5 A) parameters.
The position of the active site was introduced and the radius was
Methyl-2-bromopropionate (0.24 ml, 2.14 mmol) was added
dropwise to a stirred solution of 23 (300 mg, 1.65 mmol), sodium
iodide (0.04 g, 0.30 mmol) and anhydrous potassium carbonate
(0.46 g, 3.29 mmol) in acetone (18 ml). The reaction was heated
at reflux for 17 h. Work up was carried out as described for 24
and the product purified by column chromatography (eluent 4 :
1 v/v petroleum ether–ethyl acetate) to afford 25 as a white solid
(0.38 g, 88%).
˚
set to 10 A, with the automatic active-site detection selected.
3-Hydroxy-4-methoxy-benzoic acid methyl ester 2317
Vanadium pentoxide (0.24 g, 1.32 mmol) was added to 30% H2O2
(14.9 ml, 0.13 mol) and the solution stirred at 0 ◦C until all of the
vanadium pentoxide dissolved and the solution became reddy–
brown. This solution was added dropwise over a period of 1 h to
an ice-cold solution of iso-vanillin (5 g, 0.03 mol) in methanol
(165 ml) containing 70% HClO4 (1.6 ml, 0.02 mol). The reaction
was stirred for 6 h. The solvent was removed in vacuo and the
resulting residue redissolved in ethyl acetate (50 ml) and applied
to a silica column (eluent 4 : 1 v/v petroleum ether–ethyl acetate)
to afford 23 as an orange solid (1.56 g, 26% isolated).
RF [3 : 1 petroleum ether : ethyl acetate] = 0.29; mmax. (ATR):
=
2957, 2946 (Ar C–H stretch), 1731, 1711 (C O), 1599, 1587
−1
1
=
(C C, ar) cm ; H NMR (CDCl3) d 1.63 (3H, d, J 6.8 Hz,
CH3), 3.73 (3H, s, CH3,), 3.84 (3H, s, CH3), 3.88 (3H, s, CH3),
4.81 (1H, q, J 6.8 Hz, CH), 6.87 (1H, d, J 8.5 Hz, ArH, H-5), 7.48
(1H, d, J 1.9 Hz, ArH, H-2), 7.68 (1H, dd, J 8.5, 1.9 Hz, ArH,
H-6); 13C NMR (CDCl3) d 16.8,50.3, 50.6, 54.4, 72.2, 109.6,
114.9, 121.0, 123.4, 144.8, 152.4, 164.9, 170.5; HRMS calcd for
C13H16O6Na: MNa+, 291.0845. Found: MNa+, 291.0828
RF [4 : 1 petroleum ether : ethyl acetate] = 0.18; mmax. (ATR):
=
3411 (br, OH stretch) 3016, 2956 (Ar C–H stretch), 1697 (C O),
−1
1
=
1612, 1589 (C C, ar) cm ; H NMR (CDCl3) d 3.85 (3H, s,
CH3), 3.90 (3H, s, CH3), 5.88 (1H, br s, OH), 6.83 (1H, d, J
8.2 Hz, ArH, H-5), 7.57 (1H, dd, J 8.2, 2.1 Hz, ArH, H-6), 7.59
(1H, d, J 2.1 Hz ArH, H-2); 13C NMR (CDCl3) d 52.3, 56.4,
110.3, 116.0, 123.2, 123.7, 145.7, 150.9, 167.3; LCMS (MH+) =
183.2 (ret. time = 3.3 min); HRMS calcd for C9H10O4Na: MNa+,
205.0477. Found: MNa+, 205.0473.
3-(1-Carboxyethoxy)-4-methoxy-benzoic acid 8
Methyl ester 25 (0.18 g, 0.67 mmol) was dissolved in THF–water
(1 : 1 v/v, 10 ml). Potassium hydroxide (0.15 g, 2.68 mmol) was
added and the reaction stirred at 40 ◦C for 2 h. Acidification
and extraction as described for 7, gave 8 as a white solid (0.16 g,
99%).
mmax. (ATR): 2989 (br, acid OH stretch) 2596 (Ar C–H stretch),
4-Methoxy-3-methoxycarbonylmethoxy-benzoic acid methyl
ester 24
−1
1
6
=
=
1718, 1676 (C O), 1603, 1583 (C C, ar) cm ; H NMR (d -
acetone) d 1.60 (3H, d, J 6.8 Hz, CH3), 3.91 (3H, s, OCH3), 4.85
(1H, q, J 6.8 Hz, CH), 7.07 (1H, d, J 8.4 Hz, ArH, H-5), 7.55
(1H, d, J 2.0 Hz, ArH, H-2), 7.69 (1H, dd, J 8.4, 2.0 Hz, ArH, H-
6); 13C NMR (d6-acetone) d 19.2, 56.7, 74.6, 112.8, 117.8, 124.0,
126.1, 148.2, 155.5, 167.7, 173.5; HRMS calcd for C11H12O6Na:
MNa+, 263.0532. Found: MNa+, 263.0529.
Methylbromoacetate (0.20 ml, 2.14 mmol) was added dropwise
to a stirred solution of 23 (300 mg, 1.65 mmol), sodium iodide
(0.04 g, 0.30 mmol) and anhydrous potassium carbonate (0.46 g,
3.29 mmol) in acetone (18 ml). The reaction was heated at reflux
for 18 h. The reaction was allowed to cool and the solvent
was removed in vacuo. The residue was redissolved in DCM
(10 ml) and washed with water (10 ml). The aqueous fraction was
back extracted with DCM (2 × 10 ml). The combined organic
fractions were dried (MgSO4), and the solvent was removed in
vacuo to afford the crude product as a brown solid. Purification
by column chromatography (eluent 4 : 1 v/v petroleum ether–
ethyl acetate) gave 24 as a white solid (0.42 g, quant).
4-Methoxy-3-(2-methoxycarbonyl-allyloxy)-benzoic acid methyl
ester 26
Methyl-2-(bromomethyl)acrylate (0.26 ml, 2.14 mmol) was
added dropwise to a stirred solution of 23 (300 mg, 1.65 mmol),
sodium iodide (0.04 g, 0.30 mmol) and anhydrous potassium car-
bonate (0.46 g, 3.29 mmol) in acetone (18 ml). The reaction was
heated at reflux for 14 h. Work up was carried out as described for
24 and the product purified by column chromatography (eluent
4 : 1 v/v petroleum ether–ethyl acetate) to afford 26 as a white
solid (0.48 g, quant.).
1
RF [4 : 1 petroleum ether : ethyl acetate] = 0.16; H NMR
(CDCl3) d 3.78 (3H, s, CH3,), 3.85 (3H, s, CH3), 3.92 (3H, s,
CH3), 4.72 (2H, s, CH2) 6.90 (1H, d, J 8.5 Hz, ArH, H-5), 7.46
(1H, d, J 1.9 Hz, ArH, H-2), 7.70 (1H, dd, J 8.5, 1.9 Hz, ArH, H-
6); 13C NMR (CDCl3) d 53.8, 54.1, 57.9, 67.9, 112.8, 116.3, 124.4,
2 2 7 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1

View Article Online
RF [3 : 1 petroleum ether : ethyl acetate] = 0.45; mmax. (NaCl
1 h at 22 ◦C. The solution was diluted with ethyl acetate
(50 ml) and washed with saturated aqueous sodium bicarbonate
solution (3 × 30 ml). The organic layer was dried (Na2SO4)
and the solvent removed in vacuo. Purification by column
chromatography (eluent: 1 : 1 v/v ethyl acetate–hexane) gave
29 as a white solid (220 mg, 70%).
