Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI) inhibitors designed to probe plasticity in the active site

Article abstract of DOI:10.1039/c2ob26736e

Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro.

Full text of DOI:10.1039/c2ob26736e

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PAPER
Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI)
inhibitors designed to probe plasticity in the active site†
Alexandra Manos-Turvey,a Katie M. Cergol,a Noeris K. Salam,a Esther M. M. Bulloch,b Gamma Chi,b
Angel Pang,c,d Warwick J. Britton,c,d Nicholas P. West,c,d Edward N. Baker,b J. Shaun Lottb and
Richard J. Payne*a
Received 4th September 2012, Accepted 16th October 2012
DOI: 10.1039/c2ob26736e
Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the
biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of
M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed
large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI
active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate
scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most
compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the
micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity
in vitro.
in the biosynthesis of tryptophan, folate and siderophores,
respectively. Given the importance of these secondary metab-
Introduction
Tuberculosis (TB), caused by infection with the bacterium Myco-
bacterium tuberculosis, is responsible for significant morbidity
and mortality worldwide. In 2010, TB caused 1.5 million deaths
and a further 8.8 million new M. tuberculosis infections were
identified.1 The emergence of multi-drug and extensively-drug
resistant strains of M. tuberculosis, resistant to first or first and
second line therapies, respectively, have now been reported on
every major continent.2 There is thus an urgent need for the
development of anti-tubercular agents with novel modes of
action.
M. tuberculosis salicylate synthase (SS), or MbtI, belongs to a
family of chorismate-utilising enzymes (Scheme 1).3 Enzymes
of this class, present in bacteria, fungi, plants and apicomplexan
parasites, are responsible for the conversion of chorismate (1)
into a range of essential aromatic building blocks. For example
anthranilate synthase (AS), 4-amino-4-deoxychorismate (ADC)
synthase and isochorismate synthase (IS) catalyse the first steps
olites, there has been significant attention directed toward the
development of inhibitors of these enzymes.4–10
MbtI, like other salicylate synthases, is responsible for the
conversion of chorismate (1) into salicylate (2) through an
enzyme bound intermediate, isochorismate (3) (Scheme 2).3,11 In
M. tuberculosis, generation of salicylate is the first committed
step towards the biosynthesis of the siderophores mycobactin
T (4) and carboxymycobactin T (not shown), responsible for
sequestration of iron from the host.12–15 Due to the essential
nature of iron for the growth and continued survival of M. tuber-
culosis, MbtI represents an appealing target for the development
of new TB drug candidates.
We recently reported the first inhibitors of MbtI (e.g. 5 and 6
in Fig. 1).16 These compounds, based on a 2,3-dihydroxybenzo-
ate scaffold, to mimic the intermediate isochorismate (3), exhib-
ited low micromolar inhibition of the enzyme (Ki = 11–21 μM).
More recently, Aldrich and co-workers have reported a different
class of non-competitive benzimidathiazole-based MbtI inhibi-
tors (IC50 = 7–9 μM) based on a high throughput screen of
>100 000 commercial compounds.17
aSchool of Chemistry, Building F11, The University of Sydney,
Camperdown, NSW 2006, Australia.
E-mail: richard.payne@sydney.edu.au; Fax: +61 2 9351 3329;
Tel: +61 2 9351 5877
bMaurice Wilkins Centre for Molecular Biodiscovery and Laboratory of
Structural Biology, School of Biological Sciences, University of
Auckland, Auckland, 1010, New Zealand
Results and discussion
cCentenary Institute, Newtown, NSW 2042, Australia
dSydney Medical School, University of Sydney, NSW 2006, Australia
†Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra of all novel compounds and molecular dockings of 25–38.
See DOI: 10.1039/c2ob26736e
Prior inhibitors synthesised in our laboratory (including 5 and 6)
were recently screened against M. tuberculosis (H37Ra strain)
in vitro. Unfortunately, all compounds exhibited poor antibacterial
activity (MIC50 > 1 mM). We hypothesised that the lack of anti-
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Scheme 1 Transformations achieved by the chorismate-utilising enzymes: salicylate synthase (SS), anthranilate synthase (AS), isochorismate
synthase (IS) and 4-amino-4-deoxychorismate (ADC) synthase.
Scheme 2 Activity of MbtI, the salicylate synthase (SS) of Mycobacterium tuberculosis, showing the enzyme bound intermediate, isochorismate 3,
and siderophore product, mycobactin T (4).
esters 7 and 8). These results provided impetus for the design
and synthesis of a new series of inhibitors that target MbtI and
perhaps other chorismate-utilising enzymes operating in
M. tuberculosis which possess antibacterial activity.
Inhibitor design
We recently obtained co-crystal structures of MbtI with inhibitors
5 and 6, bearing 3-methylacrylate- and 3-phenylacrylate side
chains bound in the active site.19 These structures have provided
Fig. 1 Previously reported inhibitors of M. tuberculosis salicylate
synthase with Ki values against MbtI and MIC50 values against M. tuber-
culosis.16 NB: 7 and 8 are prodrugs of 5 and 6 and were screened
against M. tuberculosis in a whole cell assay.
new insights into the binding mode of these compounds, notably
through the observation of large inhibitor-induced rearrange-
ments of the protein structure around the active site. It is impor-
tant to note that this flexibility of active site residues was not
taken into account in previous rigid-receptor docking predictions
used for guiding prior inhibitor design and, as such, led to a pre-
dicted binding mode (from prior docking studies) which was sig-
nificantly different to that observed in the co-crystal structure
(see Fig. 2 for comparison).16
For example, the inhibitor-bound MbtI crystal structures
clearly showed the C3 side chain carboxylate oriented towards
the metal binding site, whilst the C1 carboxylate is positioned
between Arg405 and Gly419 to form favourable ionic and
hydrogen bonding interactions with these residues, respectively
tubercular activity was owing to the highly hydrophilic nature of
the molecules together with the negative charge from the di-
carboxylate moieties present in these compounds, thus making it
difficult for these to efficiently penetrate the waxy mycobacterial
cell wall.18 We therefore evaluated methyl ester variants of these
compounds in the whole cell assays against the bacterium
(designed as prodrugs). This led to a significant enhancement in
potency, compared with the free acids, with MIC50 values in the
micromolar range (see Fig. 1 for MIC50 values of dimethyl
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(Fig. 3a–c).19 Relative to the MbtI structure used for docking
studies, there were changes in the sidechain positions for resi-
dues Glu252, Thr361 and Arg405 that helped accommodate the
extended enol sidechain of 5 in this binding mode. Binding of 6
(bearing the bulky 3-phenylacrylate side chain) as the E or Z
isomer required much more extensive rearrangements of the
MbtI structure. This was achieved by significant movement of
the peptide backbone in regions adjacent to the phenyl substitu-
ent (Fig. 3).19 Specifically, residues 268–270 (within a strand of
a β-sheet) were significantly displaced by the larger side chain
containing compounds, with the greatest displacement observed
in the 3-phenylacrylate-based inhibitors (Fig. 3a–c). The co-
crystal structures of MbtI with inhibitor 6 bound showed that
both E and Z isomers could be accommodated in the active site
cleft (Fig. 3b and c).19 The side chains of these two diastereo-
isomers occupied very different regions of the active site. The
most striking observation was that a three-stranded β-sheet that
lines the enol-pyruvyl binding pocket was displaced from the
active site to accommodate the phenyl side chains of both the
E- and the Z-isomers. The local effect of this rearrangement was
the opening of a cavity in a region previously occupied by the
Fig. 2 Predicted binding mode of inhibitor 5 in the active site of MbtI
(pink) overlaid with the binding mode observed in the co-crystal struc-
ture (blue, PDB ID: 3VEH). NB: The Mg2+ ion (yellow) was not
present in the crystal structures and was modelled into the active site.
Fig. 3 Co-crystal structure of MbtI with (a) inhibitor 5 bound, (b) inhibitor (Z)-6 bound, (c) inhibitor (E)-6 bound and (d) aromatic inhibitor bearing
native enol-pyruvyl side chain bound.19 PDB ID: (a) 3VEH, both (b) and (c) 3RV6, (d) 3ST6.
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β-strand. The Z-isomer further induces movement of Glu252 and
Thr361 again facilitated by changes in the position of the
peptide backbone. The Z-isomer also revealed a further alterna-
tive positioning for Arg405, in which it twists around to form a
direct salt-bridge to the C1 carboxylate. This interaction is not
observed in the E-isomer or the crystal structure with 5 (bearing
a 3-methylacrylate side chain) bound. Taken together, these obser-
vations indicate significant plasticity in the active site of MbtI.
Interestingly, the binding mode (and movement of active site
residues) seen for inhibitors 5 and 6 is not observed when the
native enol-pyruvyl side chain (present in chorismate and iso-
chorismate) is incorporated into an inhibitor (see Fig. 3d for co-
crystal structure). In this case the inhibitor binds in the same
mode predicted by our previous docking studies (with the C1
carboxylate interacting with the Mg2+ ion and the C3 side chain
carboxylate forming interactions with the side chains of Arg405
and Tyr305). This compound is over an order of magnitude less
potent as an inhibitor of MbtI compared to 5 and 6 (with
extended side chains) suggesting that there are significantly
improved interactions with active site residues when compounds
bind in the alternate mode observed for compounds 5 and 6 pos-
sessing extended enol side chains at C3.
Given these observations we were interested in exploiting the
plasticity of the active site and the inducible cavities by design-
ing inhibitors bearing a range of meta-, ortho- or para-substitu-
ents around the aryl ring of the 3-phenylacrylate side chain of
inhibitor 6. This would enable a more detailed understanding of
which functional groups could be incorporated to facilitate
additional interactions with the induced pocket and hopefully
provide inhibitors with increased potency against the enzyme.
Use of substituted aromatic side chains in the proposed library
was further justified by the resulting increase in sidechain lipo-
philicity, which was expected to increase anti-tubercular activity
as has been reported previously.20
The proposed inhibitor library was docked into the active site
of the co-crystal structure of MbtI with (Z)-6 and (E)-6 bound
(PDB ID: 3RV6) using Glide® (Schrödinger).21 Bromo-, chloro-
, hydroxyl-, methyl- or trifluoromethyl substituents on the aryl
ring of the C3 side chain were chosen to investigate the effect of
ortho-, meta- and para-substitution (see ESI†). In the case of the
para-substituted trifluoromethylphenylacrylate side chain,
in silico studies suggested a number of unfavourable steric in-
teractions and therefore this compound was removed from the
final target library. The remainder of the inhibitors (14 in total in
both E- and Z-diastereomeric configurations) showed favourable
binding modes in the active site as gauged by visual inspection
and docking scores (see ESI†). We therefore chose to embark on
the synthesis of these compounds.
Synthesis
Synthesis of the inhibitors began from key phosphonate 10.16
This was obtained via a rhodium-carbenoid insertion between
diazophosphonoacetate and methyl 2,3-dihydroxybenzoate 9
(Scheme 3). The key phosphonate 10 was then reacted with a
range of substituted benzaldehydes via a Horner–Wadsworth–
Emmons (HWE) reaction. Specifically, phosphonate 10 was
deprotonated with lithium hexamethyldisilazide (LiHMDS) at
−78 °C and reacted with the range of aldehydes to give the
desired dimethyl esters 11–24 in good to excellent yields
(50–87%). It is important to note that 11–24 were obtained as
mixtures of Z- and E-diastereoisomers and, in most cases, could
not be easily separated via column chromatography. Nonetheless,
it was possible in some cases to isolate fractions of either pure
Z (compounds 12, 13, 15, and 24) or Z-rich fractions, however,
E-rich fractions could not be obtained.
Saponification of methyl esters 11–24 with aqueous potassium
hydroxide followed by acidification with 1 M hydrochloric acid
Scheme 3 Synthesis of inhibitors 25–38.
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gave the desired inhibitors 25–38 in 38–86% yield (Scheme 3).
It should be noted that in some cases the Z : E isomer ratios
observed do not directly reflect the ratios of the corresponding
di-methyl esters used in the preceding saponification reactions.
This change in diastereomeric ratio was owing to acid-catalysed
olefin isomerisation during work-up (see diastereomeric ratios in
parentheses).
Interestingly, the hydroxyl-substituted compounds 34–36 pos-
sessed almost identical inhibition of the enzyme irrespective of
substitution around the aryl ring of the phenylacrylate side chain.
The most potent of the inhibitors, the o-Cl and o-Br substituted
compounds (29 and 26) may be indicative of the size, electronics
and positioning of the substituent which is best tolerated in the
ligand-induced pocket.
