Competitive inhibition of aristolochene synthase by phenyl-substituted farnesyl diphosphates: Evidence of active site plasticity

Article abstract of DOI:10.1039/b713301b

Analogues of farnesyl diphosphate (FPP, 1) containing phenyl substituents in place of methyl groups have been prepared in syntheses that feature use of a Suzuki-Miyaura reaction as a key step. These analogues were found not to act as substrates of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti (AS). However, they were potent competitive inhibitors of AS with K_I-values ranging from 0.8 to 1.2 M. These results indicate that the diphosphate group contributes the largest part to the binding of the substrate to AS and that the active sites of terpene synthases are sufficiently flexible to accommodate even substrate analogues with large substituents suggesting a potential way for the generation of non-natural terpenoids. Molecular mechanics simulations of the enzyme bound inhibitors suggested that small changes in orientations of active site residues and subtle alterations of the conformation of the backbones of the inhibitors are sufficient to accommodate the phenyl-farnesyl-diphosphates. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b713301b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Competitive inhibition of aristolochene synthase by phenyl-substituted
farnesyl diphosphates: evidence of active site plasticity†
David J. Miller, Fanglei Yu, Neil J. Young and Rudolf K. Allemann*
Received 29th August 2007, Accepted 3rd September 2007
First published as an Advance Article on the web 14th September 2007
DOI: 10.1039/b713301b
Analogues of farnesyl diphosphate (FPP, 1) containing phenyl substituents in place of methyl groups
have been prepared in syntheses that feature use of a Suzuki–Miyaura reaction as a key step. These
analogues were found not to act as substrates of the sesquiterpene cyclase aristolochene synthase from
Penicillium roqueforti (AS). However, they were potent competitive inhibitors of AS with KI-values
ranging from 0.8 to 1.2 lM. These results indicate that the diphosphate group contributes the largest
part to the binding of the substrate to AS and that the active sites of terpene synthases are sufficiently
flexible to accommodate even substrate analogues with large substituents suggesting a potential way for
the generation of non-natural terpenoids. Molecular mechanics simulations of the enzyme bound
inhibitors suggested that small changes in orientations of active site residues and subtle alterations of
the conformation of the backbones of the inhibitors are sufficient to accommodate the
phenyl-farnesyl-diphosphates.
tural variety, all terpenes are derived from simple linear precursors
Introduction
such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP),
Terpenoids are the largest group of natural products with
and geranyl geranyl diphosphate (GGPP). Cyclisation of GPP to
immense diversity in their structure and function.1 There has
monoterpenes, FPP to sesquiterpenes and GGPP to diterpenes is
been significant interest in terpenoids as antifungal, antibacterial
accomplished by terpene synthases. These enzymes, many of which
and anticancer agents for the treatment of human disease with
multibillion-dollar sales worldwide. Despite their enormous struc-
share the mainly a-helical class I terpene fold, serve as high fidelity
templates that subtly channel conformation and stereochemistry
during the cyclisation reactions and are key to the generation of the
wide diversity in structure and stereochemistry found in terpenoids
(Fig. 1). They bind their respective substrates together with the
obligatory Mg2+-cofactor, catalyse the loss of the diphosphate
group and chaperone the reaction intermediates along complex
reaction pathways; often with exquisite specificity. Terpene syn-
thases catalyse highly regio- and stereospecific cyclisations, hydride
School of Chemistry, Main Building, Cardiff University, Park Place, Cardiff,
CF10 3AT, UK. E-mail: allemannrk@cardiff.ac.uk; Fax: +44 29 2087 4030
† Electronic supplementary information (ESI) available: General experi-
mental procedures and protocols for diphosphorylation of target alcohols.
Enzyme preparation, purification and kinetic characterisation of inhibitors
as well as plots of [I] versus apparent KM for FPP turnover by AS in the
presence of 19, 29, and 36. See DOI: 10.1039/b713301b
Fig. 1 Structure of FPP (1) and some examples of the complexity of natural products derived from it. The parent sesquiterpenes such as 2, 4, 6 and 8
may be further transformed by downstream metabolic processes into a diverse set of sesquiterpenoids such as PR toxin (3), artemisinin (5), parthenolide
(7), and gossypol (9).
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and methyl transfers as well as deprotonation reactions while at
the same time excluding solvent from the active site to prevent
premature quenching by water of the extremely reactive cationic
reaction intermediates. The majority of the characterised terpene
synthases form only one or a few products; however, there are some
enzymes that form a variety of products from a single substrate.
The d-selinene and c-humulene synathases from Abies grandis (the
grand fir) produce 34 and 52 sesquiterpenes, respectively.2,3
Clearly the exact arrangement of the amino acid residues in
the active site of individual terpene cyclases within a common
protein fold is crucial for the outcome of the reaction. Site
directed replacement of amino acids in and around the active
site of several terpene synthases has indicated that the product
distribution can often be altered.4–13 While these experiments
indicated the high adaptability of these enzymes, the products
generated by the mutant enzymes were always known natural
terpenoids. However, the plasticity of terpene synthases suggests
that they have the potential for the generation of novel “unnatural”
terpenes from unnatural prenyl-diphosphate analogues. While
biosynthetic restrictions imply that all prenyl-diphosphates carry
methyl substituents only, a synthetic approach to analogues of the
substituents of terpene cyclases currently allows the introduction
of a wide variety of substituents.
To test whether the active sites of terpene synthases can
accommodate alternate substrates we report here syntheses of
the FPP analogues containing phenyl substituents in place of the
methyl groups on C3 and C11 (Scheme 1). While (2E,6E,10E)-3,
7-dimethyl-11-phenyldodeca-2,6,10-trien-1-yl diphosphate (19),
(2E,6E,10Z)-3,7-dimethyl-11-phenyldodeca-2,6,10-trien-1-yl di-
phosphate (29) and (2Z,6E)-7,11-dimethyl-3-phenyldodeca-2,
6,10-trien-1-yl diphosphate (36) were not converted to products
by AS from Penicillium roqueforti, all three FPP analogues acted
as potent competitive inhibitors indicating that they bound to
the active site of this sesquiterpene cyclase in a fashion similar to
the natural substrate, thereby highlighting the enormous plasticity
of these enzymes and their potential for the production of novel
terpenoid analogues that might have superior properties for many
applications including the treatment of human disease.
disconnection used here. Previous syntheses of analogues of
farnesol concentrated on modification of the C15 methyl group14–18
and to some extent the C1419 with little attention to the C12 and
C13 methyl groups of FPP. These syntheses have largely used the
elegant and robust chain extension methodology of Weiler and
Sum as a key step.20 For the syntheses of the compounds required
here, a synthesis allowing changes to all the methyl groups of FPP
was required and it seemed that a disconnection that exploits the
oligomeric nature of FPP would be both appropriate and efficient.
Hence two series of monomers were envisaged with one group
being composed of 1,3-dienes representing the C-terminal end of
the farnesyl group and the other set comprising various E-crotonyl
iodides. The key forward reaction to connect these two monomers
would involve transformation of the terminal alkenyl group with
a hindered borane such as 9-BBN followed by Suzuki–Miyaura
coupling with the appropriate iodide.21–25
Synthesis of E- and Z-11-phenyl farnesyl diphosphate analogues
Both of these compounds were prepared using novel Suzuki–
Miyaura coupling methodology. The synthesis of the E-11-phenyl
FPP analogue 19 is shown in Scheme 2. The C-terminal 1,3-diene,
compound 12b, was prepared from acetophenone 10 in four steps.
Horner–Emmons modification using triethyl phosphonoacetate
and sodium hydride followed by DIBAL-H reduction yielded
the E-cinnamyl alcohol 11, then oxidation using PCC and then
a Wittig reaction using methyltriphenylphosphonium bromide
gave the required E-phenyl diene 12b. This compound was then
hydroborated using crystalline 9-BBN and immediately coupled
to iodide 13 using PdCl2dppf as catalyst, triphenylarsine as co-
ligand and aqueous sodium hydroxide as base.24 The E-phenyl-
geranyl ester product 14 was isolated in 59% yield under these
conditions. Ester 14 was then homologated to a new 1,3-diene
derivative 16b in a very similar manner to the preparation of
12b and a second Suzuki coupling to iodide 13 under identical
conditions gave the farnesyl ester analogue 17 in 54% yield for the
coupling step. Ester 17 was then transformed into the diphosphate
19 by DIBAL-H reduction, bromination of the resulting alcohol
and diphosphorylation using the methodology of Poulter et al.26
Preparation of the Z-11-phenyl FPP analogue was achieved
in much the same fashion (Scheme 3). Preparation of the initial
Z-1,3-diene 23b was a little more complex because Horner–
Emmons or Wittig modification of acetophenone 10 will only
yield a small amount of the Z-ester 21. It was therefore made
by Suzuki–Miyaura coupling of phenylboronic acid to Z-crotonyl
iodide 20, which was an intermediate in the preparation of 13.27
Hence treatment of 20 with phenylboronic acid in the presence of
Pd(OAc)2, triphenylarsine and K3PO4 in toluene at 90 ◦C yielded
ester 21 in 44% yield after chromatography.28 This ester was then
transformed into the 1,3-diene 23b and then to the Z-11-phenyl
FPP analogue 29 using almost identical chemistry as used for the
preparation of 19. The two crucial Suzuki–Miyaura coupling steps
took place in approximately 40% yield in each case.