=
plate): 2952 (br, acid OH stretch), 1719, 1655 (C O), 1438
−1
1
=
(C C) cm ; H NMR (CDCl3) d 3.79 (3H, s, CH3), 3.87 (3H, s,
=
CH3), 3.91 (3H, s, CH3), 4.83 (2H, s, CH2), 6.20 (1H, s, C C–H),
6.40 (1H, s, C C–H), 6.88 (1H, d, J 8.5 Hz, ArH, H-5), 7.54
=
(1H, d, J 2.0 Hz, ArH, H-2), 7.68 (1H, dd, J 8.5, 2.0 Hz, ArH,
H-6); 13C NMR (CDCl3) d 52.4, 56.4, 67.6, 111.2, 114.9, 123.1,
124.7, 127.2, 135.8, 147.7, 154.0, 166.2, 167.1; HRMS calcd for
C14H16O6Na: MNa+, 303.0845. Found: MNa+, 303.0846.
RF [1 : 1 hexane : ethyl acetate] = 0.69; mmax 3340 (NH), 1709,
−1
=
=
=
1680 (C O), 1624 (C C), 1593 (C C, ar) cm ;
1H NMR (CDCl3) d 3.81 (3 H, s, OCH3), 3.85 (3 H, s, OCH3),
=
4.13 (2H, bs, NH2), 4.81 (2H, s, CH2), 5.95 (1 H, s, CHH),
6.41 (1 H, s, CHH), 6.67 (1 H, d, J 8.2 Hz, H-5), 7.49 (1 H,
=
3-(2-Carboxy-allyloxy)-4-methoxy-benzoic acid 9
d, J 1.7 Hz, H-2), 7.57 (1 H, dd, J 8.2, 1.7 Hz, H-6); 13C NMR
(100 MHz, DEPT, CDCl3), 52.1 (CH3), 52.5 (CH3), 67.6 (CH2),
114.0 (CH), 114.1 (CH), 120.0 (C), 125.2 (CH), 127.6 (CH2),
136.1 (C), 142.0 (C), 145.0 (C), 166.3 (C), 167.5 (C); LC/MS
(ret. time = 3.7 min) (ESI+) m/z 266 [MH+].
Methyl ester 26 (0.15 g, 0.53 mmol) was dissolved in THF–water
(1 : 1 v/v, 8 ml). Potassium hydroxide (0.12 g, 2.13 mmol) was
added and the reaction stirred at 40 ◦C for 2 h. Acidification
and extraction as described for 7, gave the desired product 9 as
a white solid (0.17 g, quant.).
mmax. (ATR): 2845 (br, acid OH stretch) 2588 (Ar C–H stretch),
−1
=
=
=
1679 (C O), 1635, 1601, 1586 (C C, ar), 1436 (C C) cm ;
Lithium 4-amino-3-[(2-carboxyallyl)-oxy]-benzoate 11
1H NMR (CD3OD) d 3.89 (3H, s, OMe), 4.77 (2H, s, CH2),
A solution of 29 (200 mg, 0.75 mmol) and lithium hydroxide
(94 mg, 2.25 mmol) in H2O–MeCN (1 : 1 v/v, 2 ml) was stirred
for 10 h at 22 ◦C. The solution was diluted with water (10 ml),
washed with ethyl acetate (2 × 10 ml) and lyophilised to give the
dicarboxylate 11 as a yellow solid (165 mg, 93%).
=
=
5.99 (1H, s, C C–H), 6.35 (1H, s, C C–H), 7.02 (1H, d, J
8.5 Hz, ArH, H-5), 7.56 (1H, d, J 2.0 Hz, ArH, H-2), 7.67
(1H, dd, J 8.5, 2.0 Hz, ArH, H-6); 13C NMR (CD3OD) d 56.9,
69.1, 112.7, 116.7, 124.5, 126.2, 127.7, 138.5, 149.1, 155.5, 169.1,
170.0; HRMS calcd for C12H12O6Na: MNa+, 275.0532. Found:
MNa+, 275.0522.
−1
1
=
=
mmax (ATR) 3042 (NH), 1591 (C O + C C, ar str) cm ; H
NMR (500 MHz, D2O) d 4.57 (2 H, s, CH2), 5.47 (1 H, s, CHH),
5.73 (1 H, s, CHH), 6.63 (1 H, d, J 8.1 Hz, H-5), 7.18 (1 H,
d, J 8.1 Hz, H-6), 7.22 (1 H, s, H-2); 13C NMR (100 MHz,
DEPT, CDCl3) d 69.8 (CH2), 115.0 (CH), 115.7 (CH), 123.2
(CH2), 124.2 (CH), 127.1 (C), 140.3 (C), 142.0 (C),145.8 (C),
174.9 (C), 175.7 (C); LC/MS (ret. time = 3.3 min) (ESO+) m/z
238 (MH+); HRMS calcd for C11H10NO5: (M–H)−, 236.0564.
Found: (M–H)−, 236.0565.
Methyl 4-amino-3-(1-methoxycarbonyl-ethoxy)-benzoate 28
Methyl-2-bromopropionate (200 ll, 1.80 mmol) was added
to a solution of methyl 3-hydroxy-4-aminobenzoate (300 mg,
1.80 mmol), potassium tert-butoxide (202 mg, 1.80 mmol) and
sodium iodide (54 mg, 0.36 mmol) in acetonitrile (2 ml) and
◦
stirred at 22 C, under argon for 6 h. The mixture was diluted
with ethyl acetate (50 ml) and washed with saturated aqueous
sodium bicarbonate solution (3 × 50 ml). The organic layer was
dried over Na2SO4, filtered and the solvent removed in vacuo.
Purification by column chromatography (eluent: 1 : 1 v/v ethyl
acetate–hexane) gave 28 as a white solid (400 mg, 87%).
Methyl 4-amino-3-(1-methoxycarbonyl-ethylamino)-benzoate 31
Methyl 2-bromopropionate (335 ll, 3.00 mmol) was added
dropwise to
a solution of methyl 3,4-diaminobenzoate
RF [1 : 1 hexane : ethyl acetate] = 0.62; mmax (ATR) 3065 (NH),
(500 mg, 3.00 mmol), anhydrous potassium carbonate (620 mg,
4.50 mmol) and potassium iodide (90 mg, 0.60 mmol) in
acetonitrile (3 ml) and the reaction was stirred under nitrogen
−1
1
=
=
1721, 1692 (C O), 1609 (C C, ar) cm ; H NMR (CDCl3) d
1.57 (3 H, d, J 6.9 Hz, CH3), 3.74 (3 H, s, CH3), 3.88 (3 H, s,
CH3), 4.68 (1 H, q, J 6.9 Hz, CH), 6.88 (1 H, d, J 8.1 Hz, H-5),
7.63 (1 H, d, J 1.7 Hz, H-2), 7.66 (1 H, dd, J 8.1, 1.7 Hz, H-
6); 13C NMR (DEPT, CDCl3) d 15.1 (CH3), 50.9 (OCH3), 52.2
(CH3), 72.1 (C), 114.2 (CH), 117.2 (CH), 123.3 (CH), 124.8 (C),
129.4 (C), 141.5 (C), 165.0 (C),167.5 (C); LC/MS (ret. time =
3.5 min) (ESI+) m/z 254 (MH+).