Inhibition of M. tuberculosis growth in vitro
MbtI inhibition studies
Having established the inhibition of MbtI by inhibitors 25–38,
we were next interested in assessing the activity of these com-
pounds against M. tuberculosis growth in vitro. This was carried
out using a resazurin reduction microplate assay, previously
described.22–24 As our prior assays with the dicarboxylate inhibi-
tors showed no activity against M. tuberculosis, thought to be
due to inefficient permeability of the mycobacterial cell wall, the
di-methyl ester precursors (11–24) were screened against the
bacterium (Table 2).18
With compounds 25–38 in hand, these were next screened
against MbtI using a lactate dehydrogenase coupled assay as
reported previously.16 All compounds exhibited competitive,
reversible inhibition of MbtI with inhibition constants (Ki)
ranging from 45–186 μM (see Table 1). As such, all compounds
possessed a reduced inhibitory potency when compared to
unsubstituted inhibitor 6 and also exhibited a relatively flat struc-
ture–activity profile against the enzyme.
These results suggest that although the aryl moiety of the side-
chain is accommodated into the active site via local movement
of the backbone and side chain moieties of the enzyme,
additional interactions with active site residues do not appear to
be facilitated through substitution. Substitution at the ortho-posi-
tion appeared to be better tolerated in the active site of MbtI than
para- or meta-substitution leading to slightly better inhibition of
the enzyme. This is highlighted by the halogen-substituted
inhibitors (25–30) where compounds bearing ortho-Cl (29) and
ortho-Br (26) substituted aryl sidechains possessed a two fold
increase in activity when compared with the meta- and para-sub-
stituted compounds (25, 27, 28 and 30).
The vast majority of these compounds were significantly more
potent than all previously screened MbtI inhibitors against
M. tuberculosis with MIC50 values ranging from 25–460 μM
(Table 2). Despite the increased potency against M. tuberculosis
in vitro, there appeared to be no significant correlation between
the potency against MbtI and the antibacterial activity of the cor-
responding di-methyl ester. This discrepancy may well be the
result of more than one enzymatic target of M. tuberculosis
being inhibited. This is most striking in the example of the
o-CF3 compound 38 which was a moderate inhibitor of MbtI (Ki =
125 μM) but was found to be the most potent inhibitor of bac-
terial growth with an MIC50 of 25 μM (for di-methyl ester 24).
It is also feasible that these compounds exert their antibacterial
activity via the inhibition of more than one chorismate-utilising
enzyme in M. tuberculosis.4–10 Given the range of enzymes that
are known to convert chorismate to their respective products via
Inhibitors possessing methyl and trifluoromethyl substituted
aryl side chains (Table 1: 31–33 and 37–38) were consistently
less potent inhibitors (albeit by a small difference) of the enzyme
with Ki values between 110–186 μM.
Table 1 Inhibition of MbtI by 3-phenylacrylate-based inhibitors 25–38a
Compound
Ki MbtI (μM)
Compound
Ki MbtI (μM)
Compound
Ki MbtI (μM)
25 (m-Br)
28 (m-Cl)
31 (m-Me)
34 (m-OH)
37 (m-CF3)
86 7.8
71 5.5
186 14
97 7.9
153 11
26 (o-Br)
29 (o-Cl)
32 (o-Me)
35 (o-OH)
38 (o-CF3)
51 3.7
45 4.3
110 8.8
92 9.7
125 13
27 (p-Br)
30 (p-Cl)
33 (p-Me)
36 (p-OH)
6 (Ph)
70 7.0
78 6.6
170 20
92 13
21
5
a Kinetic constants: MbtI KM = 2.2 0.2 μm, kcat = 0.8 0.1 min−1
.
Table 2 Inhibition of M. tuberculosis by 3-phenylacrylate-based methyl ester prodrugs 11–24a
Compound
MIC50 (μM)
Compound
MIC50 (μM)
Compound
MIC50 (μM)
11 (m-Br)
14 (m-Cl)
17 (m-Me)
20 (m-OH)
23 (m-CF3)
248 26
204 18
263 51
460 36
180 19
12 (o-Br)
15 (o-Cl)
18 (o-Me)
21 (o-OAc)
24 (o-CF3)
95 29
118 32
140 47
89 15
13 (p-Br)
16 (p-Cl)
19 (p-Me)
22 (p-OAc)
8 (Ph)
188 14
203 24
270 33
120 18
277 19
25
5
a Compounds were tested in an Alamar blue (Resazurin) assay, rifampicin MIC50 = 1.3 0.4 nM, isoniazid MIC50 = 198 4.4 nM. Data represent
mean SD from three replicate assays for M. tuberculosis.
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similar mechanistic pathways it is possible that these are inhib-
ited by the compounds synthesised here.8,25 This theory may be
further supported by the recent characterisation of a 4-amino-
4-deoxychorismate lyase and an anthranilate synthase in M. tuber-
culosis.26,27 Furthermore, previously investigated lipophilic
inhibitors have exhibited greater potency against an anthranilate
synthase (from Serratia marcescens) compared with MbtI.16
Work is ongoing to investigate if inhibition of multiple biosyn-
thetic pathways is occurring.
It should be noted that we conducted an additional screen of a
selection of compounds against the H37Ra strain of M. tubercu-
losis under iron limiting conditions, where MbtI is known to be
highly over-expressed (see ESI†). Under these conditions each
of the inhibitors were significantly less effective at inhibiting the
growth of the bacillus. MbtI is expected to be essential for viabi-
lity under these iron limiting conditions. One interpretation of
these results is that they stem from the over-expression of MbtI
under iron limitation which would therefore require more com-
pound for inhibition of growth. A more detailed investigation of
these effects will be the subject of future work in our
laboratories.
recorded on a Bruker 7T Fourier Transform Ion Cyclotron Reso-
nance (FTICR) mass spectrometer. Melting points were
recorded using a Stanford Research Systems OptiMelt Auto-
mated Melting Point System. Infrared (IR) absorption spectra
were recorded on a Bruker Alpha Spectrometer with attenuated
total reflection (ATR) capability and were processed with OPUS
6.5 software.
Analytical reversed-phase HPLC was performed on a Waters
System 2695 separations module with an Alliance series column
heater at 30 °C and 2996 photodiode array detector. Inhibitors
25–38 were analysed using a Waters Sunfire 5 μm, 2.1 ×
150 mm column (C18) at a flow rate of 0.2 mL min−1 using a
mobile phase of 0.1% TFA in H2O (Solvent A) and 0.1% TFA
in CH3CN (Solvent B) and a linear gradient of 0 to 100% B over
40 min. Results were analyzed with Waters Empower software.
The purity of the final inhibitors was shown to be >97% by
analytical HPLC.
Materials. Analytical thin-layer chromatography (TLC) was
performed on commercially prepared silica plates (Merck Kiesel-
gel 60 0.25 mm F254). Flash column chromatography was per-
formed using 230–400 mesh Kieselgel 60 silica eluting with
distilled solvents as described. Ratios of solvents used for TLC
and column chromatography are expressed in v/v as specified.
Compounds were visualized by UV light at λ 254 nm or by
using vanillin or cerium molybdate stain. Commercial materials
were used as received, unless otherwise noted. Dichloromethane
and methanol were distilled from calcium hydride and THF and
diethyl ether were distilled over sodium/benzophenone. Anhy-
drous DMF was purchased from Sigma–Aldrich.
Conclusions
Recent co-crystal structures of MbtI were used to guide the
design of a second generation library of novel phenylacrylate-
derived inhibitors bearing a range of substituted aromatic side
chains in order to probe for additional interactions within an
inducible pocket. These compounds proved to be slightly less
potent against MbtI (inhibition constants in the mid-micromolar
range) compared with previous inhibitors synthesised in our lab-
oratory suggesting that, although these large groups could be
accommodated by movement of active site residues, no further
interactions were made. Dimethyl ester variants of these com-
pounds displayed significantly improved activity against the
growth of M. tuberculosis in vitro. The most potent compound in
this series was o-CF3-derived compound 24 which exhibited an
MIC50 of 25 μM against M. tuberculosis and will serve as a lead
for the development of analogues. Future work in our labora-
tories will aim to assess the inhibition of other chorismate-utilis-
ing enzymes present in M. tuberculosis by these and other
compounds.
Procedure A: Horner–Wadsworth–Emmons reaction
Lithium bis(trimethylsilyl)amide (2 equiv. 1.0 M soln. in THF)
was added dropwise over 5 min to a stirred solution of phospho-
nate 10 (1 equiv.) in THF (6 mL mmol−1) at −78 °C. The
mixture was stirred for a further 5 min before dropwise addition
of aldehyde (2 equiv.). The mixture was stirred for 2–6 h at
−78 °C before warming to rt. The reaction was quenched
through the dropwise addition of saturated aqueous NH4Cl sol-
ution (1–2 mL) followed by extraction of the aqueous layer with
EtOAc (3 × 50 mL). The combined organic fractions were dried
(Na2SO4) and the solvent was removed in vacuo. The product
was purified via column chromatography to afford diesters as
mixtures of diastereomers.
Experimental
General synthesis procedures
Methyl 3-(1-(3-bromophenyl)-3-methoxy-3-oxoprop-1-en-2-yloxy)-
2-hydroxybenzoate 11. Phosphonate 10 (150 mg, 0.43 mmol)
and m-bromobenzaldehyde (100 μL, 0.86 mmol) were reacted
for 2 h at −78 °C according to Procedure A. The product was
purified by column chromatography (eluent: hexane–EtOAc
90 : 10 v/v) to afford diester 11 as a mixture of diastereomers as
a yellow oil (147 mg, 84%, 50 : 50 Z/E). Rf [hexane : EtOAc 5 : 1
v/v] = 0.29; IR νmax (ATR): 3118 (br), 2953, 1729, 1677, 1438,
1248 cm−1; Z: 1H NMR (400 MHz, CDCl3) δ 11.05 (1H, s,
OH), 7.86 (1H, s, CvCHPh), 7.71 (1H, app. d, J 7.9 Hz, Ar-H),
7.55 (1H, dd, J 1.2, 8.0 Hz, H-6), 7.45–7.38 (1H, m, Ar-H),
7.27–7.17 (2H, m, Ar-H), 6.96 (1H, app. d, J 8.0 Hz, H-4), 6.74
(1H, t, J 8.0 Hz, H-5), 3.96 (3H, s, CO2Me), 3.76 (3H, s,
NMR spectra were recorded at 300 K using Bruker Avance
DRX200, DRX300, DPX400, or 500 spectrometers at a fre-
quency of 200.1, 300.2, 400.2 or 500.2 MHz, respectively. H
NMR chemical shifts are reported in parts per million (ppm) and
are referenced to solvent residual signals: CDCl3 (δ = 7.26 ppm),
1
1
MeOD (δ = 3.31 ppm) or D2O (δ = 4.79 ppm). H NMR data
are reported as chemical shift (δH), relative integral, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of
doublets, ddd = doublet of doublet of doublets), coupling con-
stant (J in Hz), and assignment where possible. Low-resolution
mass spectra were recorded on a Finnigan LCQ Deca ion trap
mass spectrometer (ESI). High-resolution mass spectra were
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1
CO2Me); E: H NMR (400 MHz, CDCl3) δ 11.10 (1H, s, OH),
13C NMR (100 MHz, CDCl3) δ 170.7 (CvO), 163.7 (CvO),
152.0 (C), 144.8 (C), 140.7 (C), 132.1 (2 × CH), 131.9 (2 ×
CH), 131.4 (C), 125.9 (CH), 124.1 (C), 123.9 (CH), 120.2 (CH),
118.6 (CH), 113.8 (C), 52.7 (CH3), 52.7 (CH3); E: Rf [hexane :
7.66 (1H, dd, J 1.4, 8.1 Hz, Ar-H), 7.45–7.38 (2H, m, Ar-H +
H-6), 7.27–7.17 (3H, m, Ar-H + H-4), 6.87 (1H, t, J 8.0 Hz,
H-5), 6.38 (1H, s, CvCHPh), 3.96 (3H, s, CO2Me), 3.73 (3H, s,
CO2Me); (E + Z mix) 13C NMR (100 MHz, CDCl3) 13C NMR
(100 MHz, CDCl3) δ 170.4 (CvO), 170.3 (CvO), 163.3
(CvO), 162.9 (CvO), 152.9 (C), 151.8 (C), 144.6 (C), 144.4
(C), 143.3 (C), 141.1 (C), 135.3 (C), 134.3 (C), 133.0 (C), 132.4
(C), 131.6 (C), 130.6 (C), 130.1 (CH), 129.4 (CH), 128.6 (CH),
127.4 (CH), 125.6 (CH), 125.3 (CH), 125.0 (CH), 123.8 (CH),
122.6 (CH), 121.9 (CH), 120.2 (CH), 119.1 (CH), 118.7 (CH),
118.3 (CH), 114.0 (C), 113.6 (C), 52.6 (CH3), 52.6 (CH3), 52.5
(CH3), 52.3 (CH3); LRMS [M + Na]+ 429.07; HRMS calcd for
C18H15O6BrNa: MNa+, 428.9950. Found: MNa+, 428.9940.