Scheme 1
Results
In order to explore the plasticity of terpene cyclases and to in-
vestigate their potential for the synthesis of non-natural terpenoid
analogues from modified prenyl diphosphates, we have synthesised
three FPP analogues in which the methyl groups on C3 and C11
were replaced by phenyl substituents. The general structure of
these analogues is shown in Scheme 1 along with the retrosynthetic
Synthesis of 3-phenyl farnesyl diphosphate 36
For the synthesis of 36 the chain extension methodology of Weiler
and Sum was found most effective (Scheme 4).20 Hence geraniol
30 was transformed into geranyl bromide and then treated with
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Scheme 2 Synthesis of the E-11-phenylfarnesyl diphosphate derivative 19. Reagents and conditions: (i) triethylphosphonoacetate, NaH, DME, 61%;
(ii) DIBAL-H, THF, −78 ◦C, 96%; (iii) PCC, CH2Cl2, 56%; (iv) CH3PPh3Br, n-BuLi, THF, 94%; (v) 9-BBN, THF then 13, PdCl2dppf, NaOH, AsPh3,
THF, 50 ◦C, 59%; (vi) TPAP, NMO, CH3CN, 89%; (vii) CH3PPh3Br, n-BuLi, THF, 81%; (viii) 9-BBN, THF then 13, PdCl2dppf, NaOH, AsPh3, THF,
50 ◦C, 54%; (ix) DIBAL-H, THF, −78 ◦C, 86%; (x) NEt3, MsCl, −45 ◦C then LiBr; (xi) (Bu4N)3HP2O7, CH3CN then cation exchange Bu4N+/NH4
,
+
31%.
Scheme 3 Synthesis of Z-11-phenylfarnesyl diphosphate 29. Reagents and conditions: (i) PhB(OH)2, Pd(OAc)2, AsPh3, K3PO4, toluene, 90 ◦C, 44%;
(ii) DIBAL-H, THF, −78 ◦C, 99%; (iii) oxalyl chloride, DMSO, CH2Cl2 −78 ◦C then NEt3, 88%; (iv) CH3PPh3Br, n-BuLi, THF, 76%; (v) 9-BBN, THF
then 13, PdCl2dppf, NaOH, AsPh3, THF, 50 ◦C, 41%; (vi) DIBAL-H, THF. −78 ◦C, 91%; (vii) TPAP, NMO, CH3CN, 87%; (viii) CH3PPh3Br, n-BuLi,
THF, 86%; (ix) 9-BBN, THF then 13, PdCl2dppf, NaOH, AsPh3, THF, 50 ◦C, 40%; (x) DIBAL-H, THF, −78 ◦C, 77%; (xi) NEt3, MsCl, −45 ◦C then
LiBr; (xii) (Bu4N)3HP2O7, CH3CN then cation exchange Bu4N+/NH4+, 36%.
the dienolate 31 derived from ethyl acetoacetate20 giving the b-
ketoester 32 in 92% yield. This was then treated with triflic
anhydride and KHMDS at −78 ◦C in THF to give the Z-enol
triflate compound 33 in 51% yield.16 This compound underwent a
Suzuki–Miyaura cross-coupling reaction with phenylboronic acid
to give 34 in 66% yield. Derivatisation of this compound to the
diphosphate analogue 36 was achieved in the usual manner.26
Studies of the kinetics of AS-catalysis in the presence of 19, 29
and 36
To test whether the active site of terpene synthases could accom-
modate the size increases resulting from the replacement of a
methyl with a phenyl group, the farnesyl diphosphate analogues
19, 29 and 36 were tested as substrates for and as inhibitors of
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Scheme 4 Synthesis of 3-phenylfarnesyl diphosphate 36. Reagents and conditions: (i) NEt3, MsCl, THF, −45 ◦C then LiBr, then 31, 92%; (ii) KHMDS,
(CF3SO2)2O, THF, −78 ◦C, 51%; (iii) PhB(OH)2, AsPh3, Pd(OAc)2, Ag2O, THF, D, 66%; (iv) DIBAL-H, THF, −78 ◦C, 85%; (v) NEt3, MsCl, −45 ◦
C
then LiBr; (vi) (Bu4N)3HP2O7, CH3CN then cation exchange Bu4N+/NH4+, 31%.
aristolochene synthase. AS is a Mg2+ dependent sesquiterpene
cyclase that catalyses the conversion of FPP to (+)-aristolochene
(2), which in the fungus is further transformed to produce a family
of toxins that includes PR toxin (3) (Fig. 1).29–31 The catalytic
mechanism for the formation of 2 in the active site of AS has been
studied extensively by classical substrate labelling studies as well
as by analysing the reaction products generated from substrate
analogues and by enzyme mutants.9–11,32,33 The reaction proceeds
through the intermediate eudesmane cation (38), the positive
charge of which is stabilised through interaction with the indole
ring of Trp 334 of AS. Hydride and methyl shifts followed by site-
specific deprotonation generate the bicyclic product (Scheme 5).
FPP).5,29 The concentrations of the phenyl-FPP analogues were
varied between 1 mM and 1 nM. 19, 29 and 36 acted as inhibitors
of AS with IC50 values ranging from 1–5 lM. Therefore full Ki
determinations were performed in order to examine the mode
of inhibition of these compounds. The Michaelis constant of
FPP was determined both in the presence and absence of each
inhibitor at various concentrations by use of a non-linear fit.‡
Double reciprocal plots for each set of fitted data indicated that the
compounds were all reversible competitive inhibitors of AS (Fig. 2)
and hence bound to the enzyme’s active site in a way similar to that
of the natural substrate. The KI-values were determined as 0.8
0.2, 1.2 0.2 and 1.2 0.1 lM for 19, 29, and 36, respectively (see
ESI†). No time dependent inactivation of the enzyme was observed
when each inhibitor was preincubated with enzyme prior to the
assay indicating that the inhibition was reversible in all three cases.
Molecular mechanics simulations of inhibitor-bound AS
Each of the FPP-analogues 19, 29 and 36 are characterised by
the replacement of a methyl group by the much bulkier phenyl
substituent. Despite the high structural similarities observed in
sesquiterpene synthases, that suggest that these enzymes have
evolved to facilitate subtle conformational changes of their
common substrate through the positional reorganisation of only
a limited number of active site residues, the observation that
the phenyl-substituted farnesyl-diphosphates acted as potent
competitive inhibitors of AS was somewhat surprising.
Each inhibitor was therefore docked to the active site of AS
using the existing molecular model of 1 bound to AS34 as a
starting point. Energy minimisations of the docked structures were
performed using the MMFF94 forcefield.35 Amino acids within
Scheme 5 Proposed catalytic mechanism of AS from P. roqueforti.
The phenyl-FPPs 19, 29 and 36 were incubated at a concentra-
tion of 200 lM with 100 nM aristolochene synthase at pH 7.5 in
the presence of the essential cofactor Mg2+ at 25 ◦C. The reaction
rate for the conversion of 5 lM FPP was proportional to the con-
centration of AS at this concentration of enzyme.5,11 Incubations
were overlayed with pentane in order to extract potential products
and to minimise product inhibition that is often observed with
these enzymes due to the hydrophobic nature of the terpenoid
products. The organic phase was concentrated carefully and the
reaction products analysed by GC-MS. While the production of
aristolochene from FPP was manifest by the strong GC signal
and by its mass spectrum after only 16 hours, even prolonged
incubation with 19, 29 and 36 of up to 7 days did not indicate
the formation of any products as judged by GC-MS analysis
suggesting that these compounds were not substrates of AS.
Each compound was examined as an inhibitor of AS using a
radiolabelled assay. Initially, IC50 values were determined with
FPP concentrations maintained at 1 lM (close to the KM of
˚
6.5 A of the inhibitor molecule were allowed to move while the co-
ordinates of all other residues were fixed. Each inhibitor appeared
to fit well into the active site of AS and only minor reorganisations
of active site residues were necessary to avoid steric clashes through
the introduction of the bulky phenyl substituents (Fig. 3). In
‡ Data were fitted using Systat Sigmaplot 10.0, 2007. Sigmaplot for
Windows Version 10.0, Build 10.0.0.54, 2006, Systat Software Inc. 1735,
Technology Drive, Ste 430, San Jose, CA 95110, USA. Molecular
Operating Environment (MOE 2004.03) Chemical Computing Group,
Inc., 1255 University St. Suite 1600, Montreal, Quebec, Canada. H3B
3 × 3.
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Fig. 2 Double reciprocal plots of initial rates versus the concentration of
substrate for AS catalysed turnover of FPP in the presence of 19, 29 and 36
are shown on panels a, b and c for increasing concentrations of inhibitor
(0 lM (᭹), 1 lM (ꢀ), 2 lM (᭜) and 3 lM (ꢁ)). Intersection of the lines
on the y-axis indicate that each compound is a competitive inhibitor of
AS. All assays were carried out at 37 ◦C and pH 7.5.
addition, some rearrangements of the inhibitorsꢀ prenyl-chains
were observed relative to the conformation calculated for FPP. In
the E-11-phenyl FPP analogue (19) an alteration in the orientation
of the C7–C11 portion of the prenyl chain and a movement of
the diphosphate group towards the Tyr 92 residue of AS was
observed. This was accompanied by an approximate 90◦ rotation
of the phenyl ring of Phe 112 and minor rotations of the rings of
Phe 178 and Tyr 92. The indole ring of Trp 334 moved slightly
away from the bound substrate analogue. The phenyl ring of 19
and that of Phe 178 appear to be in reasonably close proximity in
this simulation.