◦
at 22 C, for 24 h. The reaction was diluted with ethyl acetate
(50 ml) and washed with saturated aqueous sodium bicarbonate
solution (3 × 50 ml). The organic layer was dried (Na2SO4)
and the solvent removed in vacuo. Purification by column
chromatography (eluent: 1 : 1 v/v ethyl acetate–hexane) gave
31 as a yellow solid (300 mg, 40%).
RF [1 : 1 hexane : ethyl acetate] = 0.31; mmax (ATR) 3352 (NH),
−1
1
6
=
=
1676 (C O), 1601 (C C, ar) cm ; H NMR (d -acetone) d 1.35
(3 H, d, J 6.7 Hz, CH3), 3.74 (3 H, s, CH3), 3.84 (3 H, s, CH3),
3.90 (1 H, q, J 6.7 Hz, CH), 6.80 (1 H, d, J 8.6 Hz, H-5), 7.34 (2
H, m, H-2 and H-6); 13C NMR (DEPT, MeOD) d 17.9 (CH3),
51.9 (CH), 52.4 (OCH3), 52.6 (OCH3), 115.7 (CH), 115.9 (CH),
122.5 (CH), 126.3 (C), 131.8 (C), 135.6 (C), 168.6 (C), 171.6 (C);
LC/MS (ret. time = 3.1 min) (ESI+) m/z 221 ([M–MeOH]H+).
Lithium 4-amino-3-(1-carboxy-ethoxy)-benzoate 10
Methyl ester 28 (100 mg, 0.39 mmol) and lithium hydroxide
(41 m◦g, 0.98 mmol) in H2O–MeCN (1 : 1 v/v, 2 ml) was stirred
at 22 C for 8 h. The solvent was removed in vacuo to give the
dicarboxylate 10 as a white solid, (88 mg, quant).
−1
=
=
mmax (ATR) 3067 (NH), 1682 (C O), 1611 (C C, ar) cm ;
1H NMR (D2O) d 1.27 (3 H, d, J 6.9 Hz, CH3), 4.52 (1 H, q, J
6.9 Hz, CH), 6.81 (1 H, d, J 8.1 Hz, H-5), 7.28 (1 H, d, J 1.9 Hz,
H-2), 7.38 (1 H, dd, J 8.1, 1.9 Hz, H-6); 13C NMR (DEPT, D2O)
d 16.5 (CH3), 72.8 (CH), 116.9 (CH), 119.9 (CH), 124.2 (CH),
130.8 (C), 137.0 (C), 142.7 (C), 175.4 (C), 174.3 (C); LC/MS
(ret. time = 3.1 min) (ESI+) m/z 226 (MH+).
Lithium 4-amino-3-(1-carboxy-ethylamino)-benzoate 12
A solution of 31 (100 mg, 0.40 mmol) and lithium hydroxide
(41 mg, 0.84 mmol) in H2O–MeCN (1 : 1 v/v, 2 ml) was stirred
at 22 ◦C for 2 h. The acetonitrile was removed in vacuo and the
remaining solution lyophilised to afford the dicarboxylate 12 as
Methyl 4-amino-3-[(2-methoxycarbonylallyl)-oxy]-benzoate 29
a white solid. (94 mg, quant.).
−1
=
=
Potassium tert-butoxide (136 mg, 1.20 mmol) was added to
a solution of methyl 3-hydroxy-4-aminobenzoate (200 mg,
1.20 mmol) and sodium iodide (36 mg, 0.24 mmol) in acetonitrile
(10 ml) and the reaction stirred at 22 ◦C, under argon. After
20 min, methyl 2-(bromomethyl)acrylate (148 ll, 1.20 mmol)
was added dropwise and the reaction was stirred for a further
mmax (ATR) 3340 (NH), 1668 (C O), 1610 (C C, ar) cm ;
1H NMR (D2O) d 1.16 (3 H, d, J 6.8 Hz, CH3), 3.74 (1 H, q, J
6.8 Hz, CH), 6.75 (1 H, d, J 8.1 Hz, H-5), 7.19 (1 H, d, J 1.8 Hz,
H-2), 7.25 (1 H, dd, J 8.1, 1.8 Hz, H-6); 13C NMR (DEPT, D2O)
d 17.1 (CH3), 51.4 (CH), 116.0 (CH), 118.1 (CH), 122.1 (CH),
131.2 (C), 133.7 (C), 134.4 (C), 175.0 (C), 175.7 (C).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1
2 2 7 7

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Methyl 4-hydroxy-3-methylbenzoate 33
oil. Purification by column chromatography (eluent 3 : 1 v/v
petroleum ether–ethyl acetate) gave 36 as a colourless oil. (40 mg,
54%).
To a suspension of 4-hydroxy-3-methyl benzoic acid (2.5 g,
0.02 mol) in freshly distilled methanol (40 ml) was added 20
drops of thionyl chloride. The reaction mixture was heated at
reflux under nitrogen for 16 h. The solution was allowed to cool
to 22 ◦C before the solvent was removed in vacuo to give the crude
product as a white solid. Purification bycolumnchromatography
(eluent 3 : 1 v/v petroleum ether–ethyl acetate) gave 33 as a white
solid. (2.08 g, 76%).
RF [3 : 1 petroleum ether : ethyl acetate] = 0.48; mmax. (NaCl
=
plate): 2953 (Ar C–H stretch), 1759, 1722 (C O), 1612, 1592
−1
1
=
(C C, ar) cm ; H NMR (CDCl3) d 2.33 (3H, s, CH3), 3.75
(3H, s, CH3), 3.90 (3H, s, CH3), 4.06 (2H, s, CH2), 4.62 (2H, s,
CH2), 7.15 (1H, d, 1H, d, J 8.4 Hz, ArH, H-5), 8.02 (1H, dd,
J 2.1, 8.4 Hz, ArH, H-6), 8.13 (1H, d, J 2.1 Hz, ArH, H-2);
13C NMR (CDCl3) d 20.8, 51.8, 52.2, 67.0, 68.1, 122.8, 128.0,
129.5, 130.8, 131.5, 152.7, 166.1, 168.8, 170.4; HRMS calcd for
C11H12O6Na: MNa+, 319.0794. Found: MNa+, 319.0803.
RF [3 : 1 petroleum ether : ethyl acetate] = 0.41; mmax. (ATR):
=
3260 (br OH stretch), 2961 (Ar C–H stretch), 1683 (C O), 1598,
−1
1
=
1509 (C C, ar) cm ; H NMR (CDCl3) d 2.27 (3H, s, CH3),
3.87 (3H, s, CH3), 5.66 (1H, br s, OH), 6.80 (1H, d, J 8.5 Hz,
ArH, H-5), 7.78 (1H, dd, J 8.5, 1.7 Hz, ArH, H-6), 7.83 (1H, d,
J 1.7 Hz, ArH); 13C NMR (CDCl3) d 13.9, 50.2, 113.0, 120.7,
122.2, 127.7, 131.1, 156.5, 165.5; HRMS calcd for C9H10O3Na:
MNa+, 189.0528. Found: MNa+, 189.0533.