1
EtOAc 5 : 1 v/v] = 0.36; H NMR (400 MHz, CDCl3) δ 11.01
(1H, s, OH), 7.66 (1H, dd, J 1.6, 8.2 Hz, H-6), 7.43 (2H, d,
J 8.6 Hz, Ar-H), 7.24 (1H, dd, J 1.6, 8.2 Hz, H-4), 7.19 (2H, d,
J 8.6 Hz, Ar-H), 6.86 (1H, app. t, J 8.2 Hz, H-5), 6.41 (1H, s,
CvCHPh), 3.97 (3H, s, CO2Me), 3.72 (3H, s, CO2Me); 13C
NMR (100 MHz, CDCl3) δ 170.5 (CvO), 163.2 (CvO), 153.0
(C), 143.9 (C), 143.8 (C) 132.3 (C), 131.3 (2 × CH), 130.7 (2 ×
CH), 125.6 (CH), 125.2 (CH), 122.1 (C), 120.6 (CH), 118.9
(CH), 114.2 (C), 52.7 (CH3), 52.5 (CH3); LRMS [M + Na]+
430.87; HRMS calcd for C18H15O6BrNa: MNa+, 428.9950.
Found: MNa+, 428.9944.
Methyl 3-(1-(2-bromophenyl)-3-methoxy-3-oxoprop-1-en-2-yloxy)-
2-hydroxybenzoate 12. Phosphonate 10 (150 mg, 0.44 mmol)
and o-bromobenzaldehyde (160 mg, 0.87 mmol) were reacted
for 2 h at −78 °C under Procedure A. The product was purified
by column chromatography (eluent: hexane–EtOAc 5 : 1 v/v) to
afford diester 12 as a mixture of diastereomers as a white solid
(140 mg, 78%, 86 : 14 Z/E). IR νmax (ATR): 3106 (br), 2953,
1731, 1679, 1464, 1438, 1250 cm−1; Z: Rf [hexane : EtOAc 5 : 1
Methyl 3-(1-(3-chlorophenyl)-3-methoxy-3-oxoprop-1-en-2-yloxy)-
2-hydroxybenzoate 14. Phosphonate 10 (150 mg, 0.43 mmol)
and m-chlorobenzaldehyde (96 μL, 0.86 mmol) were reacted for
2 h at −78 °C according to Procedure A. The product was
purified by column chromatography (eluent: hexane–EtOAc
90 : 10 v/v) to afford diester 14 as a mixture of diastereomers as
a yellow oil (126 mg, 87%, 56 : 44 Z/E). Rf [hexane : EtOAc 5 : 1
v/v] = 0.29; IR νmax (ATR): 3115 (br), 2955, 1729, 1677, 1438,
1249 cm−1; Z: 1H NMR (400 MHz, CDCl3) δ 11.04 (1H, s,
OH), 7.71 (1H, s, CvCHPh), 7.67–7.62 (1H, m, Ar-H),
7.28–7.15 (4H, m, Ar-H + H-6), 6.97 (1H, app. d, J 7.9 Hz,
H-4), 6.73 (1H, t, J 7.9 Hz, H-5), 3.95 (3H, s, CO2Me), 3.74
(3H, s, CO2Me); E: 1H NMR (400 MHz, CDCl3) δ 11.08 (1H, s,
OH), 7.67–7.62 (1H, m, Ar-H), 7.54 (1H, app. d, J 8.0 Hz, H-6),
7.28–7.15 (4H, m, Ar-H + H-4), 6.85 (1H, t, J 7.9 Hz, H-5),
6.38 (1H, s, CvCHPh), 3.95 (3H, s, CO2Me), 3.71 (3H, s,
CO2Me); (E + Z mix) 13C NMR (100 MHz, CDCl3) δ 170.4
(CvO), 170.3 (CvO), 163.3 (CvO), 162.9 (CvO), 152.9 (C),
151.9 (C), 144.6 (C), 144.4 (C), 143.4 (C), 141.0 (C), 135.0 (C),
134.4 (C), 134.0 (C), 133.7 (C), 130.1 (C), 129.9 (C), 129.5
(CH), 129.2 (CH), 128.8 (CH), 128.3 (CH), 127.8 (CH), 127.0
(CH), 125.6 (CH), 125.3 (CH), 125.1 (CH), 123.8 (CH), 120.2
(CH), 119.3 (CH), 118.7 (CH), 118.3 (CH), 114.0 (C), 113.6
(C), 52.6 (CH3), 52.6 (CH3), 52.5 (CH3), 52.3 (CH3); LRMS
[M + Na]+ 385.07; HRMS calcd for C18H15O6ClNa: MNa+,
385.0455. Found: MNa+, 385.0445.
1
v/v] = 0.35; H NMR (400 MHz, CDCl3) δ 11.05 (1H, s, OH),
8.03 (1H, dd, J 1.4, 7.7 Hz, Ar-H), 7.70 (1H, s, CvCHPh), 7.58
(1H, d, J 7.7 Hz, Ar-H), 7.52 (1H, dd, J 1.1, 8.0 Hz, H-6), 7.23
(1H, app. t, J 7.7 Hz, Ar-H), 7.13 (1H, dt, J 1.4, 7.7, 7.7 Hz, Ar-
H), 6.98 (1H, dd, J 1.1, 8.0 Hz, H-4), 6.73 (1H, app. t, J 8.0 Hz,
H-5), 3.95 (3H, s, CO2Me), 3.78 (3H, s, CO2Me); 13C NMR
(100 MHz, CDCl3) δ 170.6 (CvO), 163.5 (CvO), 152.0 (C),
145.0 (C), 141.4 (C), 133.0 (CH), 132.2 (C), 131.2 (CH), 130.7
(CH), 127.7 (CH), 125.2 (C), 124.8 (CH), 123.8 (CH), 120.5
(CH), 118.5 (CH), 113.7 (C) 52.8 (CH3), 52.6 (CH3); E: Rf
1
[hexane : EtOAc 5 : 1 v/v] = 0.27; H NMR (400 MHz, CDCl3)
δ 11.04 (1H, s, OH), 7.67 (1H, dd, J 1.3, 8.0 Hz, H-6), 7.55
(1H, d, J 7.9 Hz, Ar-H), 7.32 (1H, app. t, J 7.7 Hz, Ar-H),
7.31–7.27 (2H, m, Ar-H + H-4), 7.16–7.11 (1H, m, Ar-H), 6.88
(1H, app. t, J 8.0 Hz, H-5), 6.51 (1H, s, CvCHPh), 3.97 (3H, s,
CO2Me), 3.66 (3H, s, CO2Me); 13C NMR (100 MHz, CDCl3) δ
170.5 (CvO), 163.5 (CvO), 153.1 (C), 144.4 (C), 143.8 (C),
134.6 (C), 130.8 (CH), 129.4 (CH), 126.9 (CH), 125.7 (CH),
125.3 (CH), 125.2 (C), 123.6 (C), 121.5 (CH), 118.9 (CH),
114.1 (C) 52.7 (CH3), 52.4 (CH3); LRMS [M + Na]+ 430.87;
HRMS calcd for C18H15O6BrNa: MNa+, 428.9950. Found:
MNa+, 428.9944.
Methyl 3-(1-(2-chlorophenyl)-3-methoxy-3-oxoprop-1-en-2-yloxy)-
2-hydroxybenzoate 15. Phosphonate 10 (150 mg, 0.44 mmol)
and o-chlorobenzaldehyde (97 μL, 0.87 mmol) were reacted for
16 h at −78 °C according to Procedure A. The product was
purified by column chromatography (eluent: hexane–EtOAc
90 : 10 v/v) to afford diester 15 as a mixture of diastereomers as
a colourless oil (89 mg, 57%, 91 : 9 Z/E); IR νmax (ATR): 3090
(br), 2954, 1729, 1677, 1465, 1438, 1249 cm−1; Z: Rf [hexane :
Methyl 3-(1-(4-bromophenyl)-3-methoxy-3-oxoprop-1-en-2-yloxy)-
2-hydroxybenzoate 13. Phosphonate 10 (150 mg, 0.44 mmol)
and p-bromobenzaldehyde (160 mg, 0.87 mmol) were reacted
for 2 h at −78 °C under Procedure A. The product was purified
by column chromatography (eluent: hexane–EtOAc 5 : 1 v/v) to
afford diester 13 as a mixture of diastereomers as a yellow oil
(110 mg, 64%, 70 : 30 Z/E). IR νmax (ATR): 3114 (br), 2953,
2923, 2851, 1729, 1679, 1464, 1439, 1249 cm−1; Z: Rf [hexane :
1
EtOAc 5 : 1 v/v] = 0.33; H NMR (400 MHz, CDCl3) δ 11.06
(1H, s, OH), 8.07 (1H, app. d, J 7.4 Hz, Ar-H), 7.75 (1H, s,
CvCHPh), 7.52 (1H, d, J 8.1 Hz, Ar-H), 7.38 (1H, app. d,
J 7.4 Hz, H-6), 7.20 (2H, app. quintet, J 7.3 Hz, Ar-H), 6.98
(1H, app. d, J 7.9 Hz, H-4), 6.73 (1H, app. t, J 7.9 Hz, H-5),
3.94 (3H, s, CO2Me), 3.77 (3H, s, CO2Me); 13C NMR
(100 MHz, CDCl3) δ 170.4 (CvO), 163.3 (CvO), 151.8 (C),
144.8 (C), 141.3 (C), 134.5 (CH), 130.9 (C), 130.3 (CH), 130.3
1
EtOAc 5 : 1 v/v] = 0.42; H NMR (400 MHz, CDCl3) δ 11.08
(1H, s, OH), 7.61 (2H, d, J 8.6 Hz, Ar-H), 7.54 (1H, dd, J 1.4,
8.0 Hz, H-6), 7.46 (2H, d, J 8.6 Hz, Ar-H), 7.30 (1H, s,
CvCHPh), 6.95 (1H, dd, J 1.4, 8.0 Hz, H-4), 6.73 (1H, app. t,
J 8.0 Hz, H-5), 3.97 (3H, s, CO2Me), 3.75 (3H, s, CO2Me);
This journal is © The Royal Society of Chemistry 2012
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(CH), 129.5 (CH), 126.9 (C), 123.7 (CH), 121.9 (CH), 120.3
3.70 (3H, s, CO2Me), 2.33 (3H, s, Ar-Me); (E + Z mix) 13C
NMR (100 MHz, CDCl3) δ 170.5 (CvO), 170.3 (CvO), 163.8
(CvO), 163.3 (CvO), 152.7 (C), 151.8 (C), 144.9 (C), 144.2
(C), 142.9 (C), 139.6 (C), 138.2 (C), 137.6 (C), 132.9 (C), 132.1
(C), 131.2 (C), 130.6 (C), 129.5 (CH), 128.8 (CH), 128.6 (CH),
127.9 (CH), 127.6 (CH), 127.4 (CH), 125.9 (CH), 125.0 (CH),
124.3 (CH), 123.3 (CH), 122.7 (CH), 119.7 (CH), 118.6 (CH),
118.3 (CH), 113.8 (C), 113.4 (C), 52.5 (CH3), 52.4 (CH3), 52.4
(CH3), 52.2 (CH3), 21.3 (CH3), 21.3 (CH3); LRMS [M + Na]+
365.13; HRMS calcd for C19H18O6Na: MNa+, 365.0996. Found:
MNa+, 365.0990.