The Z-11-phenyl FPP analogue (29) showed an entirely different
set of movements. In this instance Trp 334 did not move at all.
Most interestingly, there appears to be a possible p–p stacking
arrangement between the phenyl ring of 29 and Phe 112 aided
by a substantial movement of the side chain of Phe 112. In this
case the overall fold of the prenyl chain was very similar to that
observed for the substrate with a more modest movement of the
diphosphate group relative to that observed for 19. Again Phe 178
and Tyr 92 show small rotations upon binding of the analogue
relative to their positions adopted upon binding of FPP.
Fig. 3 Sketches from molecular mechanics simulations of the active sites
of AS complexed with (2E,6E,10E)-3,7-dimethyl-11-phenyldodeca-2,6,
10-trien-1-yl diphosphate (19) (a), (2E,6E,10Z)-3,7-dimethyl-11-phenyl-
dodeca-2,6,10-trien-1-yl diphosphate (29) (b) and (2Z,6E)-7,11-dimethyl-
3-phenyldodeca-2,6,10-trien-1-yl diphosphate (36) (c). The substrate and
the inhibitors as well as key amino acid residues are shown in gold for the
original structure and in blue for the energy minimised structures of the
inhibitor complexes of AS.
movement for the compounds in this study. Trp 334, Phe 178 and
Tyr 92 displayed only minor rearrangements; the phenyl ring of
Phe 178 was rotated by approximately 45◦ relative to its position in
the substrate complex. The overall fold of the prenyl chain in the
inhibitor complex was similar to that observed for the substrate
with the exception of the C4–C7 portion of the chain where the
apex of the phenyl group of 36 adopts an almost identical position
to that of C3 of FPP, thereby forcing the C4–C7 chain downwards
(Fig. 3) to accommodate the rest of the bulky phenyl residue in
the active site.
The simulation of the binding of 36 indicated that the binding
of this compound to FPP requires the least amount of active site
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The results described here show that the active site geometry of
terpene synthases is sufficiently flexible to accommodate substrate
analogues even when these carry large pendant groups. The
plasticity of the terpene cyclases appears not only to provide
the framework for the combinatorial production of many natural
terpenoids through subtle alterations in the composition of the
active site during evolution but may also allow modifications of
the active site residues by site directed or random mutagenesis
in vitro or in vivo for the production of functional enzymes that
convert FPP analogues to unnatural “terpenoids”.
Discussion
Three analogues of farnesyl diphosphate have been prepared
using novel methodology that involves
a Suzuki–Miyaura
cross-coupling reaction as a key step in the synthesis. These
coupling reactions proceed in moderate to good yield. It should be
possible to use this technology to prepare combinatorial libraries
of FPP analogues in the future. Some members of such libraries
may act as substrates for AS and other terpene cyclases thereby
opening up the possibility of short economical routes to complex,
synthetic unnatural analogues of terpenoids.
While the replacement of the methyl groups on C11 and C3
of FPP with phenyl substituents did not produce substrates for
AS, compounds 19, 29 and 36 proved to be potent competitive
inhibitors of AS that acted in a reversible fashion. The most
potent of these inhibitors was the E-11-phenyl-FPP analogue
19 with a KI of 0.8 lM, which is comparable to the KM of
the natural substrate5,9 and to the inhibitory constant of 12,13-
difluoro-FPP.36 Given the relatively size neutral substitutions in
12,13-difluoro-FPP, the tight binding of the phenyl substituted
FPP analogues to AS clearly indicate the remarkable plasticity of
this enzyme’s active site. Furthermore, farnesyl thiodiphosphate,
an analogue of FPP where the oxygen attached directly to the
farnesyl chain was replaced by a sulfur, was a much more
weakly bound inhibitor of this enzyme (KI = 10 lM) (Beyer
and Allemann, unpublished). Replacing the oxygen with sulfur
in the diphosphate group clearly reduces the binding energy more
significantly than a change from a methyl to a phenyl group in
the prenyl chain. The majority of the binding energy seems to
stem from the interaction of the charged diphosphate group to the
Mg2+ binding site while the interaction of the aliphatic chain with
the hydrophobic pocket of the active site contributes a smaller
amount.
Experimental
For general experimental procedures, final synthesis of diphos-
phates, enzyme preparation and purification and kinetic charac-
terisation of inhibitors see ESI.†
(E)-3-Phenylbut-2-en-1-ol (11)37
To a stirred solution of sodium hydride (4.40 g, 110 mmol)
in anhydrous DME (200 cm3) at room temperature under N2,
triethyl phosphonoacetate (21.8 cm3, 110 mmol) and a solution
of acetophenone (11.7 cm3, 100 mmol) in anhydrous DME were
sequentially added, dropwise. After 3 h, water (50 cm3) was added
and the organic layer was separated. The aqueous layer was
extracted with diethyl ether (3 × 50 cm3). The combined organic
phases were washed with brine (50 cm3), dried over MgSO4,
filtered and then concentrated under reduced pressure to give the
intermediate ester as a pale yellow oil (11.6 g, 61%); TLC Rf 0.39
(hexane–EtOAc = 9 : 1); HRMS (ES+, [M + H]+) found 191.1067,
C12H15O2 requires 191.1067; mmax(thin film)/cm−1 2980.1, 1713.0,
1628.4, 1576.4, 1494.0, 1446.0, 1365.9, 1343.8, 1272.6, 1171.3,
1044.1, 872.4, 766.9 and 694.9; dH (500 MHz, C2HCl3) 1.23 (3 H,
t, J 7.5, CH3CH2O), 2.50 (3 H, d, J 1.5, CH3CPh), 4.13 (2 H, q,
Analysis of the X-ray crystal structure of AS indicates that the
indole ring of Trp 334 is positioned close to C3 of FPP.7,34 The
GC-MS analysis of the sesquiterpenes produced by mutants of AS
in which this residue was replaced by non aromatic amino acids
indicated that the indole ring was involved in the stabilisation
of the positive charge build up on C3 during formation of the
eudesmane cation (38).7 The tight binding of 36, where the methyl
group on C3 was replaced with a phenyl substitutent was therefore
most interesting. No steric clash between the two aromatic groups
appeared to prevent tight binding of the inhibitor. The precise
geometry of binding of these compounds in the active site of
AS is currently being studied by X-ray crystallography of the
inhibitor complexes. However, our molecular modelling studies
of AS bound to the 19, 29 and 36 suggest possible binding modes
for these competitive inhibitors. AS appears to be sufficiently
flexible to accommodate the extra bulk of the phenyl groups
in the active site. The reorganisation of the active site residues,
which is accompanied by alteration of the conformation of the
prenyl chain, does however lead to loss of catalytic activity.
This may be a consequence of changes in the position of the
diphosphate group in each simulation thereby preventing the
initial diphosphate loss and hence cyclisation of FPP. In addition,
the active site conformation of the prenyl chain has been shown
to be critical for AS catalysis6,36 and the rearrangement of the
substrate analogue necessary to accommodate the phenyl ring,
may lead to an unreactive conformation.
=
J 7.5, CH3CH2O), 6.05 (1 H, q, J 1.5, PhC CH) and 7.27–7.40
(5 H, m, Ar–H); dC (125 MHz, C2HCl3) 14.4 (CH3CH2O), 18.0
=
=
(CH3C CH), 59.8 (CH3CH2O), 117.2 (PhC CH), 126.3, 128.4
and 129.0 (Ar–CH), 142.3 and 155.5 (quaternary C) and 166.9
+
+
=
(C O); m/z (ES ) 191.1 (100%, [M + H] ).
The ester (9.08 g, 47.8 mmol)◦was dissolved in anhydrous
THF (50 cm3) and cooled to −78 C (acetone–dry ice bath). To
this stirred solution, under N2, was added diisobutylaluminium
hydride (1.0 M solution in hexanes, 152 cm3, 152 mmol) dropwise
over 10 min. This solution was stirred for 2 h at −78 ◦C then
allowed to warm to 0 ◦C. Saturated potassium sodium tartrate
solution (50 cm3) and diethyl ether (50 cm3) were added. The
mixture was stirred at room temperature for another 30 min, and
the organic layer was separated. The aqueous layer was extracted
with diethyl ether (2 × 50 cm3). The combined ethereal extracts
were washed with brine (150 cm3), dried over MgSO4, filtered and
then concentrated under reduced pressure. Purification by flash
chromatography on silica gel with hexane and ethyl acetate (2 :
1) gave 11 as a light yellow oil (6.7 g, 96%); Rf 0.27 (hexane–
EtOAc = 2 : 1); HRMS: (EI+, M+) found 148.0890, C10H12O
requires 148.0888; mmax(thin film)/cm−1 3347.2, 2922.2, 2597.9,
1493.8, 1444.5, 1379.9, 1003.0, 758.1 and 696.1; dH (500 MHz,
C2HCl3) 2.11 (3 H, s, CH3CPh), 4.40 (2 H, d, J 6.5, CH2OH),
=
6.01 (1 H, t, J 6.5, PhC CH) and 7.28–7.45 (5 H, m, Ar–H);
dC (125 MHz, C2HCl3) 16.1 (CH3CPh), 60.0 (CH2OH), 126.5
3292 | Org. Biomol. Chem., 2007, 5, 3287–3298
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=
(PhC CH), 125.8, 127.3 and 128.3 (Ar–CH) and 137.8 and 142.9
PdCl2dppf (0.37 g, 0.45 mmol) and aqueous NaOH (6.0 M.