3-Carboxymethoxymethyl-4-hydroxy benzoic acid 13
Potassium hydroxide (91 mg, 1.62 mmol) was added to a solution
of 36 (20 mg, 0.07 mmol) in THF–water (1 : 1 v/v, 2 ml) and the
reaction was heated to 50 ◦C for 12 h. The reaction was allowed
to cool to 22 ◦C before dilution with water (2 ml). The aqueous
layer was washed with ethyl acetate (4 ml) before acidifying to
pH 1 with 1 M HCl. The aqueous fraction was extracted with
ethyl acetate (2 × 5 ml). The organic fraction was dried (MgSO4)
and the solvent removed in vacuo to afford the desired diacid 13
as a white solid. (13 mg, 85%).
4-Acetoxy-3-methyl-benzoic acid methyl ester 34
Methyl ester 33 (2.03 g, 0.012 mol) in dry pyridine (10 ml) was
cooled to 0 ◦C. Acetyl chloride (1.30 ml, 0.018 mol) was added
dropwise at 0 ◦C and the reaction stirred at 22 ◦C for 6 h.
The reaction was diluted with ethyl acetate (30 ml), washed
with saturated ammonium chloride solution (2 × 20 ml), brine
(20 ml), dried (MgSO4), and solvent removed in vacuo to afford
the crude product as an orange oil. Purification by column
chromatography (eluent 5 : 1 v/v petroleum ether–ethyl acetate)
gave the acetate 34 as a white solid (1.95 g, 76%).
mmax. (ATR): 3289, (br, acid OH stretch and ar OH), 2897, 2567
−1
=
=
(Ar C–H stretch), 1676 (C O str), 1617, 1586 (C C, ar) cm ;
1H NMR (d6-acetone) d 4.26 (2H, s, CH2), 4.69 (2H, s, CH2),
6.92 (1H, d, J 8.4 Hz, ArH, H-5), 7.89 (1H, dd, J 2.1, 8.5 Hz,
ArH, H-6), 7.97 (1H, d, J 2.1 Hz, ArH, H-2); 13C NMR (d6-
acetone) d 65.6, 68.2, 114.6, 120.1, 123.3, 130.5, 130.6, 159.2,
166.0, 172.4; HRMS calcd for C10H10O6Na: MNa+, 249.0375.
Found: MNa+, 249.0367.
RF [5 : 1 petroleum ether : ethyl acetate] = 0.50; mmax. (ATR)
−1
=
=
2954 (Ar C–H stretch), 1686 (C O), 1610, 1591 (C C, ar) cm ;
1H NMR (CDCl3) d 2.27 (3H, s, CH3) 3.87 (3H, s, CH3), 6.79
(1H, d, J 8.4 Hz, ArH, H-5), 7.77 (1H, dd, J 8.4, 1.5 Hz, ArH,
H-6), 7.83 (1H, d, J 1.5 Hz, ArH, H-2); 13C NMR (CDCl3) d
17.6, 20.5, 51.9, 123.0, 127.0, 129.1, 130.9, 135.6, 149.0, 166.0,
168.7; HRMS calcd for C11H12O4Na: MNa+, 231.0633. Found:
MNa+, 231.0623.
4-Acetoxy-3-(1-methoxycarbonyl-ethoxy-(S)-methyl)-benzoic
acid methyl ester 37
Methyl-(S)-lactate (30 ll, 0.31 mmol) was added dropwise to a
solution of 35 (100 mg, 0.35 mmol) in freshly distilled diethyl
ether (1.5 ml). Silver oxide (161 mg, 0.67 mmol) was added and
◦
4-Acetoxy-3-bromomethyl-benzoic acid methyl ester 35
the reaction heated at reflux (42 C) under nitrogen. After 1 h
of stirring additional silver oxide (30 mg) was added and the
reaction stirred at reflux for a further 2.5 h before stirring at
22 ◦C for 29 h. Work up was carried out as described for 36.
The product was purified by column chromatography (eluent 3 :
1 v/v petroleum ether–ethyl acetate) to afford 37 as a colourless
oil (50 mg, 53%).
To a solution of 34 (1.90 g, 9.08 mmol) in benzene (70 ml) under
nitrogen was added N-bromosuccinimide (1.62 g, 9.08 mmol)
and AIBN (0.08 g, 0.45 mmol). The reaction was heated at
reflux (85 ◦C) for 6 h. The reaction was allowed to cool to
22 ◦C before dilution with DCM (50 ml). The organic fraction
was washed with aqueous saturated sodium carbonate solution
(70 ml), water (70 ml), dried (MgSO4) and the solvent removed
in vacuo. The crude product was recrystallised from petroleum
ether–ethyl acetate to afford bromide 35 as colourless needles.
(2.05 g, 79%).
RF [3 : 1 petroleum ether : ethyl acetate] = 0.33; mmax. (NaCl
=
plate): 2953, 2921 (Ar C–H stretch), 1747, 1722 (C O, ar), 1614,
−1
1
=
1592 (C C, ar) cm ; H NMR (CDCl3) d 1.41 (3H, d, J 6.9 Hz,
CH3), 2.33 (3H, s, CH3), 3.75 (3H, s, CH3), 3.90 (3H, s, CH3),
4.00 (1H, q, J 6.9 Hz, CH), 4.38 (1H, d, J 12.2 Hz, CHH), 4.70
(1H, d, J 12.2 Hz, CHH), 7.15 (1H, d, J 8.4 Hz, ArH, H-5), 8.01
(1H, dd, J 2.1, 8.4 Hz, ArH, H-6), 8.13 (1H, d, J 2.1 Hz, ArH, H-
2); 13C NMR (CDCl3) d 19.0, 21.3, 52.4, 52.6, 67.2, 74.4, 123.2,
128.4, 130.4, 131.0, 131.9, 153.0, 166.6, 169.2, 173.7; HRMS
calcd for C15H18O7: MH+, 310.1053. Found: MH+, 310.1051.
=
mmax. (NaCl plate): 2954 (Ar C–H stretch), 1767, 1724 (C O),
−1
1
=
1613, 1595 (C C, ar) cm ; H NMR (CDCl3) d 2.38 (3H, s,
CH3), 3.90 (3H, s, CH3), 4.43 (2H, s, CH2) 7.22 (1H, d, J 8.5 Hz,
ArH, H-5), 8.01 (1H, dd, J 2.1, 8.5 Hz, ArH, H-6), 8.10 (1H, d, J
2.1 Hz, ArH, H-2); 13C NMR (CDCl3) d 22.7, 28.5, 54.0, 125.0,
129.8, 131.6, 132.9, 134.0, 154.2, 167.5, 170.0; HRMS calcd for
C11H11O4BrNa: MNa+, 308.9758. Found: MNa+, 308.9741.
3-(1-Carboxy-ethoxy-(S)-methyl)-4-hydroxy benzoic acid 14
4-Acetoxy-3-methoxycarbonyl-methoxymethyl)-benzoic acid
methyl ester 36
Potassium hydroxide (108 mg, 1.92 mmol) was added to a
solution of 37 (25 mg, 0.08 mmol) in THF–water (1 : 1 v/v,
2 ml) and the reaction was heated to 50 ◦C for 12 h. Acidification
and extraction were carried out as described for 13 to afford the
desired diacid 14 as a white solid. (17 mg, 88%).