(CH), 118.3 (CH), 113.5 (C) 52.6 (CH3), 52.4 (CH3); E: Rf
1
[hexane : EtOAc 5 : 1 v/v] = 0.30; H NMR (500 MHz, CDCl3)
δ 11.04 (1H, s, OH), 7.67 (1H, dd, J 1.5, 8.1 Hz, H-6),
7.38–7.36 (1H, m, Ar-H), 7.33–7.31 (2H, m, Ar-H), 7.24–7.18
(2H, m, Ar-H + H-4), 6.87 (1H, app. t, J 8.0 Hz, H-5), 6.54 (1H,
s, CvCHPh), 3.97 (3H, s, CO2Me), 3.67 (3H, s, CO2Me); 13C
NMR (125 MHz, CDCl3) δ 170.4 (CvO), 162.7 (CvO), 153.0
(C), 145.0 (C), 143.9 (C), 133.3 (C), 132.6 (CH), 130.7 (CH),
129.1 (CH), 129.0 (CH), 126.2 (CH), 125.5 (C), 125.2 (C),
119.1 (CH), 118.8 (CH), 114.1 (C) 52.5 (CH3), 52.2 (CH3);
LRMS [M + Na]+ 385.07; HRMS calcd for C18H15O6ClNa:
MNa+, 385.0455. Found: MNa+, 385.0447.
Methyl 2-hydroxy-3-(3-methoxy-3-oxo-1-o-tolylprop-1-en-2-
yloxy)benzoate 18. Phosphonate 10 (150 mg, 0.44 mmol) and
o-tolualdehyde (100 μL, 0.87 mmol) were reacted for 3 h at
−78 °C according to Procedure A. The product was purified by
column chromatography (eluent: hexane–EtOAc 90 : 10 v/v) to
afford diester 18 as a mixture of diastereomers as a colourless oil
(88 mg, 60%, 91 : 9 Z : E) IR νmax (ATR): 3125 (br), 2954, 1728,
1676, 1465, 1438, 1248 cm−1; Z: Rf [hexane : EtOAc 5 : 1 v/v] =
Methyl 3-(1-(4-chlorophenyl)-3-methoxy-3-oxoprop-1-en-2-yloxy)-
2-hydroxybenzoate 16. Phosphonate 10 (150 mg, 0.44 mmol)
and p-chlorobenzaldehyde (120 mg, 0.87 mmol) were reacted
for 2 h at −78 °C according to Procedure A. The product was
purified by column chromatography (eluent: hexane–EtOAc
90 : 10 v/v) to afford diester 16 as a mixture of diastereomers as
a yellow oil (110 mg, 70%, 67 : 33 Z/E). IR νmax (ATR): 3136
(br), 2957, 1727, 1677, 1464, 1438, 1249 cm−1; Z: Rf [hexane :
1
0.26; H NMR (400 MHz, CDCl3) δ 11.06 (1H, s, OH), 7.91
(1H, app. d, J 7.8 Hz, Ar-H), 7.60 (1H, s, CvCHPh), 7.50 (1H,
dd, J 1.5, 8.1 Hz, Ar-H), 7.25 (1H, dd, J 1.5, 7.3 Hz, H-6),
7.22–7.12 (2H, m, Ar-H), 6.95 (1H, dd, J 1.5, 8.0 Hz, H-4),
6.72 (1H, app. t, J 8.0 Hz, H-5), 3.96 (3H, s, CO2Me), 3.77 (3H,
s, CO2Me), 2.45 (3H, s, Ar-Me); 13C NMR (100 MHz, CDCl3) δ
170.5 (CvO), 162.9 (CvO), 151.8 (C), 145.1 (C), 140.0 (C),
135.9 (CH), 130.2 (C), 129.5 (CH), 128.8 (CH), 126.2 (CH),
125.2 (C), 124.3 (CH), 124.1 (CH), 123.3 (CH), 119.9 (CH),
113.4 (C) 52.5 (CH3), 52.4 (CH3), 20.0 (CH3); E: Rf [hexane :
1
EtOAc 5 : 1 v/v] = 0.34; H NMR (500 MHz, CDCl3) δ 11.06
(1H, s, OH), 7.68 (1H, app. d, J 8.6 Hz, Ar-H), 7.55 (1H, dd,
J 1.4, 8.1 Hz, H-6), 7.31 (1H, s, CvCHPh), 7.29–7.26 (3H, m,
Ar-H), 6.96 (1H, dd, J 1.0, 8.0 Hz, H-4), 6.73 (1H, app. t,
J 8.0 Hz, H-5), 3.97 (3H, s, CO2Me), 3.75 (3H, s, CO2Me); 13C
NMR (125 MHz, CDCl3) δ 170.5 (CvO), 163.6 (CvO), 151.9
(C), 144.7 (C), 140.5 (C), 135.6 (C), 131.6 (2 × CH), 129.0 (2 ×
CH), 128.2 (CH), 125.6 (CH), 123.7 (CH), 120.1 (CH), 118.4
(CH), 113.7 (C), 52.5 (CH3), 52.5 (CH3); E: Rf [hexane : EtOAc
1
EtOAc 5 : 1 v/v] = 0.18; H NMR (400 MHz, CDCl3) δ 11.04
1
5 : 1 v/v] = 0.31; H NMR (500 MHz, CDCl3) δ 11.01 (1H, s,
(1H, s, OH), 7.65 (1H, dd, J 1.6, 8.1 Hz, H-6), 7.22–7.12 (5H,
m, Ar-H + H-4), 6.87 (1H, app. t, J 8.0 Hz, H-5), 6.61 (1H, s,
CvCHPh), 3.98 (3H, s, CO2Me), 3.64 (3H, s, CO2Me), 2.25
(3H, s, Ar-Me); 13C NMR (125 MHz, CDCl3) δ 170.3 (CvO),
163.8 (CvO), 152.8 (C), 144.1 (C), 143.2 (C), 137.6 (C), 132.8
(CH), 130.8 (CH), 129.5 (CH), 129.4 (CH), 127.9 (CH), 125.0
(C), 122.2 (C), 118.7 (CH), 118.3 (CH), 113.8 (C) 52.5 (CH3),
52.1 (CH3), 20.2 (CH3); LRMS [M + Na]+ 365.07; HRMS calcd
for C19H18O6Na: MNa+, 365.0996. Found: MNa+, 365.0989.
OH), 7.67 (1H, app. d, J 8.9 Hz, Ar-H), 7.66 (1H, dd, J 1.5,
8.1 Hz, H-6), 7.29–7.26 (3H, m, Ar-H), 7.25 (1H, dd, J 1.5,
8.0 Hz, H-4), 6.86 (1H, app. t, J 8.0 Hz, H-5), 6.44 (1H, s,
CvCHPh), 3.97 (3H, s, CO2Me), 3.72 (3H, s, CO2Me); 13C
NMR (125 MHz, CDCl3) δ 170.4 (CvO), 163.1 (CvO), 152.9
(C), 143.8 (C), 143.8 (C) 133.8 (C), 131.7 (2 × CH), 130.8 (2 ×
CH), 130.3 (CH), 125.5 (CH), 125.0 (CH), 120.5 (CH), 118.7
(CH), 114.1 (C), 52.6 (CH3), 52.3 (CH3); LRMS [M + Na]+
385.33; HRMS calcd for C18H15O6ClNa: MNa+, 385.0455.
Found: MNa+, 385.0449.
Methyl 2-hydroxy-3-(3-methoxy-3-oxo-1-p-tolylprop-1-en-2-
yloxy)benzoate 19. Phosphonate 10 (140 mg, 0.41 mmol) and
p-tolualdehyde (100 μL, 0.83 mmol) were reacted for 3 h at
−78 °C under Procedure A. The product was purified by column
chromatography (eluent: hexane–EtOAc 5 : 1 v/v) to afford
diester 19 as a mixture of diastereomers as a white solid (71 mg,
50%, 67 : 33 Z/E). Rf [hexane : EtOAc 5 : 1 v/v] = 0.38; IR νmax
(ATR): 3137 (br), 2954, 2923, 1726, 1678, 1464, 1439,
1249 cm−1; Z: 1H NMR (400 MHz, CDCl3) δ 11.05 (1H, s,
OH), 7.64 (2H, d, J 8.1 Hz, Ar-H), 7.52 (1H, dd, J 1.5, 8.0 Hz,
H-6), 7.38 (1H, s, CvCHPh), 7.14 (2H, d, J 8.1 Hz, Ar-H),
6.96 (1H, dd, J 1.5, 8.0 Hz, H-4), 6.71 (1H, app. t, J 8.0 Hz,
H-5), 3.96 (3H, s, CO2Me), 3.75 (3H, s, CO2Me), 2.32 (3H, s,
Ar-Me); 13C NMR (100 MHz, CDCl3) δ 170.7 (CvO), 164.0
(CvO), 152.0 (C), 145.1 (C), 140.3 (C), 139.2 (C), 130.6 (2 ×
CH), 129.6 (2 × CH), 127.8 (CH), 123.5 (CH), 119.8 (CH),
118.5 (CH), 113.7 (C), 52.6 (CH3), 52.6 (CH3), 21.6 (CH3) 1
signal obscured; E: 1H NMR (400 MHz, CDCl3) δ 11.00 (1H, s,
Methyl 2-hydroxy-3-(3-methoxy-3-oxo-1-m-tolylprop-1-en-2-
yloxy)benzoate 17. Phosphonate 10 (70 mg, 0.20 mmol) and
m-tolualdehyde (47 μL, 0.40 mmol) were reacted for 2 h at −78 °C
according to Procedure A. The product was purified by column
chromatography (eluent: hexane–EtOAc 90 : 10 v/v) to afford
diester 17 as a mixture of diastereomers as a yellow oil (56 mg,
82%, 56 : 44 Z/E). Rf [hexane : EtOAc 5 : 1 v/v] = 0.31; IR νmax
1
(ATR): 3099 (br), 2953, 1727, 1677, 1438, 1249 cm−1; Z: H
NMR (400 MHz, CDCl3) δ 11.10 (1H, s, OH), 7.57 (1H, app d,
J 7.8 Hz, Ar-H), 7.53 (1H, s, CvCHPh), 7.37 (1H, s, Ar-H),
7.26–7.08 (3H, m, Ar-H + H-6), 6.96 (1H, dm, J 8.0 Hz, H-4),
6.72 (1H, t, J 8.0 Hz, H-5), 3.96 (3H, s, CO2Me), 3.76 (3H, s,
1
CO2Me), 2.31 (3H, s, Ar-Me); E: H NMR (400 MHz, CDCl3)
δ 11.04 (1H, s, OH), 7.63 (1H, dm, J 8.1 Hz, Ar-H), 7.52 (1H,
dm, J 8.0 Hz, H-6), 7.26–7.08 (4H, m, Ar-H + H-4), 6.84 (1H, t,
J 8.0 Hz, H-5), 6.56 (1H, s, CvCHPh), 3.96 (3H, s, CO2Me),
9230 | Org. Biomol. Chem., 2012, 10, 9223–9236
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OH), 7.62 (1H, dd, J 1.5, 8.0 Hz, H-6), 7.26 (2H, d, J 8.2 Hz,
Ar-H), 7.24–7.21 (1H, m, H-4), 7.14–7.11 (2H, m, Ar-H), 6.83
(1H, app. t, J 8.0 Hz, H-5), 6.59 (1H, s, CvCHPh), 3.96 (3H, s,
CO2Me), 3.70 (3H, s, CO2Me), 2.34 (3H, s, Ar-Me); 13C NMR
(100 MHz, CDCl3) δ 170.5 (CvO), 163.5 (CvO), 152.8 (C),
144.7 (C), 142.4 (C), 138.3 (C), 130.1 (C), 129.1 (2 × CH),
128.9 (2 × CH), 124.9 (CH), 124.1 (CH), 123.8 (CH), 118.8
(CH), 114.0 (C), 52.7 (CH3), 52.3 (CH3), 21.4 (CH3); LRMS
[M + Na]+ 364.87; HRMS calcd for C19H18O6Na: MNa+,
365.1001. Found: MNa+, 365.0994.
(C), 130.7 (C), 130.7 (CH), 126.5 (CH), 125.2 (CH), 123.8
(CH), 122.5 (CH), 120.5 (CH), 119.5 (CH), 118.6 (CH), 113.7
(C), 52.7 (CH3), 52.6 (CH3), 21.1 (CH3); E: 1H NMR
(400 MHz, CDCl3) δ 11.02 (1H, s, OH), 7.68 (1H, dd, J 1.3, 8.0
Hz, H-6), 7.31–7.27 (2H, m, Ar-H), 7.26 (1H, dd, J 1.4, 8.0 Hz,
Ar-H), 7.19 (1H, dt, J 0.8, 7.6, 8.0 Hz, Ar-H), 7.06 (1H, dd,
J 1.4, 8.0 Hz, H-4) 6.87 (1H, t, J 8.0 Hz, H-5), 6.20 (1H, s,
CvCHPh), 3.97 (3H, s, CO2Me), 3.69 (3H, s, CO2Me), 2.23
(3H, s, COMe); 13C NMR (100 MHz, CDCl3) δ 170.5 (CvO),
169.1 (CvO), 163.0 (C), 153.3 (C), 148.2 (C), 145.5 (C), 143.4
(C), 130.4 (CH), 129.0 (CH), 127.2 (CH), 127.2 (C), 126.0
(CH), 125.8 (CH), 122.2 (CH), 119.0 (CH), 114.5 (CH), 114.3
(C), 52.7 (CH3), 52.4 (CH3), 20.9 (CH3); LRMS [M + Na]+
408.87; HRMS calcd for C20H18O8Na: MNa+, 409.0899. Found:
MNa+, 409.0895.