9.92 cm3, 39.7 mmol) were added in quick succession, the complete
solution was then stirred at 50 ◦C for 15 h. After cooling to
room temperature, aqueous hydrogen peroxide solution (30%,
15 cm3) was carefully added and the solution was stirred for a
further 30 min. Water (30 cm3) and diethyl ether (30 cm3) were
added, and the organic layer was separated. The aqueous layer was
extracted with diethyl ether (2 × 25 cm3). The combined ethereal
extracts were washed with water (2 × 20 cm3) and brine (20 cm3),
dried over MgSO4, filtered and then concentrated under reduced
pressure. Purification by flash chromatography on silica gel with
hexane and ethyl acetate gave 14 as a light yellow oil (1.51 g,
59%); Rf 0.27 (hexane–EtOAc = 19 : 1); HRMS: (ES+, [M +
NH4]+) found 276.1959, C17H26NO2 requires 276.1958; mmax(thin
film)/cm−1 2978.8, 2930.2, 1714.9, 1647.6, 1493.8, 1444.3, 1381.4,
1327.6, 1272.3, 1222.8, 1146.1, 1098.9, 1049.6, 864.4, 757.9 and
696.4; dH (500 MHz, C2HCl3) 1.20 (3 H, t, J 7.0, CH3CH2O), 1.96
(quaternary C); m/z (EI+) 148.1 (13%, M+) and 115.1 (100).
(E)-3-Phenylbut-2-enal (12a)
Pyridinium chlorochromate (12.3 g, 56.0 mmol) was suspended in
anhydrous CH2Cl2 (50 cm3) then a solution of 11 (6.7 g, 45 mmol)
in CH2Cl2 (50 cm3) was added in one portion to the stirred
suspension. After 4 h, dry diethyl ether (100 cm3) was added and
the supernatant liquid was decanted from the resulting black gum.
The insoluble residue was washed with diethyl ether (100 cm3) and
became a black granular solid. The organic phases were combined,
washed with brine (300 cm3), dried over MgSO4, filtered and then
concentrated under reduced pressure to give 12a as a yellow oil
(3.7 g, 56%); Rf 0.37 (hexane–EtOAc = 4 : 1); HRMS: (EI+, M+)
found: 146.0730, C10H10O requires 146.0732; mmax(thin film)/cm−1
1722.2, 1659.7, 1446.2, 1377.3, 1248.1, 1144.5, 865.5 and 758.6; dH
(500 MHz, C2HCl3) 1.94 (3 H, d, J 1.0, CH3CPh), 6.41 (1 H, dq,
=
(3 H, d, J 1.0, CH3C CHCO2Et), 2.13 (3 H, d, J 1.0, CH3CPh),
=
J 7.5, J 1.0, PhC CH), 7.10–7.18 (5 H, m, Ar–H) and 10.05 (1 H,
2.19–2.32 (4 H, m, CH2CH2), 4.07 (2 H, q, J 7.0, CH3CH2O),
d, J 7.5, CHO); dC (125 MHz, C2HCl3) 15.5 (CH3CPh), 127.4
=
5.64 (2 H, m, 2 × C CH) and 7.13–7.30 (5 H, m, Ar–H); dC
=
(PhC CH), 126.2, 128.5 and 129.5 (Ar–CH), 140.8 and 155.8
2
=
(125 MHz, C HCl3) 14.4 (CH3CH2O), 15.9 (CH3C CHCO), 18.9
(quaternary C) and 189.8 (CHO). m/z (EI+) 146.1 (46%, M+) and
(CH3CPh), 26.8 and 40.6 (CH2CH2), 59.6 (CH3CH2O), 115.9
145.1 (100).
=
=
(C CHCO), 126.66 (PhC CH), 125.7, 126.73 and 128.2 (Ar–
=
CH), 135.8, 144.7, 159.3 (quaternary C) and 166.9 (C O); m/z
(E)-Penta-2,4-dien-2-ylbenzene (12b)
(CI+) 276.2 (100%, [M + NH4]+).
A stirred suspension of methyltriphenylphosphonium bromide
(7.61◦g, 21.3 mmol) in anhydrous THF (50 cm3) was cooled to
−78 C then n-BuLi (2.5 M, 8.52 cm3, 21.3 mmol) was added
dropwise under argon. The reaction mixture was allowed to warm
to 0 ◦C giving a clear deep yellow solution. After stirring at
(2E,6E)-3-Methyl-7-phenylocta-2,6-dien-1-ol (15)
To a stirred solution of 14 (1.46 g, 5.67 mmol) in anhydrous
THF (60 cm3) at −78 ◦C (acetone–dry ice bath) was added
diisobutylaluminium hydride (1.0 M solution in hexanes, 13.6 cm3,
13.6 mmol) dropwise. The resulting mixture was stirred at −78 ◦C
for 2 h and then allowed to warm to 0 ◦C at which time
the reaction was judged complete by TLC analysis. Saturated
potassium sodium tartrate solution (50 cm3) and diethyl ether
(50 cm3) were added. The mixture was stirred at room temperature
for another 30 min, and the organic layer was separated. The
aqueous layer was extracted with diethyl ether (2 × 30 cm3).
The combined ethereal extracts were washed with brine (30 cm3),
dried over MgSO4, filtered and then concentrated under reduced
pressure. Purification by flash chromatography on silica gel with
hexane and ethyl acetate (2 : 1) as eluent gave 15 as a light yellow oil
(1.2 g, 96%); Rf 0.27 (hexane–EtOAc = 2 : 1); HRMS: (ES+, [M +
NH4]+) found 234.1851, C15H24NO requires 234.1852; mmax(thin
0
◦C for 30 min, the aldehyde 12a (1.83 g, 12.6 mmol) was
added dropwise and the complete reaction mixture was stirred for
16 h whilst slowly warming to room temperature. Water (20 cm3)
and diethyl ether (20 cm3) were added and the organic layer was
separated. The aqueous layer was extracted with diethyl ether (2 ×
15 cm3). The combined ethereal extracts were washed with water
(2 × 20 cm3) and brine (20 cm3), dried over MgSO4, filtered and
then concentrated under reduced pressure. Purification by flash
chromatography on silica gel with hexane and ethyl acetate (9 :
1) as eluent gave 12b as a light yellow oil (1.70 g, 94%); Rf 0.57
(hexane–EtOAc = 19 : 1); HRMS: (EI+, M+) found 144.0938,
C11H12 requires 144.0939; mmax(thin film)/cm−1 3029.5, 2923.2,
1803.9, 1627.6, 1594.2, 1493.3, 1445.8, 1380.1, 1175.1, 1027.4,
985.3, 903.6, 759.8 and 694.8; dH (400 MHz, C2HCl3) 2.09 (3 H, s,
film)/cm−1 3363.0, 2923.4, 1493.3, 1444.0, 999.4, 756.3 and 695.8;
=
CH3CPh), 5.10 (1 H, d, J 10.0, CHCH CHtransHcis), 5.22 (1 H, d, J
2
=
dH (500 MHz, C HCl3) 1.65 (3 H, s, CH3C CHCH2OH), 1.96
=
=
17.0, CHCH CHtransHcis), 6.37 (1 H, d, J 11.0, CHCH CH2), 6.68
(3 H, d, J 0.9, CH3CPh), 2.09–2.29 (4 H, m, CH2CH2), 4.09 (2 H,
d, J 10.0, CH2OH), 5.40 (1 H, tq, J 10.0, J 1.5, CHCH2OH),
=
(1 H, dt, J 17.0, J 10.5, CHCH CH2) and 7.14–7.26 (5 H, m, Ar–
H); dC (100 MHz, C HCl3) 16.1 (CH3CPh), 117.7 (CHCH CH2),
127.8 (CHCH CH2), 125.7, 127.2 and 128.3 (Ar–CH), 133.6
2
=
5.68 (1H, tq, J 7.0, J 1.5, CHCH2CH2) and 7.13–7.31 (5 H,
=
2
=
m, Ar–H); dC (125 MHz, C HCl3) 15.9 (CH3C CHCH2OH),
+
=
(CHCH CH2) and 136.8 and 143.0 (quaternary C); m/z (EI )
16.4 (CH3CPh), 27.1 and 39.2 (CH2CH2), 59.4 (CH2OH), 123.7
144.1 (35%, M+) and 129.1 (100, [M − CH3]+).