Methyl glycolate (20 ll, 0.25 mmol) was added dropwise to a
solution of 35 (80 mg, 0.28 mmol) in freshly distilled diethyl
ether (1.5 ml). Silver oxide (129 mg, 0.56 mmol) was added and
◦
the reaction heated at reflux (42 C) under nitrogen. After 1 h
mmax. (ATR): 3290 (br, acid OH stretch and ar OH), 2926, 2547
−1
=
=
of stirring, additional silver oxide (50 mg) was added and the
reaction stirred at reflux for a further 2.5 h before stirring at
(Ar C–H stretch), 1731 (C O), 1673, 1616, 1587 (C C, ar) cm ;
1H NMR (d6-acetone) d 1.43 (3H, d, J 7.0 Hz, CH3), 4.24 (1H,
q, J 7.0 Hz, CH), 4.59 (1H, d, J 11.2 Hz, CHH), 4.72 (1H, d,
J 11.2 Hz, CHH), 6.91 (1H, d, J 8.5 Hz, ArH, H-5), 7.87 (1H,
dd, J 2.2, 8.5 Hz, ArH, H-6), 7.94 (1H, d, J 2.2 Hz, ArH, H-
2); 13C NMR (d6-acetone) d 20.3, 70.5, 75.9, 118.0, 123.9, 126.0,
◦
22 C for 18 h. The reaction was filtered through a small plug
of silica and the silica washed with diethyl ether (30 ml). The
combined organic fractions were dried (MgSO4), and the solvent
removed in vacuo to afford the crude product as a colourless
2 2 7 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1

View Article Online
133.8, 162.8, 168.9, 178.1; HRMS calcd for C11H12O6Na: MNa+,
263.0532. Found: MNa+, 263.0539.
and extraction was carried out as described for 13 to afford the
desired diacid 16 as a white solid (50 mg, 88%).
mmax. (ATR): 3191, 2987 (br, acid OH stretch and ar OH), 2947,
−1
=
=
2886 (Ar C–H stretch), 1680 (C O str), 1610 (C C, ar) cm ;
1H NMR (d6-acetone) d 1.18 (3H, d, J 7.1 Hz, CH3), 2.79 (1H,
ddd, J 5.7, 7.0, 7.1 Hz, CH), 3.63 (1H, dd, J 5.7, 9.2 Hz, CHH),
3.75 (1H, dd, J 7.0, 9.2 Hz, CHH), 4.62 (2H, s, CH2), 6.91 (1H,
d, J 8.5 Hz, ArH, H-5), 7.83 (1H, dd, J 2.2, 8.5 Hz, ArH, H-6),
7.99 (1H, d, J 2.2 Hz, ArH, H-2), 9.05 (1H, brs, OH); 13C NMR
(d6-acetone) d 12.4, 38.6, 67.2, 71.4, 114.0, 125.2, 123.7, 129.8
(× 2), 158.3, 170.9, 179.1; HRMS calcd for C12H14O6Na: MNa+,
277.0688. Found: MNa+, 277.0677.
4-Acetoxy-3-(1-methoxycarbonyl-ethoxy-(R)-methyl)-benzoic
acid methyl ester 38
Compound 38 was synthesised in an identical manner to 37,
above, using methyl-(R)-lactate in place of methyl-(S)-lactate.
Purification by column chromatography (eluent 3 : 1 v/v
petroleum ether–ethyl acetate) gave 38 as a colourless oil (30 mg,
32%).
RF [3 : 1 petroleum ether : ethyl acetate] = 0.33; mmax. (NaCl
=
plate): 2953, 2921 (Ar C–H stretch), 1747, 1722 (C O), 1614,
−1
1
=
1592 (C C, ar) cm ; H NMR (CDCl3) d 1.40 (3H, d, J 6.9 Hz,
CH3), 2.33 (3H, s, CH3), 3.75 (3H, s, CH3), 3.90 (3H, s, CH3),
4.00 (1H, q, J 6.9 Hz, CH), 4.39 (1H, d, J 12.2 Hz, CHH), 4.70
(1H, d, J 12.2 Hz, CHH), 7.15 (1H, d, J 8.1 Hz ArH, H-5),
8.00 (1H, dd, J 2.1, 8.1 Hz, ArH, H-6), 8.13 (1H, d, J 2.1 Hz,
ArH, H-2); 13C NMR (CDCl3) d 19.0, 21.3, 52.4, 52.6, 67.2,
74.4, 123.2, 128.4, 130.4, 131.0, 131.9, 153.0, 166.6, 169.2, 173.7;
LCMS (MH+ = 311.2) (ret. time = 3.73 min); HRMS calcd for
C15H18O7Na: MNa+, 333.0950. Found: MNa+, 333.0942.
4-Acetoxy-3-(2-methoxycarbonyl-propoxy-(R)-methyl)-benzoic
acid methyl ester 40
Methyl (R)-3-hydroxy-2-methylpropionate (52 ll, 0.47 mmol)
was added dropwise to a solution of 35 (150 mg, 0.52 mmol)
in freshly distilled diethyl ether (2 ml). Silver oxide (242 mg,
1.05 mmol) was added and the reaction heated at reflux (42 ◦C)
under nitrogen. After 1 h of stirring additional silver oxide
(75 mg) was added and the reaction stirred at reflux for a further
2.5 h before stirring at 22 ◦C for 20 h. Work up was carried
out as described for 36 and the product was purified by column
chromatography (eluent 3 : 1 v/v petroleum ether–ethyl acetate)
to afford 40 as a colourless oil (20 mg, 12%).
3-(1-Carboxy-ethoxy-(R)-methyl)-4-hydroxy benzoic acid 15
Potassium hydroxide (91 mg, 1.62 mmol) was added to a solution
of 38 (20 mg, 0.06 mm◦ol) in THF–water (1 : 1 v/v, 2 ml) and the
reaction heated at 50 C for 12 h. Acidification and extraction
were carried out as described for 13 to afford the desired diacid
15 as a white solid. (15 mg, 97%).