Methyl 3-(1-(3-acetoxyphenyl)-3-methoxy-3-oxoprop-1-en-2-
yloxy)-2-hydroxybenzoate 20. Phosphonate 10 (150 mg,
0.43 mmol) and m-formylphenyl acetate (140 mg, 0.86 mmol)
were reacted for 6 h at −78 °C under Procedure A. The product
was purified by column chromatography (eluent: hexane–EtOAc
5 : 1 v/v) to afford diester 20 as a mixture of diastereomers as a
yellow oil (94 mg, 56%, 41 : 59 Z/E). Rf [hexane : EtOAc 5 : 1
v/v] = 0.21; IR νmax (ATR): 3046 (br), 2954, 1765, 1729, 1677,
Methyl 3-(1-(4-acetoxyphenyl)-3-methoxy-3-oxoprop-1-en-2-
yloxy)-2-hydroxybenzoate 22. Phosphonate 10 (150 mg,
0.43 mmol) and p-formylphenyl acetate (140 mg, 0.86 mmol)
were reacted for 6 h at −78 °C under Procedure A. The product
was purified by column chromatography (eluent: hexane–EtOAc
5 : 1 v/v) to afford diester 22 as a mixture of diastereomers as a
yellow oil (100 mg, 61%, 56 : 44 Z/E). IR νmax (ATR): 3144
(br), 2957, 1765, 1727, 1677, 1463, 1439, 1195 cm−1; Z: Rf
1
1439, 1199 cm−1; Z: H NMR (400 MHz, CDCl3) δ 11.06 (1H,
s, OH), 7.61 (1H, app. d, J 7.8 Hz, Ar-H), 7.54 (1H, dd, J 1.4,
8.0 Hz, H-6), 7.48 (1H, app. t, J 2.2 Hz, Ar-H), 7.34 (1H, t,
J 8.0 Hz, Ar-H), 7.32 (1H, s, CvCHPh), 7.08 (1H, ddd, J 1.0,
2.2, 7.8 Hz, Ar-H), 6.97 (1H, dd, J 1.4, 8.0 Hz, H-4), 6.73 (1H,
t, J 8.0 Hz, H-5), 3.97 (3H, s, CO2Me), 3.75 (3H, s, CO2Me),
2.27 (3H, s, COMe); 13C NMR (100 MHz, CDCl3) δ 170.7
(CvO), 169.3 (CvO), 163.2 (C), 152.1 (C), 150.8 (C), 144.9
(C), 141.0 (C), 133.9 (C), 129.7 (CH), 127.9 (CH), 125.8 (CH),
123.9 (CH), 123.4 (CH), 123.0 (CH), 120.5 (CH), 118.5 (CH),
113.7 (C), 52.6 (CH3), 52.4 (CH3), 21.2 (CH3); E: 1H NMR
(400 MHz, CDCl3) δ 11.01 (1H, s, OH), 7.66 (1H, dd, J 1.6,
8.0 Hz, H-6), 7.31 (1H, t, J 7.8 Hz, Ar-H), 7.25 (1H, dd, J 1.6,
8.0 Hz, H-4), 7.16 (1H, dt, J 0.8, 1.9, 7.8 Hz, Ar-H), 7.08 (1H,
app. t, J 1.9 Hz, Ar-H), 6.99 (1H, ddd, J 0.8, 1.9, 7.8 Hz, Ar-H),
6.85 (1H, t, J 8.0 Hz, H-5), 6.45 (1H, s, CvCHPh), 3.96 (3H, s,
CO2Me), 3.71 (3H, s, CO2Me), 2.28 (3H, s, COMe); 13C NMR
(100 MHz, CDCl3) δ 170.4 (CvO), 169.4 (CvO), 163.6 (C),
153.1 (C), 150.4 (C), 144.3 (C), 143.8 (C), 134.8 (C), 129.0
(CH), 126.6 (CH), 125.6 (CH), 125.3 (CH), 122.3 (CH), 121.1
(CH), 120.3 (CH), 118.8 (CH), 114.1 (C), 52.6 (CH3), 52.6
(CH3), 21.2 (CH3); LRMS [M + Na]+ 408.87; HRMS calcd for
C20H18O8Na: MNa+, 409.0899. Found: MNa+, 409.0896.
1
[hexane : EtOAc 5 : 1 v/v] = 0.45, H NMR (400 MHz, CDCl3)
δ 11.06 (1H, s, OH), 7.77 (2H, d, J 8.6 Hz, Ar-H), 7.53 (1H, dd,
J 1.3, 8.1 Hz, H-6), 7.36 (1H, s, CvCHPh), 7.07 (2H, d,
J 8.6 Hz, Ar-H), 6.95 (1H, dd, J 1.3, 8.1 Hz, H-4), 6.72 (1H, t,
J 8.1 Hz, H-5), 3.96 (3H, s, CO2Me), 3.75 (3H, s, CO2Me), 2.28
(3H, s, COMe); 13C NMR (100 MHz, CDCl3) δ 170.7 (CvO),
169.4 (CvO), 163.9 (C), 152.1 (C), 151.6 (C), 145.0 (C), 140.1
(C), 131.8 (2 × CH), 130.2 (CH), 126.3 (CH), 123.8 (CH),
122.1 (2 × CH), 120.1 (CH), 118.6 (CH), 113.8 (C), 52.7 (CH3),
52.6 (CH3), 21.3 (CH3); E: Rf [hexane : EtOAc 5 : 1 v/v] = 0.36;
1H NMR (400 MHz, CDCl3) δ 11.01 (1H, s, OH), 7.64 (1H, dd,
J 1.5, 8.0 Hz, H-6), 7.36 (2H, d, J 7.8 Hz, Ar-H), 7.23 (1H, dd,
J 1.5, 8.0 Hz, H-4), 7.04 (2H, d, J 7.8 Hz, Ar-H), 6.85 (1H, t,
J 8.0 Hz, H-5), 6.52 (1H, s, CvCHPh), 3.96 (3H, s, CO2Me),
3.70 (3H, s, CO2Me), 2.29 (3H, s, COMe); 13C NMR
(100 MHz, CDCl3) δ 170.5 (CvO), 169.3 (CvO), 163.4 (C),
153.0 (C), 150.5 (C), 144.3 (C), 143.5 (C), 130.9 (CH), 130.4 (2
× CH), 125.4 (CH), 124.7 (CH), 121.8 (CH), 121.4 (2 × CH),
118.9 (CH), 114.1 (C), 52.6 (CH3), 52.4 (CH3), 21.2 (CH3);
LRMS [M + Na]+ 408.87; HRMS calcd for C20H18O8Na:
MNa+, 409.0899. Found: MNa+, 409.0896.
Methyl 3-(1-(2-acetoxyphenyl)-3-methoxy-3-oxoprop-1-en-2-
yloxy)-2-hydroxybenzoate 21. Phosphonate 10 (150 mg,
0.43 mmol) and o-formylphenyl acetate (140 mg, 0.86 mmol)
were reacted for 6 h at −78 °C under Procedure A. The product
was purified by column chromatography (eluent: hexane–EtOAc
5 : 1 v/v) to afford diester 21 as a mixture of diastereomers as a
yellow oil (99 mg, 60%, 67 : 33 Z/E). Rf [hexane : EtOAc 5 : 1
v/v] = 0.31; IR νmax (ATR): 3094 (br), 2952, 1768, 1731, 1678,
Methyl 3-(1-(3-trifluoromethylphenyl)-3-methoxy-3-oxoprop-
1-en-2-yloxy)-2-hydroxybenzoate 23. Phosphonate 10 (150 mg,
0.43 mmol) and m-trifluoromethylbenzaldehyde (140 mg,
0.86 mmol) were reacted for 6 h at −78 °C according to Pro-
cedure A. The product was purified by column chromatography
(eluent: hexane–EtOAc 90 : 10 v/v) to afford diester 23 as a
mixture of diastereomers as a yellow oil (114 mg, 67%, 59 : 41
Z/E). Rf [hexane : EtOAc 5 : 1 v/v] = 0.33; IR νmax (ATR): 3117
1
1464, 1251 cm−1; Z: H NMR (400 MHz, CDCl3) δ 11.04 (1H,
s, OH), 8.11 (1H, dd, J 1.6, 8.0 Hz, Ar-H), 7.52 (1H, dd, J 1.6,
8.0 Hz, H-6), 7.43 (1H, s, CvCHPh), 7.35–7.31 (1H, m, Ar-H),
7.18 (1H, dt, J 0.6, 7.2, 7.8 Hz, Ar-H), 7.09 (1H, dd, J 0.6,
7.8 Hz, Ar-H), 6.95 (1H, dd, J 1.6, 8.0 Hz, H-4), 6.72 (1H, t,
J 8.0 Hz, H-5), 3.96 (3H, s, CO2Me), 3.75 (3H, s, CO2Me), 2.41
(3H, s, COMe); 13C NMR (100 MHz, CDCl3) δ 170.7 (CvO),
169.3 (CvO), 163.7 (C), 152.1 (C), 149.2 (C), 145.0 (C), 141.4
1
(br), 2957, 1730, 1678, 1439, 1327, 1121 cm−1; Z: H NMR
(400 MHz, CDCl3) δ 11.10 (1H, s, OH), 7.97 (1H, app. d,
J 7.9 Hz, Ar-H), 7.94 (1H, s, CvCHPh), 7.56–7.40 (2H, m, H-6
+ H-4), 7.48 (1H, app. t, J 2.2 Hz, Ar-H), 7.35 (1H, app. s, Ar-
H), 6.97 (1H, app. d, J 7.7 Hz, Ar-H), 6.73 (1H, t, J 8.1 Hz,
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H-5), 3.96 (3H, s, CO2Me), 3.77 (3H, s, CO2Me); 13C NMR
removed in vacuo prior to acidification to pH 5–6 with 1 M HCl
and extraction into DCM (3 × 10 mL). The organic layers were
dried (Na2SO4), and solvent removed in vacuo to afford the
desired acids. Method B: THF removed in vacuo prior to acidifi-
cation to pH 5–6 with 1 M HCl. The solution was filtered and
purified immediately using reverse phase HPLC (gradient:
0–100% MeCN with 0.1% formic acid, from 100% H2O with
0.1% formic acid over 40 minutes). The fractions containing the
desired diacid were lyophilised.
(100 MHz, CDCl3) δ 170.5 (CvO), 163.3 (CvO), 151.9 (C),
2
144.5 (C), 141.4 (C), 134.1 (CH), 133.1 (C), 131.0 (C, q, JC–F
3
3
32.7 Hz), 127.1 (CH, q, JC–F 3.8 Hz), 127.1 (CH, q, JC–F 3.7
1
Hz), 125.7 (CH), 123.9 (CH), 123.7 (C, q, JC–F 272 Hz),
120.2 (CH), 118.4 (CH), 113.6 (C), 52.6 (CH3), 52.5 (CH3)
1
1 signal obscured; E: H NMR (400 MHz, CDCl3) δ 11.07 (1H,
s, OH), 7.67 (1H, dd, J 1.0, 8.1 Hz, H-6), 7.56–7.40 (4H, m,
Ar-H), 7.27 (1H, app. d, J 7.9 Hz, H-4), 6.88 (1H, t, J 8.0 Hz,
H-5), 6.42 (1H, s, CvCHPh), 3.96 (3H, s, CO2Me), 3.72 (3H, s,
CO2Me); 13C NMR (100 MHz, CDCl3) δ 170.3 (CvO), 162.9
(CvO), 153.0 (C), 144.8 (C), 143.2 (C), 133.0 (C), 132.1 (CH),
3-(2-(3-Bromophenyl)-1-carboxyvinyloxy)-2-hydroxybenzoic
acid 25. Potassium hydroxide (1.2 mL) was added to a solution
of diester 11 (48 mg, 0.12 mmol) in THF (1.5 mL). Water
(0.60 mL) was then added and the reaction stirred at rt for 48 h.