=
(CHCH2OH), 127.7 (PhC CH), 125.6, 126.6 and 128.2 (Ar–CH)
and 135.0, 139.4, 143.9 (quaternary C); m/z (CI+) 234.2 (100%,
(2E,6E)-Ethyl 3-methyl-7-phenylocta-2,6-dienoate (14)
[M + NH4]+).
A mixture of 12b (1.45 g, 9.92 mmol) and 9-BBN (3.63 g,
14.9 mmol) dissolved in anhydrous THF (50 cm3) was stirred at
room temperature under N2 until all the starting material had
been consumed as judged by TLC (approx. 2 h). The iodide
1327 (2.38 g, 9.92 mmol), triphenylarsine (0.30 g, 0.99 mmol),
(2E,6E)-3-Methyl-7-phenylocta-2,6-dienal (16a)
A mixture of 15 (0.98 g, 4.52 mmol), N-methylmorpholine-N-
oxide (074 g, 6.33 mmol) and freshly activated powdered 4 A
˚
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molecular sieves (0.35 g) in anhydrous acetonitrile (35 cm3)
was stirred for 10 min whereupon tetra-n-propylammonium
perruthenate (81 mg, 0.23 mmol) was added. The reaction became
warm and was then stirred at room temperature for 16 h. The
(2E,6E,10E)-3,7-Dimethyl-11-phenyldodeca-2,6,10-trien-1-ol (18)
This compound was prepared from 17 in a manner identical to
that for the alcohol 15; purification by flash chromatography
using hexane and ethyl acetate (2 : 1) as eluent gave 18 as a light
yellow oil (0.41 g, 86%); Rf 0.31 (hexane–EtOAc = 2 : 1); HRMS
(EI+, M+) found 284.2141, C20H28O requires 284.2140; mmax(thin
film)/cm−1 3321.5, 2921.0, 1666.9, 1597.7, 1493.7, 1444.1, 1381.1,
1000.4, 846.3, 756.6 and 696.1; dH (500 MHz, C2HCl3) 1.57 (3 H, s,
R
mixture was filtered through a short pad of Celiteꢀ and the
solvent was concentrated under reduced pressure. Purification
by flash chromatography on silica gel with hexane and ethyl
acetate (4 : 1) as eluent gave 16a as a light yellow oil (0.86 g,
89%); Rf 0.33 (hexane–EtOAc = 4 : 1); HRMS: (EI+, M+) found
214.1350, C15H18O requires 214.1352; mmax(thin film)/cm−1 1672.0,
=
=
CH3C CH), 1.60 (3 H, s, CH3C CHCH2OH), 1.96 (3 H, d, J 1.0,
PhCCH3), 1.97–2.25 (8 H, m, 2 × CH2CH2), 4.04 (2 H, d, J 7.0,
1493.5, 1443.9, 1193.1, 1124.0, 757.7 and 696.2; dH (500 MHz;
=
CH2OH), 5.08 (1 H, dt, J 7.0, J 1.0, CH3C CH), 5.33 (1 H, m,
2
=
C6 H6) 1.62 (3 H, d, J 1.1, CH3C CHCHO), 1.92 (3 H, d, J 1.0,
=
=
C CHCH2OH), 5.68 (1 H, dt, J 7.0, J 1.5, PhC CH) and 7.12–
7.30 (5 H, m, Ar–H); dC (125 MHz, C2HCl3) 15.8, 16.1 and 16.3
(3 × CH3), 26.3, 27.4, 39.4 and 39.5 (2 × CH2CH2), 59.4 (CH2OH),
CH3CPh), 1.86–2.10 (4 H, m, CH2CH2), 5.64 (1 H, tq, J 7.0, J 1.0,
=
C CHCH2CH2), 5.95 (1 H, dq, J 7.5, J 1.0, CHCHO), 7.21–7.44
(5 H, m, Ar–H) and 9.98 (1 H, d, J 7.5, CHO); dC (125 MHz;
=
=
123.4 (CHCH2OH), 124.3 (CH3C CH) and 128.2 (PhC CH),
125.6, 126.5 and 128.2 (Ar–CH) and 134.6, 135.0, 139.7 and 144.0
(quaternary C); m/z (EI+) 284.2 (1%, M+), 131.1 (100), 266.2 (2,
[M − H2O]+).
2
=
C6 H6) 15.7 (CH3CPh), 16.7 (CH3C CHCHO), 26.3 and 39.8
=
(CH2CH2), 126.3 (PhC CH), 127.5 (CHCHO), 125.8, 127.0 and
128.4 (Ar–CH), 136.0, 143.8 and 160.9 (quaternary C) and 189.6
(CHO); m/z (CI+) 232.2 (100%, [M + NH4]+).
(Z)-Ethyl 3-phenylbut-2-enoate (21)27,28
(2E,7E)-8-Phenyl-4-methyl-nona-1,3,7-triene (16b)
To a stirred solution of the iodide 20 (7.20 g, 30.0 mmol) in
anhydrous toluene (100 cm3) under N2 was added palladium(II)
acetate (0.34 g, 1.50 mmol), triphenylarsine (0.79 g, 3.00 mmol),
tripotassium orthophosphate (19.1 g, 90.0 mmol) and phenyl-
boronic acid (5.49 g, 45.0 mmol). The complete reaction mixture
This compound was prepared from 16a in a manner identical
to that for the 12b; purification by flash chromatography using
hexane and ethyl acetate (9 : 1) as eluent gave 16b as a light
yellow oil (0.68 g, 81%); Rf 0.68 (hexane–EtOAc = 9 : 1); HRMS:
(EI+, M+) found 212.1559, C16H20 requires 212.1560; mmax(thin
film)/cm−1 2919.4, 1649.7, 1597.7, 1493.5, 1443.8, 1379.7, 987.8,
896.6, 756.3 and 695.3; dH (500 MHz, C2HCl3) 1.73 (3 H, s,
◦
was then stirred at 90 C for 6 h. Water (50 cm3) and diethyl
ether (50 cm3) were added, and the organic layer was separated.
The aqueous layer was extracted with diethyl ether (2 × 30 cm3).
The combined ethereal extracts were washed with water (2 ×
30 cm3) and brine (30 cm3), dried over MgSO4, filtered and
then concentrated under reduced pressure. Purification by flash
chromatography on silica gel with hexane and ethyl acetate (9 : 1)
as eluent gave 21 as a light yellow oil (2.49 g, 44%); Rf 0.30 (hexane–
EtOAc = 9 : 1); HRMS (ES+, [M + H]+) found 191.1067, C12H15O2
requires 191.1067; mmax (thin film)/cm−1 2979.6, 1725.4, 1639.5,
1492.5, 1442.6, 1374.8, 1277.2, 1230.2, 1162.1, 1095.6, 1076.6,
1047.3, 867.6, 768.4 and 698.1 cm−1; dH (500 MHz, C2HCl3) 1.14
(3 H, t, J 7.5, CH3CH2O), 2.23 (3 H, d, J 1.5, CH3CPh), 4.05 (2 H,
=
=
CH3C CHCH CH2), 1.92 (3 H, s, CH3CPh), 2.11–2.29 (4 H, m,
=
CH2CH2), 4.92 (1 H, d, J 10.0, CH CHCHtransHcis), 5.02 (1 H, dd,
=
=
J 17.0, J 1.5, CH CHtransHcis), 5.68 (1 H, t, J 7.0, C CHCH2CH2),
5.82 (1 H, d, J 11.0, CHCH CH2), 6.52 (1 H, dt, J 17.0, J
10.5, CH CH2) and 7.12–7.30 (5 H, m, Ar–H); dC (125 MHz,
C HCl3) 15.9 (CH3CPh), 16.8 (CH3C CHCH2CH2), 27.3 and
=
=
2
=
=
=
39.6 (CH2CH2), 114.9 (CH CH2), 125.8 (CHCH CH2), 127.8
=
=
(PhC CH), 125.7, 126.6 and 128.2 (Ar–CH), 133.4 (CH CH2)
and 135.0, 139.1 and 143.9 (quaternary C); m/z (CI+) 213.1 (100%,
[M + H]+).
=
q, J 7.5, CH3CH2O), 6.05 (1 H, q, J 1.5, PhC CH) and 7.26–7.42
(2E,6E,10E)-Ethyl-3,7-dimethyl-11-phenyldodeca-
2,6,10-trienoate (17)
(5 H, m, Ar–H); dC (125 MHz, C2HCl3) 14.0 (CH3CH2O), 27.2
=
(CH3CPh), 59.8 (CH3CH2O), 117.8 (PhC CH), 126.9, 127.8 and
127.9 (Ar–CH), 140.9 and 155.4 (quaternary C) and 165.9 (C O);
m/z (CI+) 208.1 (100%, [M + NH4]+) and 191.0 (35%, [M + H]+).