RF [3 : 1 petroleum ether : ethyl acetate] = 0.39; mmax. (NaCl
=
plate): 2953, 2878 (Ar C–H stretch), 1765, 1725 (C O), 1610,
−1
1
=
1591 (C C, ar) cm ; H NMR (CDCl3) d 1.15 (3H, d, J 7.1 Hz,
CH3), 2.30 (3H, s, CH3), 2.74 (1H, ddd, J 5.8, 7.1, 7.3 Hz, CH),
3.45 (1H, dd, J 5.8, 9.0 Hz, CHH), 3.61 (1H, dd, J 7.3, 9.0 Hz,
CHH), 3.68 (3H, s, CH3), 3.89 (3H, s, CH3), 4.42 (1H, d, J
12.6 Hz, CHH), 4.48 (1H, d, J 12.6 Hz, CHH), 7.12 (1H, d,
J 8.4 Hz, ArH, H-5), 7.98 (1H, dd, J 2.1, 8.4 Hz, ArH, H-
6), 8.08 (1H, d, J 2.1 Hz, ArH, H-2); 13C NMR (CDCl3) d 13.8,
20.7, 39.9, 51.6, 52.0, 67.9, 72.2, 122.4, 127.8, 130.2, 130.4, 130.9,
152.2, 166.1, 168.5, 175.0; HRMS calcd for C16H20O7Na: MNa+,
347.1107. Found: MNa+, 347.1097.
mmax. (ATR): 3290, 2987 (br, acid OH stretch and ar OH), 2930,
=
=
2547 (Ar C–H stretch), 1731 (C O), 1674, 1615, 1587 (C C,
ar) cm−1; 1H NMR (d6-acetone) d 1.43 (3H, d, J 7.0 Hz, CH3),
4.24 (1H, q, J 7.0 Hz, CH), 4.59 (1H, d, J 11.2 Hz, CHH), 4.72
(1H, d, J 11.2 Hz, CHH), 6.91 (1H, d, J 8.5 Hz, ArH, H-5), 7.87
(1H, dd, J 2.2, 8.5 Hz, ArH, H-6), 7.94 (1H, d, J 2.2 Hz, ArH, H-
2); 13C NMR (d6-acetone) d 20.3, 70.5, 75.9, 118.0, 123.9, 126.0,
133.8 (× 2), 162.8, 168.9, 178.1;HRMS calcd for C11H12O6Na:
MNa+, 263.0532. Found: MNa+, 263.0540.
3-(2-Carboxy-propoxy-(R)-methyl)-4-hydroxy-benzoic acid 17
4-Acetoxy-3-(2-methoxycarbonyl-propoxy-(S)-methyl)-benzoic
acid methyl ester 39
Potassium hydroxide (58 mg, 1.03 mmol) was added to a solution
of 40 (21 mg, 0.01 mmol) in THF–water (1 : 1 v/v, 2 ml).
The reaction was heated at 50 ◦C for 12 h. Acidification and
extraction was carried out as described for 13 to afford the
desired diacid 17 as a white solid. (14 mg, 85%).
Methyl (S)-3-hydroxy-2-methylpropionate (52 ll, 0.47 mmol)
was added dropwise to a solution of 35 (150 mg, 0.52 mmol)
in freshly distilled diethyl ether (2 ml). Silver oxide (242 mg,
1.05 mmol) was added and the reaction heated at reflux (42 ◦C)
under nitrogen. After 1 h of stirring additional silver oxide
(75 mg) was added and the reaction stirred at reflux for a further
2.5 h before stirring at 22 ◦C for 20 h. Work up was carried
out as described for 36 and the product was purified by column
chromatography (eluent 3 : 1 v/v petroleum ether–ethyl acetate)
to afford 39 as a colourless oil (76 mg, 50%).
mmax. (ATR): 3185 (br, acid OH stretch and ar OH), 2948, 2879
−1
1
=
=
(Ar C–H stretch), 1682 (C O, str), 1610 (C C, ar) cm ; H
NMR (d6-acetone) d 1.18 (3H, d, J 7.1 Hz, CH3), 2.79 (1H, ddd,
J 5.7, 7.0, 7.1 Hz, CH), 3.63 (1H, dd, J 5.7, 9.2 Hz, CHH), 3.75
(1H, dd, J 7.0, 9.2 Hz, CHH), 4.62 (2H, s, CH2), 6.91 (1H, d,
J 8.5 Hz, ArH, H-5), 7.83 (1H, dd, J 2.2, 8.5 Hz, ArH, H-6),
7.99 (1H, d, J 2.2 Hz, ArH, H-2), 9.05 (1H, brs, OH); 13C NMR
(d6-acetone) d 12.4, 38.6, 67.2, 71.4, 114.0, 125.2, 123.7, 129.8
(× 2), 158.3, 170.9, 179.1; HRMS calcd for C12H14O6Na: MNa+,
277.0688. Found: MNa+, 277.0678.
RF [3 : 1 petroleum ether : ethyl acetate] = 0.40; mmax. (NaCl
=
plate): 2980, 2953, 2878 (Ar C–H stretch), 1766, 1730 (C O),
−1
1
=
1614, 1593 (C C, ar) cm ; H NMR (CDCl3) d 1.15 (3H, d, J
7.1 Hz, CH3), 2.30 (3H, s, CH3), 2.74 (1H, ddd, J 5.8, 7.1, 7.3 Hz,
CH), 3.45 (1H, dd, J 5.8, 9.0 Hz, CHH), 3.61 (1H, dd, J 7.3,
9.0 Hz, CHH), 3.68 (3H, s, CH3), 3.89 (3H, s, CH3), 4.42 (1H, d,
J 12.6 Hz, CHH), 4.48 (1H, d, J 12.6 Hz, CHH), 7.12 (1H, d, J
8.4 Hz, ArH, H-5), 7.98 (1H, dd, J 2.1, 8.4 Hz, ArH, H-6), 8.08
(1H, d, J 2.1 Hz, ArH, H-2); 13C NMR (CDCl3) d 13.8, 20.7,
39.9, 51.6, 52.0, 67.9, 72.2, 122.4, 127.8, 130.2, 130.4, 130.9,
152.2, 166.1, 168.5, 175.0; HRMS calcd for C16H20O7Na:MNa+,
347.1107. Found: MNa+, 347.1109.
4-Acetoxy-3-(2-methoxycarbonyl-1-methyl-allyloxymethyl)-
benzoic acid methyl ester 41
Methyl-3-hydroxy-2-methylenebutyrate (57 ll, 0.47 mmol) was
added to a solution of 35 (150 mg, 0.52 mmol) in freshly distilled
diethyl ether (2 ml). Silver oxide (242 mg, 1.05 mmol) was added
and the reaction heated at reflux (42 ◦C) under nitrogen. After
1 h of stirring additional silver oxide (75 mg) was added and
the reaction stirred at reflux for a further 2.5 h before stirring at
22 ◦C for 20 h. Work up was carried out as described for 36 and
the product was purified by column chromatography (eluent 3 :
1 v/v petroleum ether–ethyl acetate) to afford 41 as a colourless
oil (60 mg, 36%).
3-(2-Carboxy-propoxy-(S)-methyl)-4-hydroxy-benzoic acid 16
Potassium hydroxide (198 mg, 3.54 mmol) was added to a
solution of 39 (72 mg, 0.22 mmol) in THF–water (1 : 1 v/v,
4 ml) and the reaction was heated at 50 ◦C for 12 h. Acidification
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1
2 2 7 9

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RF [3 : 1 petroleum ether:ethyl acetate] = 0.43; mmax. (NaCl
NMR (d6-acetone) d 36.0, 68.2, 73.3, 114.7, 120.4, 122.4, 130.6,
130.6, 159.4, 166.0, 169.8, 172.6; HRMS calcd for C12H12O8Na:
MNa+, 307.0430. Found: MNa+, 307.0438.