The mixture was worked up according to Method A to afford the
desired diacid 25 as a mixture of diastereomers as a colourless
oil (24 mg, 54%, 63 : 37 Z : E). IR νmax (ATR): 3075 (br), 2570,
2
130.3 (C, q, JC–F 35.3 Hz), 125.9 (CH), 125.7 (CH), 125.7
3
(CH, q, 3JC–F 4.9 Hz), 124.8 (CH), 124.3 (CH, q, JC–F 3.8 Hz),
1
123.9 (C, q, JC–F 272 Hz), 118.8 (CH), 118.7 (CH), 114.1 (C),
52.6 (CH3), 52.3 (CH3); LRMS [M + Na]+ 418.67; HRMS calcd
for C19H15O6F3Na: MNa+, 419.0713. Found: MNa+, 419.0719.
1
1686, 1467, 1237 cm−1; Z: H NMR (400 MHz, MeOD) δ 7.95
(1H, s, CvCHPh), 7.68 (1H, app. d, J 7.8 Hz, Ar-H), 7.55 (1H,
dd, J 1.6, 8.0 Hz, H-6), 7.45 (1H, dm, J 8.0 Hz, Ar-H), 7.32
(1H, s, Ar-H), 7.24 (1H, t, J 8.0 Hz, Ar-H), 6.98 (1H, dd, J 1.4,
8.0 Hz, H-4), 6.75 (1H, t, J 8.0 Hz, H-5); 13C NMR (100 MHz,
MeOD) δ 173.6 (CvO), 165.7 (CvO), 153.2 (C), 146.0 (C),
143.1 (C), 136.2 (C), 133.8 (C), 133.4 (CH), 132.7 (CH), 130.0
(CH), 125.7 (CH), 125.2 (CH), 123.6 (CH), 120.9 (CH), 119.3
Methyl 3-(1-(2-trifluoromethylphenyl)-3-methoxy-3-oxoprop-
1-en-2-yloxy)-2-hydroxybenzoate 24. Phosphonate 10 (150 mg,
0.44 mmol) and o-trifluoromethylbenzaldehyde (100 μL,
0.87 mmol) were reacted for 16 h at −78 °C according to Pro-
cedure A. The product was purified by column chromatography
(eluent: hexane–EtOAc 90 : 10 v/v) to afford diester 24 as a
mixture of diastereomers as a colourless oil (115 mg, 67%, 91 : 9
Z : E) IR νmax (ATR): 3099 (br), 2956, 1732, 1677, 1464, 1439,
1
(CH), 115.2 (C); E: H NMR (400 MHz, MeOD) δ 7.69 (1H,
1
1249 cm−1; Z: Rf [hexane : EtOAc 5 : 1 v/v] = 0.27; H NMR
dd, J 1.6, 8.0 Hz, Ar-H), 7.51 (1H, br t, J 5.0 Hz, Ar-H), 7.38
(2H, dm, J 7.8 Hz, Ar-H + H-6), 7.27 (1H, dd, J 1.6, 8.0 Hz,
H-4), 7.18 (1H, t, J 7.8 Hz, Ar-H), 6.89 (1H, t, J 8.0 Hz, H-5),
6.33 (1H, s, CvCHPh); 13C NMR (100 MHz, MeOD) δ 173.4
(CvO), 165.7 (CvO), 154.5 (C), 146.9 (C), 144.7 (C), 137.4
(C), 131.4 (C), 131.4 (CH), 130.7 (CH), 128.9 (CH), 127.3
(CH), 126.4 (CH), 122.8 (CH), 119.7 (CH), 118.9 (CH), 115.8
(C); LRMS [M + Na]+ 400.53; HRMS calcd for C16H11O6BrNa:
MNa+, 400.9637. Found: MNa+, 400.9636.
(400 MHz, CDCl3) δ 11.06 (1H, s, OH), 8.07 (1H, app. d,
J 7.7 Hz, Ar-H), 7.65 (1H, app. d, J 7.6 Hz, Ar-H) 7.65 (1H, s,
CvCHPh), 7.52 (1H, app. d, J 8.5 Hz, H-6), 7.46 (1H, app. d,
J 7.7 Hz, Ar-H), 7.37 (1H, app. d, J 7.6 Hz, Ar-H), 6.95 (1H, app.
d, J 7.9 Hz, H-4), 6.72 (1H, app. t, J 8.0 Hz, H-5), 3.94 (3H, s,
CO2Me), 3.78 (3H, s, CO2Me); 13C NMR (100 MHz, CDCl3) δ
170.5 (CvO), 163.2 (CvO), 151.8 (C), 144.9 (C), 141.9 (C),
3
131.8 (CH), 131.1 (C), 130.3 (C, q, JC–F 2.4 Hz), 128.8 (CH),
2
3
128.5 (C, q, JC–F 29.7 Hz), 125.8 (CH, q, JC–F 5.6 Hz), 123.9
1
Z-3-(2-(2-Bromophenyl)-1-carboxyvinyloxy)-2-hydroxybenzoic
acid 26. Potassium hydroxide (0.17 mL) was added to a solution
of pure Z diester 12 (7 mg, 0.02 mmol) in THF. Water (0.83 mL)
was then added and the reaction stirred at rt for 48 h. The
mixture was worked-up according to Method B, affording the
desired pure Z diacid 26 as a white solid (7 mg, quant). IR νmax
(ATR): 3081 (br), 2578, 1682, 1466, 1237, 753 cm−1; retention
(C, q, JC–F 274 Hz), 123.8 (CH), 121.4 (CH), 120.5 (CH),
118.4 (CH), 113.5 (C), 52.7 (CH3), 52.5 (CH3); E: Rf [hexane :
1
EtOAc 5 : 1 v/v] = 0.25; H NMR (500 MHz, CDCl3) δ 11.02
(1H, s, OH), 7.68–7.65 (2H, m, Ar-H + H-6), 7.49 (1H, app. t,
J 8.3 Hz, Ar-H), 7.39 (1H, app. t, J 7.5 Hz, Ar-H), 7.32 (1H,
app. d, J 7.6 Hz, H-4), 7.27 (1H, dd, J 1.4, 7.6 Hz, Ar-H), 6.88
(1H, app. t, J 8.0 Hz, H-5), 6.64 (1H, s, CvCHPh), 3.98 (3H, s,
CO2Me), 3.61 (3H, s, CO2Me); 13C NMR (125 MHz, CDCl3)
δ 170.4 (CvO), 162.4 (CvO), 153.0 (C), 144.9 (C), 143.6 (C),
132.9 (C, q, 3JC–F 2.2 Hz), 131.2 (CH), 130.8 (CH), 127.9 (C, q,
1
time: 33.53 min; mp 211–213 °C; H NMR (300 MHz, MeOD)
δ 8.00 (1H, dd, J 1.7, 7.8 Hz, Ar-H), 7.67 (1H, s, CvCHPh),
7.63 (1H, dd, J 1.1, 7.9 Hz, Ar-H), 7.53 (1H, dd, J 1.5, 8.0 Hz,
H-6), 7.29 (1H, dt, J 1.1, 7.6, 7.9 Hz, Ar-H), 7.19 (1H, dt, J 1.7,
7.6, 7.8 Hz, Ar-H), 6.98 (1H, dd, J 1.5, 8.0 Hz, H-4), 6.74 (1H,
t, J 8.0 Hz, H-5); 13C NMR (75 MHz, MeOD) δ 173.7 (CvO),
165.9 (CvO), 153.2 (C), 146.3 (C), 143.8 (C), 134.0 (CH),
133.8 (C), 132.1 (CH), 131.7 (CH), 128.7 (CH), 125.7 (C),
125.2 (CH), 124.7 (CH), 121.0 (CH), 119.1 (CH), 115.9 (C);
LRMS [M + Na]+ 402.60; HRMS calcd for C16H11O6BrNa:
MNa+, 400.9637. Found: MNa+, 400.9628.
3
2JC–F 29.9 Hz), 127.6 (CH), 125.7 (CH), 125.6 (CH, q, JC–F
1
5.4 Hz), 125.4 (C), 121.8 (C, q, JC–F 290 Hz), 118.8 (CH),
118.3 (CH), 114.1 (C) 52.5 (CH3), 52.2 (CH3); LRMS
[M + Na]+ 418.9; HRMS calcd for C19H15O6F3Na: MNa+,
419.0713. Found: MNa+, 419.0718.
Procedure B: saponification reaction of dimethyl esters
Aqueous potassium hydroxide (2 M soln., 20 eq.) was added to
a solution of diester in THF (1 mL). Water was then added to
make the solution up to 2 mL (or 2.6 mL when the base volume
exceeded 1 mL) and the reaction stirred at rt for 48 h. Work-up
was carried out using one of two methods; Method A: THF
Z-3-(2-(4-Bromophenyl)-1-carboxyvinyloxy)-2-hydroxybenzoic
acid 27. Potassium hydroxide (0.29 mL) was added to a solution
of pure Z diester 13 (12 mg, 0.03 mmol) in THF. Water
(0.71 mL) was then added and the reaction stirred at rt for 48 h.
The mixture was worked-up according to Method B, affording
9232 | Org. Biomol. Chem., 2012, 10, 9223–9236
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the desired pure Z diacid 27 as a white solid (9 mg, 79%). IR
the desired diacid 30 as a mixture of diastereomers as a colour-
less oil (11 mg, 86%, 83 : 17 Z : E). IR νmax (ATR): 3446 (br),
νmax (ATR): 3097 (br), 2571, 1686, 1466, 1240, 753 cm−1
;
1
1
retention time: 33.50 min; H NMR (300 MHz, MeOD) δ 7.67
(2H, d, J 8.6 Hz, Ar-H), 7.55 (1H, dd, J 1.5, 8.0 Hz, H-6), 7.51
(2H, d, J 8.6 Hz, Ar-H), 7.34 (1H, s, CvCHPh), 6.98 (1H, dd,
J 1.5, 8.0 Hz, H-4), 6.75 (1H, t, J 8.0 Hz, H-5); 13C NMR
(75 MHz, MeOD) δ 173.8 (CvO), 166.0 (CvO), 153.2 (C),
146.1 (C), 146.1 (C), 142.8 (C), 133.3 (C), 132.9 (2 × CH),
132.9 (2 × CH), 126.0 (CH), 125.2 (CH), 124.5 (C), 120.6
(CH), 119.1 (CH); LRMS [M − H]− 376.73; HRMS calcd for
C16H11O6BrNa: MNa+, 400.9637. Found: MNa+, 400.9628.
2554, 1676, 1467, 1240 cm−1; retention time: 32.67 min; Z: H
NMR (400 MHz, MeOD) δ 7.74 (2H, app. d, J 8.6 Hz, Ar-H),
7.55 (1H, dd, J 1.3, 8.0 Hz, H-6), 7.35 (1H, s, CvCHPh), 7.34
(2H, app. d, J 8.9 Hz, Ar-H), 6.99 (1H, dd, J 1.3, 7.9 Hz, H-4),
6.75 (1H, t, J 8.0 Hz, H-5); 13C NMR (100 MHz, MeOD) δ
173.6 (CvO), 165.9 (CvO), 153.2 (C), 146.1 (C), 142.5 (C),
136.4 (C), 132.8 (CH), 132.7 (2 × CH), 129.9 (2 × CH), 126.1
1
(CH), 125.1 (CH), 120.8 (CH), 119.3 (CH), 115.4 (C); E: H
NMR (400 MHz, MeOD) δ 7.69 (1H, dd, J 1.6, 8.1 Hz, Ar-H),
7.35–7.26 (5H, m, Ar-H + H-6 + H-4), 6.88 (1H, t, J 8.0 Hz,
H-5), 6.37 (1H, s, CvCHPh); 13C NMR (100 MHz, MeOD) δ
132.8 (CH), 131.6 (2 × CH), 129.1 (2 × CH), 127.1 (CH), 126.2
(CH), 119.7 (CH), 119.7 (CH) 7 quaternary signals obscured;
LRMS [M − H]− 349.53; HRMS calcd for C16H11O6ClNa:
MNa+, 357.0142. Found: MNa+, 357.0136.
3-(2-(3-Chlorophenyl)-1-carboxyvinyloxy)-2-hydroxybenzoic
acid 28. Potassium hydroxide (1.4 mL) was added to a solution
of diester 14 (50 mg, 0.14 mmol) in THF (1.5 mL). Water
(0.70 mL) was then added and the reaction stirred at rt for 48 h.