=
This compound was prepared from 16b in a manner identical to
that for the ester 14; purification by flash chromatography using
hexane and ethyl acetate (25 : 1) as eluent gave 17 as a light yellow
oil (0.56 g, 54%); Rf 0.32 (hexane–EtOAc = 25 : 1); HRMS (ES+,
[M + H]+) found 327.2320, C22H31O2 requires 327.2319; mmax(thin
film)/cm−1 2928.8, 1714.9, 1647.2, 1444.1, 1381.4, 1221.1, 1143.0,
757.1 and 695.9; dH (500 MHz, C2HCl3) 1.21 (3 H, t, J 6.5,
(Z)-3-Phenylbut-2-ene-1-ol (22)
This compound was prepared from 21 in a manner identical to
that for the alcohol 15; purification by flash chromatography using
hexane and ethyl acetate (2 : 1) as eluent gave 22 as a colourless oil
(4.12 g, 99%); Rf 0.26 (hexane–EtOAc = 2 : 1); HRMS (CI+, [M +
NH4]+) found 166.1229, C10H16NO requires 166.1226; mmax (thin
film)/cm−1 3331.7, 3055.2, 2969.9, 1656.1, 1600.0, 1493.6, 1434.8,
1376.0, 1246.4, 1065.2, 1002.0, 764.1 and 700.8; dH (500 MHz,
C2HCl3) 1.46 (1 H, b, OH), 2.02 (3 H, d, J 1.0, CH3CPh), 3.99 (2 H,
=
CH3CH2O), 1.50, 1.58 and 1.96 (3 × 3 H, s, 3 × CH3C CH),
2.02–2.24 (8H, m, 2 × CH2CH2), 4.07 (2 H, q, J 6.5, CH3CH2O),
=
5.08 and 5.67 (2 H, m, 2 × C CHCH2CH2), 5.60 (1 H, d, J 1.0,
CHCO2Et) and 7.13–7.30 (5 H, m, Ar–H); dC (125 MHz, C2HCl3)
=
14.4 (CH3CH2O), 15.8, 16.1 and 18.9 (3 × CH3C CH), 26.0, 27.4,
39.4 and 41.0 (2 × CH2CH2), 59.5 (CH3CH2O), 115.6 (CHCO2Et),
=
=
123.3 and 128.1 (C CH), 125.6, 127.0 and 128.2 (Ar–CH), 131.6,
dd, J 7.0, J 1.0, CH2OH), 5.64 (1 H, tq, J 7.0, J 1.5, PhC CH) and
+
7.09–7.28 (5 H, m, Ar–H); dC (125 MHz, C2HCl3) 25.4 (CH3CPh),
=
135.8, 144.0 and 159.8 (quaternary C) and 166.9 (C O); m/z (CI )
344.3 (100%, [M + NH4]+), 327.3 (65, [M + H]+).
60.3 (CH2OH), 126.1 (PhC CH), 127.2, 127.8 and 128.2
=
3294 | Org. Biomol. Chem., 2007, 5, 3287–3298
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(Ar–CH), 140.3 and 140.8 (quaternary C); m/z (CI+) 166.1 (20%,
[M + NH4]+), 148.1 (50, M+) and 131.0 (100, [M − OH]+).
m, C CHCH2CH2), 5.52 (1 H, d, J 0.5, C CHCO2Et) and 7.07–
7.30 (5 H, m, Ar–H); dC (125 MHz, C2HCl3) 14.4 (CH3CH2O), 18.7
=
=
=
(CH3CPh), 25.7 (CH3C CHCO2Et), 26.9 and 41.2 (CH2CH2),
59.5 (CH3CH2O), 115.7 (C CHCO2Et), 125.8 (PhC CH), 126.6,
127.9 and 128.2 (Ar–CH), 137.5, 141.8 and 159.4 (quaternary C)
=
=
(Z)-3-Phenylbut-2-ene-1-al (23a)
To a stirred solution of oxalyl chloride (2.69 cm3, 31.3 mmol)
in anhydrous CH2Cl2 (80 cm3) at −78 ◦C under N2, was added
anhydrous dimethylsulfoxide (4.44 cm3, 62.6 mmol). The reaction
mixture was stirred for 5 min then a solution of 22 (3.85 g,
26.1 mmol) in CH2Cl2 was added over 5 min. Stirring was
continued at −78 ◦C for an additional 15 min. Triethylamine
(18.2 cm3, 130 mmol) was added and the reaction mixture was
stirred for 5 min and then allowed to warm to room temperature.
Water (50 cm3) was then added, and the organic layer was
separated. The aqueous layer was extracted with CH2Cl2 (2 ×
30 cm3). The combined organic extracts were washed with water
(2 × 30 cm3) and brine (30 cm3), dried over MgSO4, filtered and
then concentrated under reduced pressure. Purification by flash
chromatography on silica gel with hexane and ethyl acetate (2 :
1) as eluent gave 23a as a light yellow oil (3.36 g, 88%); Rf 0.48
(hexane–EtOAc = 2 : 1); HRMS (ES+, [M − H]+) found 145.0645,
C10H9O requires 145.0648; mmax (thin film)/cm−1 2357.8, 1668.3,
+
+
=
and 166.9 (C O); m/z (CI ) 276.2 (100%, [M + NH4] ), 259.2 (95,
[M + H]+).
(2E,6Z)-3-Methyl-7-phenylocta-2,6-dien-1-ol (25)
This compound was prepared from 24 in a manner identical to
that for the alcohol 15; purification by flash chromatography on
silica gel using hexane and ethyl acetate (2 : 1) as eluent gave 25
light yellow oil (0.816 g, 91%); Rf 0.31 (hexane–EtOAc = 2 : 1);
HRMS (ES+, [M + NH4]+) found 234.1850, C15H24NO requires
234.1852; mmax (thin film)/cm−1 3335.5, 2965.2, 2914.6, 1667.9,
1492.8, 1436.1, 1376.6, 999.9, 763.1 and 700.1; dH (500 MHz,
C HCl3) 1.22 (1 H, b, OH), 1.50 (3 H, s, CH3C CHCH2OH),
1.95 (3 H, d, J 1.0, CH3CPh), 1.97–2.04 (4 H, m, CH2CH2), 4.04
(2 H, d, J 7.0, CH2OH), 5.28 (1 H, dt, J 7.0, J 1.0, C CHCH2OH),
5.36 (1 H, dt, J 7.0, J 1.5, C CHCH2CH2) and 7.10–7.28 (5 H,
m, Ar–H); dC (125 MHz, C HCl3) 16.2 (CH3C CHCH2OH),
25.6 (CH3CPh), 27.3 and 39.8 (CH2CH2), 59.4 (CH2OH), 123.5
(C CHCH2OH), 126.8 (PhC CH), 126.5, 127.9 and 128.1 (Ar–
CH) and 136.6, 139.5 and 142.1 (quaternary C); m/z (CI+) 234.2
(100%, [M + NH4]+), 216.2 (80, M+), 199.1 (40, [M − OH]+).
2
=
=
=
2
=
1614.1, 1433.3, 1388.2, 1136.6, 767.0 and 701.5; dH (500 MHz,
2
=
=
=
C HCl3) 1.81 (3 H, s, CH3CPh), 6.09 (1 H, d, J 8.0, PhC CH),
6.95–7.13 (5 H, m, Ar–H) and 9.71 (1 H, dd, J 8.0, J 3.5, CHO);
dC (125 MHz, C2HCl3) 25.6 (CH3CPh), 128.3, 128.4 and 128.7
=
(Ar–CH), 129.4 (PhC CH), 138.5 and 160.1 (quaternary C) and
191.6 (CHO); m/z (CI+) 164.1 (95%, [M + NH4]+), 161.1 (100%)
(2E,6Z)-3-Methyl-7-phenylocta-2,6-dienal (26a)
and 146.1 (30%, M+).
This compound was prepared from 25 in a manner identical to
that for the compound 16a; purification by flash chromatography
using hexane and ethyl acetate (2 : 1) as eluent to gave 26a as a light
yellow oil (0.86 g, 87%); Rf 0.45 (hexane–EtOAc = 2 : 1); HRMS
(EI+, M+) found 214.1352, C15H18O requires 214.1358; mmax (thin
film)/cm−1 2912.7, 2851.7, 2360.2, 1672.8, 1630.3, 1492.6, 1439.9,
(Z)-4-Phenyl-penta-1,3-diene (23b)
This compound was prepared from 23a in a manner identical
to that for the diene 12b; purification by flash chromatography
using hexane and ethyl acetate (9 : 1) as eluent gave 23b as a
light yellow oil (1.80 g, 76%); Rf 0.63 (hexane–EtOAc = 9 : 1);
HRMS (EI+, M+) found 144.0938, C11H12 requires 144.0939; mmax
(thin film)/cm−1 3080.3, 2960.4, 2856.8, 1805.6, 1636.2, 1601.6,
1492.1, 1433.6, 1414.1, 1375.3, 1024.7, 995.6, 898.4, 766.1 and
700.2; dH (400 MHz, C2HCl3) 2.04 (3 H, s, CH3CPh), 4.86
1379.2, 1193.2, 1124.0, 1025.8, 831.0, 764.1, 702.0 and 668.1; dH
2
=
(500 MHz, C HCl3) 1.49 (3 H, d, J 1.0, CH3C CHCHO), 1.85–
2.08 (4 H, m, CH2CH2), 2.03 (3 H, d, J 1.0, CH3CPh), 5.30
=
(1 H, td, J 7.0, J 1.0, C CHCH2CH2), 5.85 (1 H, dd, J 8.0, J
=
1.0, C CHCHO), 7.17–7.29 (5 H, m, Ar–H) and 9.91 (1 H, d, J
2
=
=
(1 H, d, J 10.0, CHCH CHtransHcis), 5.08 (1 H, d, J 17.0,
8.0, CHO); dC (125 MHz, C HCl3) 16.4 (CH3C CHCHO), 25.5
(CH3CPh), 26.7 and 40.4 (CH2CH2), 126.0 (PhC CH), 127.6
=
=
=
CHCH CHtransHcis), 6.06 (1 H, d, J 11.0, CHCH CH2), 6.32
=
=
(1 H, dt, J 17.0, J 10.5, CHCH CH2) and 7.14–7.28 (5 H, m, Ar–
H); dC (100 MHz, C HCl3) 25.5 (CH3CPh), 116.1 (CHCH CH2),
(CH CHO), 126.9, 127.9 and 128.3 (Ar–CH), 137.6, 141.8 and
160.8 (quaternary C) and 189.6 (CHO); m/z (EI+) 214.1 (2%, M+),
2
=
=
127.9 (CHCH CH2), 127.1, 128.1 and 128.3 (Ar–CH), 134.6
131.1 (100).