=
plate): 2980, 2952 (Ar C–H stretch), 1765, 1720 (C O), 1630,
−1
1
=
=
1599 (C C, ar), 1438 (C C) cm ; H NMR (CDCl3) d 1.33
(3H, d, J 6.4 Hz, CH3), 2.29 (3H, s, CH3), 3.76 (3H, s, CH3),
3.89 (3H, s, CH3), 4.39 (1H, q, J 6.4 Hz, CH), 4.35 (1H, d, J
12.3 Hz, CHH), 4.47 (1H, d, J 12.3 Hz, CHH), 5.91 (1H, d, J
2-(2-Acetoxy-5-methoxycarbonyl-(R)-benzyloxy)-succinic acid
dimethyl ester 43
=
=
1.2 Hz, C CHH), 6.29 (1H, d, J 1.2 Hz, C CHH), 7.13 (1H, d,
J 8.4 Hz, ArH, H-5), 7.98 (1H, dd, J 2.2, 8.4 Hz, ArH, H-6), 8.14
(1H, d, J 2.2 Hz, ArH, H-2); 13C NMR (CDCl3) d 21.2, 22.2,
52.2, 52.6, 66.0, 74.1, 122.9, 125.0, 128.4, 130.6, 131.2, 131.5,
142.5, 152.6, 166.7, 167.0, 169.0; HRMS calcd for C17H20O7Na:
MNa+, 359.1107. Found: MNa+, 347.1104.
Dimethyl-(R)-malate (62 ll 0.47 mmol) was added dropwise to
a solution of 35 (150 mg, 0.52 mmol) in freshly distilled diethyl
ether (2 ml). Silver oxide (242 mg, 1.05 mmol) was added and
◦
the reaction heated at reflux (42 C) under nitrogen. After 1 h
of stirring additional silver oxide (75 mg) was added and the
reaction stirred at reflux for a further 2.5 h before stirring at
22 ◦C for 42 h. Work up was carried out as described for 36 and
the product was purified by column chromatography (eluent 2 :
1 v/v petroleum ether–ethyl acetate) to afford 43 as a colourless
oil (64 mg, 37%).
3-[1-(1-Carboxy-ethyl)-vinyloxymethyl]-4-hydroxy-benzoic
acid 18
Potassium hydroxide (107 mg, 1.90 mmol) was added to a
solution of 41 (40 mg, 0.19 mmol) in THF–water (1 : 1 v/v,
2 ml) and the reaction heated to 50 C for 12 h. Acidification
and extraction was carried out as described for 13 to afford the
desired diacid 18 as a white solid (20 mg, 63%).
mmax. (ATR): 3183 (br, acid OH stretch and ar OH), 2948 (Ar C–
RF [2 : 1 petroleum ether : ethyl acetate] = 0.37; mmax. (NaCl
◦
=
plate): 2956, 2918 (Ar C–H stretch), 1758, 1739 (C O), 1613
−1
1
=
(C C, ar) cm ; H NMR (CDCl3) d 2.32 (3H, s, CH3), 2.77
(2H, dd, J 5.1, 7.6 Hz, CH2), 3.66 (3H, s, CH3), 3.75 (3H, s,
CH3), 3.89 (3H, s, CH3) 4.34 (1H, dd, J 5.1, 7.6 Hz, CH), 4.48
(1H, d, J 11.9 Hz, CHH), 4.75 (1H, d, J 11.9 Hz, CHH), 7.14
(1H, d, J 8.4 Hz, ArH, H-5), 7.99 (1H, dd, J 2.1, 8.4 Hz, ArH, H-
6), 8.12 (1H, d, J 2.1 Hz, ArH, H-2); 13C NMR (CDCl3) d 23.0,
39.8, 54.1, 54.4, 54.4, 69.8, 76.7, 124.8, 130.1, 131.8, 132.9, 133.9,
154.7, 168.3, 170.9, 172.5, 173.6; HRMS calcd for C17H20O9Na:
MNa+, 391.1005. Found: MNa+, 391.1019.
−1
=
=
=
H stretch), 1650 (C O, str), 1586 (C C, ar), 1427 (C C) cm ;
1H NMR (d6-acetone) d 1.36 (3H, d, J 6.4 Hz, CH3), 4.48 (1H,
q, J 6.4 Hz, CH), 4.53 (1H, d, J 12.5 Hz, CHH), 4.62 (1H, d, J
=
12.5 Hz, CHH), 5.98 (1H, d, J 1.4 Hz, C CHH), 6.30 (1H, d, J
1.4 Hz, C CHH), 6.91 (1H, d, J 8.4 Hz, ArH, H-5), 7.83 (1H,
dd, J 2.1, 8.4 Hz, ArH, H-6), 8.05 (1H, d, J 2.1 Hz, ArH, H-2);
13C NMR (d6-acetone) d 22.3, 67.2, 75.1, 116.2, 123.0, 125.1,
126.3, 132.0 (× 2), 144.0, 160.5, 168.0, 168.1; HRMS calcd for
C13H14O6Na: MNa+, 289.0688. Found: MNa+, 289.0677.
=
2-(2-Acetoxy-5-carboxy-(R)-benzyloxy)-succinic acid 20
Potassium hydroxide (160 mg, 2.82 mmol) was added to a
solution of 43 (65 mg, 0.18 mmol) in THF–water (1 : 1 v/v,
2 ml) and the reaction heated to 50 C for 12 h. Acidification
and extraction was carried out as described for 13 to afford the
desired triacid 20 as a white solid (42 mg, 85%).
mmax. (ATR): 3251, 2900 (br, acid OH stretch and ar OH), 2948,
2-(2-Acetoxy-5-methoxycarbonyl-(S)benzyloxy)-succinic acid
dimethyl ester 42
◦
Dimethyl-(S)-malate (62 ll, 0.47 mmol) was added dropwise to
a solution of 35 (150 mg, 0.52 mmol) in freshly distilled diethyl
ether (2 ml). Silver oxide (242 mg, 1.05 mmol) was added and
=
=
2869 (Ar C–H stretch), 1728, 1691 (C O, str), 1670, 1618 (C C,
ar) cm−1; 1H NMR (d6-acetone) d 2.78 (1H, dd, J 8.5, 16.3 Hz,
CH), 2.93 (1H, dd, J 3.8, 16.3 Hz, CH), 4.54 (1H, dd, J 3.8,
8.5 Hz, CH), 4.66 (1H, d, J 11.4 Hz, CHH), 4.84 (1H, d, J
11.4 Hz, CHH), 6.91 (1H, d, J 8.4 Hz, ArH, H-5), 7.98 (1H, dd,
◦
the reaction heated at reflux (42 C) under nitrogen. After 1 h
of stirring additional silver oxide (75 mg) was added and the
reaction stirred at reflux for a further 2.5 h before stirring at
22 ◦C for 42 h. Work up was carried out as described for 36 and
the product was purified by column chromatography (eluent 2 :
1 v/v petroleum ether–ethyl acetate) to afford 42 as a colourless
oil (90 mg, 52%).
J 1.8, 8.4 Hz, ArH, H-6), 7.95 (1H, d, J 1.8 Hz, ArH, H-2); 13
C
NMR (d6-acetone) d 36.0, 68.2, 73.3, 114.7, 120.4, 122.4, 130.6,
130.6, 159.4, 166.0, 169.8, 172.6; HRMS calcd for C12H12O8Na:
MNa+, 307.0430. Found: MNa+, 307.0420.