The mixture was worked up according to Method A, affording
the desired diacid 28 as a mixture of diastereomers as a colour-
less oil (31 mg, 68%, 59 : 41 Z : E). IR νmax (ATR): 3064 (br),
3-(1-Carboxy-2-m-tolylvinyloxy)-2-hydroxybenzoic acid 31.
Potassium hydroxide (0.9 mL) was added to a solution of diester
17 (29 mg, 0.08 mmol) in THF (1.0 mL). Water (0.45 mL) was
then added and the reaction stirred at rt for 48 h. The mixture
was worked-up according to Method A, affording the desired
diacid 31 as a mixture of diastereomers as a colourless oil
(19 mg, 76%, 63 : 37 Z : E). IR νmax (ATR): 3043 (br), 2559,
1
2582, 1682, 1466, 1232 cm−1; Z: H NMR (400 MHz, MeOD)
δ 7.80 (1H, s, CvCHPh), 7.54 (1H, app. d, J 8.0 Hz, Ar-H),
7.37–7.22 (4H, m, Ar-H + H-6), 6.98 (1H, app. d, J 8.0 Hz,
H-4), 6.76 (1H, t, J 8.0 Hz, H-5); 13C NMR (100 MHz, MeOD)
δ 173.6 (CvO), 165.7 (CvO), 153.3 (C), 146.0 (C), 143.1 (C),
136.0 (C), 135.6 (C), 131.2 (CH), 130.8 (CH), 129.6 (CH),
128.5 (CH), 125.7 (CH), 125.2 (CH), 121.0 (CH), 119.3 (CH),
1
1681, 1466, 1236, 755 cm−1; retention time: 29.22 min; Z: H
1
115.2 (C); E: H NMR (400 MHz, MeOD) δ 7.69 (1H, app. d,
NMR (400 MHz, MeOD) δ 7.56–7.51 (2H, m, Ar-H + H-6),
7.38 (1H, s, CvCHPh), 7.20 (1H, t, J 7.6 Hz, Ar-H), 7.16–7.12
(2H, m, Ar-H), 6.94 (1H, dd, J 1.2, 8.0 Hz, H-4), 6.73 (1H, t,
J 8.0 Hz, H-5), 2.28 (3H, s, Ar-Me); 13C NMR (100 MHz,
MeOD) δ 173.7 (CvO), 166.2 (CvO), 153.1 (C), 146.3 (C),
141.5 (C), 139.5 (C), 133.8 (CH), 132.0 (CH), 131.5 (CH),
130.6 (CH), 129.6 (CH), 128.5 (CH), 124.8 (CH), 120.4 (CH),
119.3 (CH), 115.1 (C), 21.4 (CH3); E: 1H NMR (400 MHz,
MeOD) δ 7.66 (1H, dd, J 1.4, 8.0 Hz, Ar-H), 7.56–7.51 (2H, m,
Ar-H + H-6), 7.24 (1H, dd, J 1.3, 7.9 Hz, H-4), 7.16–7.12 (1H,
m, Ar-H), 7.06–7.05 (1H, m, Ar-H), 6.87 (1H, t, J 8.0 Hz, H-5),
6.43 (1H, s, CvCHPh), 2.29 (3H, s, Ar-Me); 13C NMR
(100 MHz, MeOD) δ 173.5 (CvO), 166.3 (CvO), 154.4 (C),
145.4 (C), 145.3 (C), 138.7 (C), 134.7 (CH), 129.5 (CH), 128.9
(CH), 128.3 (CH), 127.1 (CH), 126.8 (CH), 125.6 (CH), 122.0
(CH), 119.6 (CH), 115.6 (C), 21.4 (CH3); LRMS [M + Na]+
336.73; HRMS calcd for C17H14O6Na: MNa+, 337.0688. Found:
MNa+, 337.0691.
J 8.0 Hz, Ar-H), 7.65 (1H, br t, J 3.7 Hz, Ar-H), 7.37–7.22 (4H,
m, Ar-H + H-6 + H-4), 6.90 (1H, t, J 8.0 Hz, H-5), 6.34 (1H, s,
CvCHPh); 13C NMR (100 MHz, MeOD) δ 173.4 (CvO),
165.8 (CvO), 154.6 (C), 147.0 (C), 144.8 (C), 137.2 (C), 134.8
(C), 130.5 (CH), 130.4 (CH), 129.8 (CH), 128.5 (CH), 127.3
(CH), 126.4 (CH), 119.7 (CH), 118.9 (CH), 115.8 (C); LRMS
[M + Na]+ 357.07; HRMS calcd for C16H11O6ClNa: MNa+,
357.0136. Found: MNa+, 357.0137.
Z-3-(2-(2-Chlorophenyl)-1-carboxyvinyloxy)-2-hydroxybenzoic
acid 29. Potassium hydroxide (0.5 mL) was added to a solution
of pure Z diester 15 (13.8 mg, 0.04 mmol) in THF (0.5 mL).
Water (0.25 mL) was then added and the reaction stirred at rt for
48 h. The mixture was worked-up according to Method B,
affording the desired pure Z diacid 29 as a white solid (13.1 mg,
quant). IR νmax (ATR): 3214 (br), 2567, 1668, 1467, 1240,
1054 cm−1; retention time: 33.53 min; 1H NMR (400 MHz,
MeOD) δ 8.04 (1H, dd, J 1.8, 7.7 Hz, Ar-H), 7.72 (1H, s,
CvCHPh), 7.53 (1H, dd, J 1.4, 8.0 Hz, Ar-H), 7.44 (1H, dd,
J 1.3, 8.0 Hz, H-6), 7.29 (1H, dt, J 1.8, 7.4 Hz, Ar-H), 7.24 (1H,
dt, J 1.1, 7.5 Hz, Ar-H), 6.98 (1H, dd, J 1.4, 8.0 Hz, H-4), 6.76
(1H, t, J 8.0 Hz, H-5); 13C NMR (100 MHz, MeOD) δ 173.6
(CvO), 165.7 (CvO), 153.2 (C), 146.2 (C), 143.6 (C), 135.3
(C), 131.9 (C), 131.8 (CH), 131.7 (CH), 130.7 (CH), 128.2
(CH), 125.2 (CH), 122.1 (CH), 121.1 (CH), 119.3 (CH), 115.3
(C); LRMS [M − H]− 333.40; HRMS calcd for C16H11O6ClNa:
MNa+, 357.0142. Found: MNa+, 357.0136.
3-(1-Carboxy-2-o-tolylvinyloxy)-2-hydroxybenzoic acid 32.
Potassium hydroxide (0.9 mL) was added to a solution of diester
18 (29 mg, 0.08 mmol) in THF (1.0 mL). Water (0.45 mL) was
then added and the reaction stirred at rt for 48 h. The mixture
was worked-up according to Method A, affording the desired
diacid 32 as a mixture of diastereomers as a colourless oil
(22 mg, 82%, 56 : 44 Z : E). IR νmax (ATR): 3057 (br), 2538,
1
1678, 1463, 1227 cm−1; retention time: 32.50 min; Z: H NMR
(400 MHz, MeOD) δ 7.84 (1H, app. d, J 7.7 Hz, Ar-H), 7.61
(1H, s, CvCHPh), 7.50 (1H, dd, J 1.4, 8.0 Hz, H-6), 7.20–7.07
(3H, m, Ar-H), 6.97 (1H, dd, J 1.4, 8.0 Hz, H-4), 6.73 (1H, t,
J 8.0 Hz, H-5), 2.41 (3H, s, Ar-Me); 13C NMR (100 MHz,
MeOD) δ 173.6 (CvO), 166.3 (CvO), 153.1 (C), 141.9 (C),
138.7 (C), 137.2 (C), 134.7 (CH), 132.3 (CH), 131.3 (CH),
3-(2-(4-Chlorophenyl)-1-carboxyvinyloxy)-2-hydroxybenzoic
acid 30. Potassium hydroxide (0.50 mL) was added to a solution
of diester 16 (14 mg, 0.04 mmol) in THF (0.50 mL). Water
(0.25 mL) was then added and the reaction stirred at rt for 48 h.
The mixture was worked-up according to Method B, affording
This journal is © The Royal Society of Chemistry 2012
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130.4 (CH), 127.1 (CH), 126.4 (CH), 124.9 (CH), 120.7 (CH),
119.2 (CH), 115.1 (C), 20.2 (CH3); E: 1H NMR (400 MHz,
MeOD) δ 7.69 (1H, dd, J 1.5, 8.0 Hz, Ar-H), 7.31 (1H, dd,
J 1.5, 7.9 Hz, H-6), 7.20–7.07 (4H, m, Ar-H + H-4), 6.90 (1H, t,
J 8.0 Hz, H-5), 6.45 (1H, s, CvCHPh), 2.21 (3H, s, Ar-Me);
13C NMR (100 MHz, MeOD) δ 173.5 (CvO), 165.9 (CvO),
154.6 (C), 146.5 (2 × C), 145.9 (C), 145.1 (CH), 130.4 (CH),
130.1 (CH), 128.7 (CH), 127.0 (CH), 126.2 (CH), 124.8 (CH),
121.0 (CH), 119.7 (CH), 115.7 (C), 20.2 (CH3); LRMS
[M + Na]+ 336.87; HRMS calcd for C17H14O6Na: MNa+,
337.0688. Found: MNa+, 337.0683.
(CH), 120.1 (CH), 119.3 (CH), 116.6 (CH), 116.3 (C) 1 signal
obscured; LRMS [M
−
H]− 314.87; HRMS calcd for
C16H12O7Na: MNa+, 339.0481. Found: MNa+, 339.0472.
3-(1-Carboxy-2-(2-hydroxyphenyl)vinyloxy)-2-hydroxybenzoic
acid 35. Potassium hydroxide (0.24 mL) was added to a solution
of Z-rich diester 21 (9 mg, 0.02 mmol) in THF. Water (0.76 mL)
was then added and the reaction stirred at rt for 48 h. The
mixture was worked-up according to Method B, affording the
desired pure Z diacid 35 as a white solid (3 mg, 38%). IR νmax
(ATR): 3257 (br), 2566, 1679, 1459, 1244, 754 cm−1; retention
1
time: 26.63 min; mp 137–139 °C; H NMR (400 MHz, MeOD)
δ 7.92 (1H, dd, J 1.6, 7.9 Hz, Ar-H), 7.90 (1H, s, CvCHPh),
7.52 (1H, dd, J 1.5, 8.0 Hz, H-6), 7.13 (1H, ddd, J 1.6, 7.5,
8.2 Hz, Ar-H), 6.92 (1H, dd, J 1.5, 8.0 Hz, H-4), 6.82 (1H, dd,
J 0.8, 8.2 Hz, Ar-H), 6.73 (1H, ddd, J 0.8, 7.5, 7.9 Hz, Ar-H),
6.70 (1H, t, J 8.0 Hz, H-5); 13C NMR (100 MHz, MeOD)
δ 174.3 (CvO), 166.9 (CvO), 157.4 (C), 153.0 (C), 146.4 (C),
140.8 (C), 132.0 (CH), 131.3 (CH), 124.7 (CH), 122.4 (CH),
121.0 (C), 120.7 (CH), 119.5 (CH), 118.8 (CH), 117.1 (C),
116.3 (CH); LRMS [M − H]− 314.87; HRMS calcd for
C16H12O7Na: MNa+, 339.0481. Found: MNa+, 339.0474.
3-(1-Carboxy-2-p-tolylvinyloxy)-2-hydroxybenzoic acid 33.
Potassium hydroxide (1.1 mL) was added to a solution of diester
19 (37 mg, 0.11 mmol) in THF. Water (0.54 mL) was then added
and the reaction stirred at rt for 48 h. The mixture was worked
up according to Method A, affording the desired diacid 33 as a
mixture of diastereomers as an oil (21 mg, 61%, 67 : 33 Z : E).