+
=
(CHCH CH2) and 139.5 and 141.5 (quaternary C); m/z (EI )
144.1 (35%, M+), 129.1 (100, [M − CH3]+).
(3E,7Z)-4-Methyl-8-phenyl-nona-1,3,7-triene (26b)
This compound was prepared from 26a in a manner identical to
that for the diene 12b; purification by flash chromatography using
hexane and ethyl acetate (2 : 1) as eluent gave 26b as a light yellow
oil (0.48 g, 86%); Rf 0.62 (hexane–EtOAc = 9 : 1); HRMS (ES+,
[M + H]+) found 212.1569, C16H20 requires 212.1565; mmax(thin
film)/cm−1 2964.8, 2358.3, 1649.8, 1598.9, 1493.0, 1436.5, 1378.1,
985.7, 896.9, 761.8 and 700.0; dH (500 MHz, C2HCl3) 1.70 (3 H, s,
(2E,6Z)-Ethyl 3-methyl-7-phenylocta-2,6-dienoate (24)
This compound was prepared from 23b in a manner identical
to that for the ester 14; the crude product was purified by flash
chromatography using hexane and ethyl acetate (9 : 1) as eluent to
give 24 as a light yellow oil (1.33 g, 41%); Rf 0.35 (hexane–EtOAc =
9 : 1); HRMS (ES+, [M + H]+) found 259.1691, C17H23O2 requires
259.1693; mmax (thin film)/cm−1 2975.8, 2359.6, 1715.0, 1647.3,
1493.4, 1442.0, 1367.2, 1221.8, 1146.1, 1099.6, 1052.2, 865.4, 763.2
and 701.0; dH (500 MHz, C2HCl3) 1.18 (3 H, t, J 7.0, CH3CH2O),
=
=
CH3C CHCH CH2), 2.07 (3 H, s, CH3CPh), 2.08–2.17 (4 H,
=
m, CH2CH2), 5.00 (1 H, dd, J 10.0, J 1.5, CH CHcisHtrans), 5.10
=
(1 H, dd, J 17.0, J 2.0, CH CHcisHtrans), 5.46 (1 H, td, J 7.0, J
=
=
=
1.95 (3 H, s, CH3C CHCO2Et), 1.98 (3 H, d, J 1.0, CH3CPh),
1.5, C CHCH2CH2), 5.83 (1 H, dd, J 11.0, J 0.5, CHCH CH2),
=
2.07 (4 H, m, CH2CH2), 4.07 (2 H, q, J 7.0, CH3CH2O), 5.32 (1 H,
6.59 (1 H, dt, J 17.0, J 10.5, CHCH CH2) and 7.21–7.39 (5 H,
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2
=
m, Ar–H); dC (125 MHz, C HCl3) 16.6 (CH3C CHCH2), 25.6
then brine (20 cm3), dried over Na2SO4 and filtered. Concentration
of the solvent gave the intermediate bromide as a light yellow oil,
which was used without further purification.
=
(CH3CPh), 27.4 and 40.1 (CH2CH2), 114.7 (CHCH CH2), 125.6
=
=
(CHCH CH2), 126.9 (PhC CH), 126.5, 128.0 and 128.2 (Ar–
=
CH), 133.4 (CH CH2) and 136.6, 139.1 and 142.1 (quaternary
To a stirred suspension of NaH (60% dispersion in mineral
oil, 1.20 g, 49.5 mmol) in anhydrous THF (100 cm3) was added
ethyl acetoacetate (5.73 cm3, 45.0 mmol) dropwise at 0 ◦C. After
10 min, n-BuLi (2.2 M, 21.5 cm3, 47.3 mmol) was added slowly
over 3 min, during which time the colourless solution gradually
C); m/z (EI+) 212.2 (5%, M+), 131.1 (100) and 91.1 (40).
(2E,6E,10Z)-Ethyl-3,7-dimethyl-11-phenyldodeca-
2,6,10-trienoate (27)
◦
turned yellow. This was stirred for an additional 10 min at 0 C,
This compound was prepared from 26b in a manner identical to
that for the ester 14; purification by flash chromatography using
hexane and ethyl acetate (19 : 1) gave 27 as a light yellow oil
(0.29 g, 40%); Rf 0.33 (hexane–EtOAc = 19 : 1); HRMS (ES+,
[M + H]+) found 327.2318, C22H31O2 requires 327.2319; mmax (thin
film)/cm−1 2926.3, 1715.4, 1647.5, 1442.7, 1366.5, 1221.5, 1144.3,
1053.9, 865.3, 762.0 and 700.5; dH (500 MHz, C2HCl3) 1.20 (3 H,
as a solution of the bromide in THF (5 cm3) was added. The
clear solution turned to a cloudy yellow suspension. After stirring
for 30 min at 0 ◦C, hydrochloric acid (3 M, 10.0 cm3) was added
followed by water (30 cm3) and diethyl ether (30 cm3) then the
organic layer was separated. The aqueous layer was extracted with
diethyl ether (2 × 20 cm3). The combined ethereal extracts were
washed with water (2 × 20 cm3) and brine (20 cm3), dried over
MgSO4 then filtered and concentrated under reduced pressure.
Purification by flash chromatography on silica gel with hexane
and ethyl acetate (4 : 1) as eluent gave 32 as a pale yellow oil
(3.61 g, 92%); Rf 0.45 (hexane–EtOAc = 2 : 1); HRMS (ES+,
[M + H]+) found 267.1954, C16H27O3 requires 267.1954; mmax (thin
film)/cm−1 2968.0, 2918.6, 1746.5, 1717.2, 1648.9, 1445.8, 1409.9,
=
t, J 7.0, OCH2CH3), 1.43 (3 H, s, CH3C CH), 1.95 (3 H, d, J 1.0,
=
=
CH3C CH), 2.08 (3 H, d, J 1.0, CH3C CHCO2Et), 1.91–2.11
(8 H, m, 2 × CH2CH2), 4.06 (2 H, q, J 7.0, OCH2CH3), 4.97 (1 H,
=
=
b, C CHCH2CH2), 5.35 (1 H, dt, J 7.0, J 1.0, C CHCH2CH2),
5.58 (1 H, s, C CHCO2Et) and 7.09–7.30 (5 H, m, Ar–H); dC
(125 MHz, C HCl3) 14.4 (OCH2CH3), 16.0 (CH3C CH), 18.9
(CH3C CHCO2Et), 25.6 (CH3C CH), 26.0, 27.5, 39.9 and 41.0
=
2
=
=
=
1367.4, 1313.3, 1235.8, 1177.2, 1035.9 and 839.8; dH (500 MHz,
=
(2 × CH2CH2), 59.5 (OCH2CH3), 115.6 (C CHCO2Et), 123.1
2
=
C HCl3) 1.30 (3 H, t, J 7.0, CH3CH2O), 1.61 (3 H, s, CH3C CH),
=
and 127.2 (2 × C CHCH2CH2), 126.4, 127.6 and 128.0 (Ar–CH),
=
=
1.63 (3 H, s, CH3C CH), 1.69 (3 H, s, CH3C CH), 1.98 (4 H, m,
(CH3)2C CHCH2CH2), 2.30 (2 H, q, J 7.5, CH2CH2C O), 2.59
(2 H, t, J 7.5, CH2CH2C O), 3.45 (2 H, s, COCH2CO), 4.20 (2 H,
=
135.7, 136.2, 142.1 and 159.9 (quaternary C) and 166.9 (C O);
=
=
m/z (CI+) 344.3 (100%, [M + NH4]+) and 327.3 (50, [M + H]+).