RF [3 : 1 petroleum ether : ethyl acetate] = 0.17; mmax. (NaCl
=
plate): 2955, 2855 (Ar C–H stretch), 1756, 1731 (C O), 1612
=
−1
1
(C C, ar) cm . H NMR (CDCl3) d 2.32 (3H, s, CH3), 2.77
(2H, dd, J 5.1, 7.6 Hz, CH2), 3.66 (3H, s, CH3), 3.75 (3H, s,
CH3), 3.89 (3H, s, CH3), 4.34 (1H, dd, J 5.1, 7.6 Hz, CH), 4.48
(1H, d, J 11.9 Hz, CHH), 4.75 (1H, d, J 11.9 Hz, CHH), 7.14
(1H, d, J 8.4 Hz, ArH, H-5), 7.99 (1H, dd, J 2.1, 8.4 Hz, ArH, H-
6), 8.12 (1H, d, J 2.1 Hz, ArH, H-2); 13C NMR (CDCl3) d 23.0,
39.8, 54.1, 54.4, 54.4, 69.8, 76.7, 124.8, 130.1, 131.8, 132.9, 133.9,
154.7, 168.3, 170.9, 172.5, 173.6; HRMS calcd for C17H20O9Na:
MNa+, 391.1005. Found: MNa+, 391.1006.
4-Benzoyl-3-(1-methoxycarbonyl-ethoxy)-benzoic acid methyl
ester 45
To
a solution of methyl-3,4-dihydroxybenzoate (1.11 g,
6.60 mmol) in dry DMF (7 ml) was added sodium hydride
(264 mg, 6.60 mmol, 60% dispersion in oil), followed by
the dropwise addition of methyl-2-bromopropionate (0.74 ml,
6.6 mmol). The reaction was stirred at 22 ◦C for 5 days at which
point the solvent was removed in vacuo. The crude residue was
purified by column chromatography (eluent: 3 : 1 v/v petroleum
ether–ethyl acetate) to give a mixture of the two mono-alkylated
products (0.28 g, 17%), which was used in the next step.
The mixture of monoalkylated products (0.28 g,◦1.12 mmol)
were dissolved in pyridine (7 ml) and cooled to 0 C. Benzoyl
chloride (0.195 ml, 1.68 mmol) was added dropwise and the
reaction was stirred at 22 ◦C for 24 h. The reaction was diluted
with ethyl acetate (15 ml) and washed sequentially with saturated
aqueous NH4Cl solution (20 ml), brine (20 ml), dried (MgSO4)
and the solvent removed in vacuo. Purification by column
chromatography (eluent 3 : 1 v/v petroleum ether–ethyl acetate)
gave the desired mono-benzoylate 50 as a white solid (0.08 g, 4%
over the two steps).
2-(2-Acetoxy-5-carboxy-(S)-benzyloxy)-succinic acid 19
Potassium hydroxide (141 mg, 2.51 mmol) was added to a
solution of 42 (58 mg, 0.16 mmol) in THF–water (1 : 1 v/v,
◦
2 ml) and the reaction heated to 50 C for 12 h. Acidification
and extraction was carried out as described for 13 to afford the
desired triacid 19 as a white solid (49 mg, quant.).
mmax. (ATR): 3254, 2900 (br, acid OH stretch and ar OH), 2948,
=
=
2607 (Ar C–H stretch), 1729, 1693 (C O, str), 1668, 1618 (C C,
ar) cm−1; 1H NMR (d6-acetone) d 2.78 (1H, dd, J 8.5, 16.3 Hz,
CH), 2.93 (1H, dd, J 3.8, 16.3 Hz, CH), 4.54 (1H, dd, J 3.8,
8.5 Hz, CH), 4.66 (1H, d, J 11.4 Hz, CHH), 4.84 (1H, d, J
11.4 Hz, CHH), 6.91 (1H, d, J 8.4 Hz, ArH, H-5), 7.98 (1H, dd,
1H NMR (CDCl3) d 1.51 (3H, d, J 6.8 Hz, CH3), 3.90 (3H, s,
CH3), 4.81 (1H, q, J 6.7 Hz CH), 7.26 (1H, d, J 8.5 Hz, ArH,
J 1.8, 8.4 Hz, ArH, H-6), 7.95 (1H, d, J 1.8 Hz, ArH, H-2); 13
C
2 2 8 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 2 7 1 – 2 2 8 1

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H-5,), 7.50 (2H, m, ArH), 7.59 (1H, d, J 1.7 Hz ArH, H-2),
7.62 (1H, m, ArH), 7.74 (1H, dd, J 8.5, 1.7 Hz, ArH, H-6,
ArH), 8.20 (2H, m, ArH); NOE: irridiation at d 4.81 ppm caused
enlargement of the signal at d 7.59 ppm and vice versa. 13C NMR
(CDCl3) d 20.3, 54.2 (× 2), 76.0, 118.0, 125.3, 125.8, 130.5, 130.7,
131.0, 132.2, 135.6, 146.8, 151.4, 166.2, 168.0, 173.5.
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10 H. Zalkin and J. L. Smith, Adv. Enzymol. Relat. Areas Mol. Biol.,
1998, 72, 87.
3-(1-Carboxy-ethoxy)-4-hydroxy-benzoic acid 21
Potassium hydroxide (38 mg, 0.67 mmol) was added to a solution
of 45 (60 mg, 0.17 mmol) in THF–water (1 : 1 v/v, 2.6 ml). The
reaction was stirred at 22 ◦C for 4 h before dilution with water
(5 ml). The aqueous fraction was washed with ethyl acetate
(10 ml), before acidifying to pH 1 with 1 M HCl. The aqueous
fraction was extracted with ethyl acetate (2 × 10 ml), before the
combined organic fractions were dried (MgSO4) and the solvent
removed in vacuo to afford the desired product and benzoic acid.
HPLC purification and lyophilisation gave the desired diacid 23
as a white solid (30 mg, 80%).
11 C. T. Walsh, M. D. Erion, A. E. Walts, J. J. Delany and G. A.
Berchtold, Biochemistry, 1987, 26, 4734–4745.
12 M. C. Kozlowski, N. J. Tom, C. T. Seto, A. M. Sefler and P. A.
Bartlett, J. Am. Chem. Soc., 1995, 117, 2128–2140.
13 A. A. Morollo and M. J. Eck, Nat. Struct. Biol., 2001, 8, 243–247.
14 H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H.
Weissig, N. Shindyalov and P. E. Bourne, Nucleic Acids Res., 2000,
28, 235–242.
1H NMR (d6-acetone) d 1.64 (3H, d, J 6.9 Hz, CH3), 4.90 (1H,
q, J 6.9 Hz, CH), 6.92 (1H, d, J 8.3 Hz, ArH, H-5), 7.60 (1H, d,
J 1.9 Hz, ArH, H-2), 7.62 (1H, dd, J 8.3, 1.9 Hz, ArH, H-6); 13
C
NMR (d6-acetone) d 17.0, 73.7, 114.4, 116.2, 120.9, 124.4, 144.5,
151.3, 165.2, 172.2; HRMS calcd for C10H10O6: M+, 226.0477.
Found: M+, 226.0484.
15 Tripos SYBYL version 6.5; Tripos, Inc., 1699, South Hanley Road,
St Louis, Missouri, 63144, USA.
16 G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, J. Mol.
Biol., 1997, 267, 727–748.
Acknowledgements
17 R. Gopinath and B. K. Patel, Org. Lett., 2000, 2(5), 577–579.
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Chem., 1976, 251, 941–944; (b) R. Bauerle, J. Hess and S. French,
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We would like to thank the Gates Cambridge Trust for PhD
funding (R. J. P. and E. M. M. B.) and the BBSRC.
19 Enzyme Kinetics! Pro (version: 2.33), 1999–2000, SynexChem, LLC,
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