1
IR νmax (ATR): 3054 (br), 2564, 1679, 1467, 1237 cm−1; Z: H
NMR (400 MHz, MeOD) δ 7.62 (2H, d, J 8.2 Hz, Ar-H), 7.53
(1H, dd, J 1.5, 8.0 Hz, H-6), 7.39 (1H, s, CvCHPh), 7.15 (2H,
d, J 8.2 Hz, Ar-H), 6.96 (1H, dd, J 1.5, 8.0 Hz, H-4), 6.73 (1H,
t, J 8.0 Hz, H-5), 2.30 (3H, s, Ar-Me); 13C NMR (100 MHz,
MeOD) δ 173.6 (CvO), 166.3 (CvO), 153.1 (C), 146.3 (C),
141.4 (C), 140.9 (C), 131.4 (2 × CH), 131.1 (C), 130.4 (2 ×
CH), 128.3 (CH), 124.8 (CH), 120.3 (C), 119.3 (CH), 115.3 (C),
3-(1-Carboxy-2-(4-hydroxyphenyl)vinyloxy)-2-hydroxybenzoic
acid 36. Potassium hydroxide (0.69 mL) was added to a solution
of diester 22 (27 mg, 0.07 mmol) in THF. Water (0.34 mL) was
then added and the reaction stirred at rt for 48 h. The mixture
was worked-up according to Method B, affording the desired
pure Z diacid 36 as a colourless oil (13 mg, 59%). IR νmax
(ATR): 3110 (br), 2562, 1690, 1604, 1466, 1242 cm−1; retention
1
21.4 (CH3); E: H NMR (400 MHz, MeOD) δ 7.66 (1H, dd,
J 1.6, 8.0 Hz, H-6), 7.26 (2H, d, J 8.0 Hz, Ar-H), 7.25 (1H, dd,
J 1.6, 8.0 Hz, H-4), 7.10 (2H, d, J 8.0 Hz, Ar-H), 6.87 (1H, t,
J 8.0 Hz, H-5), 6.46 (1H, s, CvCHPh), 2.30 (3H, s, Ar-Me);
13C NMR (100 MHz, MeOD) δ 173.5 (CvO), 166.3 (CvO),
154.3 (C), 145.6 (C), 144.9 (C), 139.0 (C), 131.8 (C), 130.1
(2 × CH), 129.7 (2 × CH), 126.6 (CH), 125.4 (CH), 122.5 (CH),
119.6 (CH), 115.7 (C), 21.3 (CH3); LRMS [M + Na]+ 336.73;
HRMS calcd for C17H14O6Na: MNa+, 337.0688. Found: MNa+,
337.0681.
1
time: 26.00 min; H NMR (400 MHz, MeOD) δ 7.61 (2H, d,
J 8.8 Hz, Ar-H), 7.52 (1H, dd, J 1.5, 8.0 Hz, H-6), 7.36 (1H, s,
CvCHPh), 6.90 (1H, dd, J 1.5, 8.0 Hz, H-4), 6.74 (2H, d,
J 8.8 Hz, Ar-H), 6.69 (1H, t, J 8.0 Hz, H-5); 13C NMR
(100 MHz, MeOD) δ 174.4 (CvO), 167.0 (CvO), 160.4 (C),
152.9 (C), 146.3 (C), 139.4 (C), 133.4 (2 × CH), 128.6 (CH),
125.4 (C), 124.7 (CH), 119.1 (CH), 118.7 (CH), 117.4 (C),
116.6 (2 × CH); LRMS [M − H]− 314.93; HRMS calcd for
C16H12O7Na: MNa+, 339.0481. Found: MNa+, 339.0473.
3-(1-Carboxy-2-(3-hydroxyphenyl)vinyloxy)-2-hydroxybenzoic
acid 34. Potassium hydroxide (0.31 mL) was added to a solution
of diester 20 (12 mg, 0.03 mmol) in THF. Water (0.69 mL) was
then added and the reaction stirred at rt for 48 h. The mixture
was worked-up according to Method B, affording the desired
diacid 34 as a mixture of diastereomers as a white solid (5.5 mg,
56%, 63 : 37 Z : E). IR νmax (ATR): 3199 (br), 2575, 1687, 1469,
1239 cm−1; retention time: 25.58 min; mp 120–122. Z: 1H NMR
(400 MHz, MeOD) δ 7.54 (1H, dd, J 1.5, 8.0 Hz, H-6), 7.31
(1H, s, CvCHPh), 7.26–7.22 (1H, m, Ar-H), 7.21–7.17 (1H, m,
Ar-H), 7.16 (1H, t, J 7.8 Hz, Ar-H), 6.96 (1H, dd, J 1.5, 8.0 Hz,
H-4), 6.78–6.75 (1H, m, Ar-H), 6.74 (1H, t, J 8.0 Hz, H-5); 13C
NMR (100 MHz, MeOD) δ 174.0 (CvO), 166.3 (CvO), 158.7
(C), 153.2 (C), 146.3 (C), 141.9 (C), 135.2 (C), 130.6 (CH),
127.9 (CH), 125.0 (CH), 123.0 (CH), 120.3 (CH), 119.0 (CH),
117.9 (CH), 117.7 (CH), 115.7 (C); E: 1H NMR (400 MHz,
MeOD) δ 7.68 (1H, dd, J 1.6, 8.0 Hz, H-6), 7.26–7.22 (1H, m,
H-4), 7.09 (1H, t, J 8.0 Hz, Ar-H), 6.85 (1H, t, J 8.0 Hz, H-5),
6.83–6.77 (2H, m, Ar-H), 6.67 (1H, dd, J 2.2, 8.0 Hz, Ar-H),
6.30 (1H, s, CvCHPh); 13C NMR (100 MHz, MeOD) δ 173.8
(CvO), 166.8 (CvO), 158.2 (C), 154.5 (C), 146.6 (C), 145.2
(C), 136.3 (C), 130.0 (CH), 127.0 (CH), 125.5 (CH), 121.4
3-(2-(3-Trifluoromethylphenyl)-1-carboxyvinyloxy)-2-hydroxy-
benzoic acid 37. Potassium hydroxide (0.75 mL) was added to a
solution of diester 23 (25 mg, 0.06 mmol) in THF (0.75 mL).
Water (0.40 mL) was then added and the reaction stirred at rt for
48 h. The mixture was worked-up according to Method B,
affording the desired diacid 37 as a mixture of diastereomers as a
colourless oil (19 mg, 80%, 77 : 23 Z : E). IR νmax (ATR): 3072
(br), 2542, 1666, 1465, 1329, 1120 cm−1; retention time:
34.11 min; Z: 1H NMR (500 MHz, MeOD) δ 8.07 (1H, s,
CvCHPh), 7.95 (1H, app. d, J = 7.8 Hz, Ar-H), 7.56–7.51 (3H,
m, Ar-H + H-6), 7.41 (1H, app. s, Ar-H), 7.00 (1H, dd, J 1.4,
8.0 Hz, H-4), 6.75 (1H, t, J 8.0 Hz, H-5); 13C NMR (125 MHz,
MeOD) δ 173.6 (CvO), 165.8 (CvO), 153.2 (C), 146.0 (C),
2
143.8 (C), 132.1 (C, q, JC–F 32 Hz), 130.5 (CH), 129.7 (CH),
3
3
127.6 (CH, q, JC–F 4.0 Hz), 126.7 (CH, q, JC–F 3.7 Hz), 125.3
1
(CH), 125.2 (CH), 125.4 (C, q, JC–F 272 Hz), 121.0 (CH),
119.2 (CH), 115.6 (C) 1 signal obscured; E: 1H NMR
(400 MHz, MeOD) δ 7.70 (1H, dd, J 1.4, 8.0 Hz, Ar-H), 7.64
(1H, s, Ar-H), 7.60 (1H, app. d, J 7.9 Hz, H-6), 7.56–7.51 (1H,
m, Ar-H), 7.46 (1H, t, J 7.8 Hz, Ar-H), 7.29 (1H, dd, J 1.4, 8.0
9234 | Org. Biomol. Chem., 2012, 10, 9223–9236
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Hz, H-4), 6.89 (1H, t, J 8.0 Hz, H-5), 6.39 (1H, s, CvCHPh);
13C NMR (125 MHz, MeOD) δ 173.5 (CvO), 165.8 (CvO),
154.6 (C), 147.7 (C), 144.7 (C), 132.2 (CH), 132.0 (CH), 131.4
80. Freshly seeded cultures were grown at 37 °C, for approxi-
mately 14 days, to mid-exponential phase (OD600 0.4–0.8) for
use in the inhibition assays. The effect of the inhibitors against
M. tuberculosis growth were measured by a resazurin reduction
microplate assay, using the procedure previously described by
Taneja and Tyagi.22 M. tuberculosis grown to mid-exponential
phase was diluted to OD600 0.002 in 7H9S media (Middlebrook
7H9 with OADC, 0.5% glycerol, 0.02% tyloxapol, 1% tryptone)
containing 0.5% DMSO; 96-well microtiter plates were set up
with 100 μL inhibitors, serially diluted into 7H9S. Diluted M.
tuberculosis (100 μL, representing ∼2 × 104 CFU mL−1) was
added to each well. Plates were incubated for 5 days at 37 °C in
a humidified incubator prior to the addition of a 0.02% resazurin
solution (30 μL) and 20% Tween-80 (12.5 μL) to each well.
Sample fluorescence was measured after 48 h on a BMG
Labtech Polarstar Omega instrument with an excitation wave-
length of 530 nm and emission at 590 nm. Changes in fluor-
escence relative to positive control wells (H37Ra with no
inhibitor) minus negative control wells (no H37Ra) were plotted
for determination of MIC50 values.
2
3
(C, q, JC–F 36 Hz), 127.4 (CH), 126.6 (CH, q, JC–F 4.0 Hz),
3
1
126.5 (CH), 125.0 (CH, q, JC–F 3.7 Hz), 125.7 (C, q, JC–F
272 Hz), 119.7 (CH), 118.2 (CH), 116.3 (C) 1 signal obscured;
LRMS [M + H]+ 367.47; HRMS calcd for C17H11F3O6Na:
MNa+, 391.0405. Found: MNa+, 391.0400.
3-(2-(2-Trifluoromethylphenyl)-1-carboxyvinyloxy)-2-hydroxy-
benzoic acid 38. Potassium hydroxide (0.65 mL) was added to a
solution of pure Z diester 24 (22 mg, 0.05 mmol) in THF
(0.65 mL). Water (0.40 mL) was then added and the reaction
stirred at rt for 48 h. The mixture was worked-up according to
Method B, affording the desired pure Z diacid 38 as a colourless
oil (16 mg, 75%). IR νmax (ATR): 3439 (br), 2517, 1647, 1466,
1314, 1113 cm−1; retention time: 32.54 min; 1H NMR
(500 MHz, MeOD) δ 8.06 (1H, app. d, J = 8.0 Hz, Ar-H), 7.71
(1H, app. d, J 7.8 Hz, Ar-H), 7.60 (1H, s, CvCHPh), 7.56–7.51
(2H, m, Ar-H + H-6), 7.45 (1H, t, J 8.0 Hz, Ar-H), 6.96 (1H,
app. d, J 8.0 Hz, H-4), 6.73 (1H, t, J 8.0 Hz, H-5); 13C NMR
(125 MHz, MeOD) δ 173.7 (CvO), 165.7 (CvO), 153.2 (C),
146.3 (C), 144.6 (C), 133.1 (CH), 132.3 (CH), 130.0 (CH),
Acknowledgements
2
3
129.4 (C, q, JC–F 30 Hz), 126.8 (2 × CH, q, JC–F 5.8 Hz),
1
125.3 (CH), 125.6 (C, q, JC–F 276 Hz), 121.3 (CH), 119.1
We thank the Australian National Health and Medical Research
Council (NHMRC project grant 632769) and the New Zealand
Health Research Council (HRC project grant 06/441) for
financial support. We also thank The University of Sydney for a
Vice-Chancellor’s Research Scholarship (A.M.-T.).
(CH), 116.1 (C) 1 signal obscured; LRMS [M + H]+ 367.53;
HRMS calcd for C17H11F3O6Na: MNa+, 391.0405. Found:
MNa+, 391.0400.
Enzyme inhibition assays
Notes and references
Over-expression and purification of MbtI was achieved using the
method previously reported.16
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Inhibition constants against MbtI were measured by activity
assays in which the production of pyruvate from chorismate by
each enzyme was coupled to the oxidation of NADH using
lactate dehydrogenase (LDH). Loss of NADH was followed at a
UV absorbance of 340 nm. Assays were performed on a Cary
4000 UV spectrophotometer (Varian) equipped with a 6 ×
6 multi-cell holder using 1 cm pathlength quartz cuvettes
and temperature control (25 °C). MbtI activity assays consisted
of 50 mM Tris·HCl (pH 8), 5 mM MgCl2, 0.25 mM
NADH, 2 mg mL−1 LDH, 40 mg mL−1 MbtI, and 1–64 mM
chorismate.
Inhibitors were dissolved in water or DMSO depending on
solubility and added to assays at concentrations ranging from 50
nM to 2 mM. Assays were pre-incubated with inhibitor present
at 25 °C for 10 min and were initiated by the addition of choris-
mate. Reaction rates were measured by least-squares fitting of
the initial decrease in absorbance with time. Kinetic parameters
were determined by nonlinear fitting of reaction rates to a com-
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