=
=
q, J 7.0, OCH2CH3) and 5.09 (2 H, dt, J 1.0, J 7.0, 2 × C CH);
(2E,6E,10Z)-3,7-Dimethyl-11-phenyldodeca-2,6,10-trien-1-ol (28)
2
=
dC (125 MHz, C HCl3) 14.1 (OCH2CH3), 16.0 (CH3C CH),
=
=
=
17.7 (CH3C CH), 22.2 (CH2CH2C O), 25.7 (CH3C CH),
26.6 and 39.4 ((CH3)2C CHCH2CH2), 43.1 (CH2CH2CO), 49.4
This compound was prepared in a manner identical to that for
the alcohol 15; purification by flash chromatography using hexane
and ethyl acetate (2 : 1) as eluent gave 28 as a light yellow oil
(0.12 g, 77%); Rf 0.26 (hexane–EtOAc = 2 : 1); HRMS (ES+, [M +
NH4]+) found 302.2477, C20H32NO requires 302.2478; mmax (thin
film)/cm−1 3344.4, 2919.3, 1666.0, 1597.4, 1493.4, 1443.8, 1381.2,
998.9, 756.6 and 696.0; dH (500 MHz, C2HCl3) 1.23 (1 H, b, OH),
=
=
(COCH2CO), 61.4 (OCH2CH3), 122.1 and 122.4 (2 × C CH),
=
131.5 and 136.8 (quaternary C), 167.3 (ester C O) and 202.7
+
+
=
(ketone C O); m/z (CI ) 284.2 (100%, [M + NH4] ), 267.2 (86,
[M + H]+).
=
=
1.43 (3 H, s, CH3C CH), 1.60 (3 H, s, CH3C CH), 1.95 (3 H,
(1Z,5E)-1-(Ethoxycarbonyl)-6,10-dimethylundeca-1,5,9-trien-2-yl
trifluoromethanesulfonate (33)16
=
d, J 1.0, CH3C CH), 1.91–2.06 (8 H, m, 2 × CH2CH2), 4.07
=
(2 H, d, J 7.0, CH2OH), 4.99 (1 H, td, J 7.0, J 1.0, C CH),
A stirred solution of 32 (546 mg, 2.05 mmol) in anhydrous
THF (15 cm3) under N2 was cooled to −78 ◦C then potassium
bis(trimethylsilyl)amide (0.5 M in THF, 4.93 cm3, 2.46 mmol) was
added. The resulting mixture was stirred at −78 ◦C for 30 min.
Trifluoromethanesulfonic anhydride (414 mm3, 2.46 mmol) was
=
5.33 (2 H, m, 2 × C CH) and 7.10–7.30 (5 H, m, Ar–H); dC
(125 MHz, C2HCl3) 16.0, 16.3 and 25.6 (3 × CH3), 26.3, 27.6, 39.5
and 40.0 (2 × CH2CH2), 59.4 (CH2OH), 123.3, 124.0 and 127.3
=
(3 × C CH), 126.4, 128.0 and 128.0 (Ar–CH) and 135.0, 136.1,
139.8 and 142.2 (quaternary C); m/z (CI+) 302.3 (100%, [M +
◦
added at −78 C and the solution stirred for 16 h whilst slowly
NH4]+), 284.3 (55, M+).
warming to room temperature. Diethyl ether (20 cm3) was added
and the solution was washed with 10% citric acid solution (2 ×
15 cm3) and water (15 cm3). The separated organic layer was dried
over MgSO4 and filtered. Evaporation of the solvent gave a yellow
oil which was purified by flash chromatography on silica gel with
hexane and ethyl acetate (4 : 1) as eluent to give 33 as light yellow
oil (0.42 g, 51%); Rf 0.41 (hexane–EtOAc = 4 : 1); mmax (thin
film)/cm−1 2975.6, 2905.3, 2855.1, 2353.0, 1731.9, 1676.1, 1427.9,
1209.1, 1141.0, 1037.6, 923.1 and 840.1; dH (500 MHz, C2HCl3)
(E)-Ethyl 7,11-dimethyl-3-oxododeca-6,10-dienoate (32)20,38
To a stirred solution of geraniol (2.60 cm3 g, 15.0 mmol) and
triethylamine (4.20 cm3, 30.0 mmol) in anhydrous THF (100 cm3)
at −45 ◦C under N2 was added methanesulfonyl chloride (1.50 cm3,
19.5 mmol). The resulting milky mixture was stirred at −45 ◦C for
45 min then a solution of lithium bromide (5.20 g, 60.0 mmol) in
THF (10 cm3) was added via a cannula at −45 ◦C. The suspension
was allowed to warm to 0◦C and stirred for an additional 1 h before
cold water (30 cm3) and hexane (30 cm3) were added to quench
the reaction. The two layers were separated, and the aqueous layer
was extracted with hexane (2 × 20 cm3). The combined organic
layers were washed with saturated NaHCO3 solution (20 cm3) and
=
1.24 (3 H, t, J 7.0, OCH2CH3), 1.53 (3 H, s, CH3C CH), 1.54
=
=
(3 H, s, CH3C CH), 1.61 (3 H, s, CH3C CH), 1.91 (4 H, m,
=
=
(CH3)2C CHCH2CH2), 2.20 (2 H, t, J 7.5, CH2CH2C O), 2.34
(2 H, t, J 7.5, CH2CH2C O), 4.17 (2 H, q, J 7.0, OCH2CH3), 4.99
(2 H, t, J 7.0, 2 × C CHCH2CH2) and 5.67 (1 H, s, O–C CH);
=
=
=
3296 | Org. Biomol. Chem., 2007, 5, 3287–3298
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©

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2
=
dC (125 MHz, C HCl3) 14.1 (OCH2CH3), 16.1 (CH3C CH),
Molecular mechanics simulations
=
=
17.7 (CH3C CH), 24.4 (CH2CH2CO), 25.7 (CH3C CH), 26.5
and 39.6 ((CH3)2C CHCH2CH2), 34.6 (CH2CH2C O), 61.3
The X-ray crystal structure of AS from P. roqueforti containing
a docked FPP molecule (PDB 1FIP) was used as the starting
structure.34 For each docking experiment, the FPP substrate was
converted to the relevant substrate analogue 19, 29 and 36,
respectively, using the molecule builder module of the software
package MOE.§ Hydrogen atoms were added to both the protein
and FPP automatically using the software. The inhibitor and
=
=
=
=
(OCH2CH3), 112.0 (O–C CH), 120.6 and 123.9 (2 × C CH),
=
=
131.7 and 138.2 (2 × C CH), 158.5 (CH COSO2CF3) and
2
+
=
162.5 (C O); dF (283 MHz, C HCl3) −74.6 (s). m/z (CI ) 398.1
(2%, M+), 358.0 (80), 314.1 (18), 267.0 (21), 190 (23) and 114 (100).
(2Z,6E)-Ethyl-7,11-dimethyl-3-phenyldodeca-2,6,10-trienoate
˚
all amino acid residues containing heavy atoms within 6.5 A of
(34)
the inhibitor were selected as the key active site atoms and the
MMFF94 molecular mechanics forcefield was applied to these
atoms only.35 The energy of the system was minimised and the
resulting coordinates used to generate Fig. 3.
To a stirred solution of 33 (0.36 g, 0.91 mmol) in anhydrous
THF (10 cm3) under N2, was added palladium(II) acetate (0.02 g,
0.09 mmol), triphenylarsine (0.11 g, 0.36 mmol), silver oxide
(0.42 g, 1.81 mmol) and phenylboronic acid (0.17 g, 1.36 mmol)
in quick succession. The complete mixture was then heated under
reflux for 15 h. Water (20 cm3) and diethyl ether (20 cm3) were
added, and the organic layer was separated. The aqueous layer
was extracted with diethyl ether (2 × 15 cm3). The combined
ethereal extracts were washed with water (2 × 10 cm3) and brine
(10 cm3), dried over MgSO4 filtered and concentrated under
reduced pressure. Purification by flash chromatography on silica
gel with hexane and ethyl acetate (9 : 1) as eluent gave 34 as
a light yellow oil (0.19 g, 66%); Rf 0.35 (hexane–EtOAc = 9 :
1); HRMS (ES+, [M + H]+) found 327.2324, C22H31O2 requires
327.2319; mmax (thin film)/cm−1 2975.7, 2926.3, 2361.9, 1727.2,
1638.2, 1442.5, 1377.2, 1276.4, 1223.8, 1159.0, 1042.6, 865.8 and
698.6; dH (500 MHz, C2HCl3) 0.99 (3 H, t, J 7.0, OCH2CH3),
Acknowledgements
This work was supported by the Engineering & Physical Sciences
Research Council. We thank the EPSRC national mass spectrom-
etry service, University College, Swansea, for some mass spectral
analysis.
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2384.
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silica gel using hexane and ethyl acetate (2 : 1) as eluent gave 35 as
a light yellow oil (0.11 g, 85%); Rf 0.38 (hexane–EtOAc = 2 : 1);
HRMS (EI+, M+) found 284.2147, C20H28O requires 284.2140; mmax
(thin film)/cm−1 3355.5, 2965.4, 2922.2, 2855.4, 1650.6, 1491.7,
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=
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=
=
(3 H, s, CH3C CH), 1.71 (3 H, s, CH3C CH), 1.97 (6 H, m,
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C CH), 5.72 (1 H, t, J 7.0, C CHCH2OH) and 7.15 (5 H, m, Ar–
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26.5, 26.7, 39.0 and 39.7 (2 × CH2CH2), 60.3 (CH2OH), 123.5
=
=
and 124.4 (2 × C CH), 125.7 (C CHCH2OH), 127.1, 128.1 and
128.2 (Ar–CH) and 131.3, 135.5, 140.0 and 144.6 (quaternary C);
m/z (EI+) 284.2 (10%, M+) and 266.2 (100, [M − H2O]+).
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