Identification of novel mammalian squalene synthase inhibitors using a three-dimensional pharmacophore

Article abstract of DOI:10.1016/S0968-0896(02)00090-1

Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerisation of farnesyl diphosphate in a [1-4] head to head fashion to form squalene, and is the first committed step in cholesterol biosynthesis. Specific inhibitors of squalene synthase would inhibit cholesterol formation and allow production of other important compounds derived from the cholesterol biosynthetic pathway, namely the ubiquinones (co-enzyme Q₁₀), dolichol, and would also allow the isoprenylation process of ras by farnesyl-protein transferase. The construction of a hypothetical squalene synthase three-dimensional pharmacophore is presented. It serves as a template for the identification of several new potential classes of inhibitors. The synthesis, anti-microbial and mammalian pig liver squalene synthase activities of analogues based on the bicyclo[3.2.0]heptane and bicyclo[3.3.0]octane ring systems are reported. Analogues of the latter system are pro-drug type inhibitors and exhibit promising biological activity.

Full text of DOI:10.1016/S0968-0896(02)00090-1

Bioorganic & Medicinal Chemistry 10 (2002) 2641–2656
Identification of Novel Mammalian Squalene Synthase Inhibitors
Using a Three-Dimensional Pharmacophore
Ian J. S. Fairlamb,a,*,y Julia M. Dickinson,a,* Rachael O’Connor,b
Seamus Higson,c Lynsey Grievesonc and Veronica Marina,d
aDepartment of Chemistry and Materials, John Dalton Building, The Manchester Metropolitan University,
Chester Street, Manchester M20 5GD, UK
bDepartment of Biological Sciences, John Dalton Building, The Manchester Metropolitan University,
Chester Street, Manchester M20 5GD, UK
cMaterials Science Centre, University of Manchester Institute of Science and Technology, Manchester, UK
dDepartamentul de Stiinte ingineresti, Divizia Chimica Filiera Engleza, Universitatea Politechnica Bucuresti,
Splaiul Independentei nr. 313, Bucuresti, Romania
Received 31 January 2002; accepted 8 March 2002
Abstract—Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerisation of farnesyl diphosphate in a [1–4] head to head
fashion to form squalene, and is the first committed step in cholesterol biosynthesis. Specific inhibitors of squalene synthase would
inhibit cholesterol formation and allow production of other important compounds derived from the cholesterol biosynthetic pathway,
namely the ubiquinones (co-enzyme Q10), dolichol, and would also allow the isoprenylation process of ras by farnesyl-protein transfer-
ase. The construction of a hypothetical squalene synthase three-dimensional pharmacophore is presented. It serves as a template for the
identification of several new potential classes of inhibitors. The synthesis, anti-microbial and mammalian pig liver squalene synthase
activities of analogues based on the bicyclo[3.2.0]heptane and bicyclo[3.3.0]octane ring systems are reported. Analogues of the latter
system are pro-drug type inhibitors and exhibit promising biological activity. # 2002 Published by Elsevier Science Ltd.
Introduction
SQS is a 47 kDa membrane-associated enzyme which
catalyses the remarkable reductive dimerisation of two
molecules FPP in a [1–4] head-to-head fashion, via pre-
squalene diphosphate (PSQPP) to form squalene (S)
(Scheme 1).1
Over the past 10 years inhibition of squalene synthase
(E.C. 2.5.1.21) (SQS) has been the focus of many aca-
demic and industrial groups in the search for new choles-
terol-lowering agents.1 Efforts to find SQS inhibitors have
been prompted as an alternative to the statin class of
HMG–CoA reductase inhibitors as cholesterol-lowering
agents. The latter class of inhibitors have the potential
to cause depletion of other essential compounds, such as
ubiquinones,1 dolichols2 and isoprenylated proteins3
(ras proteins). Inhibition of SQS after farnesyl diphos-
phate (FPP) is considered advantageous, as it is the first
step committed solely to the synthesis of cholesterol.
The SQS mediated reaction proceeds via two distinct
steps. In the first step, two molecules of FPP react to form
the stable cyclopropylcarbinyl diphosphate intermediate,
PSQPP. Mutagenesis data identifying the residues that
affect substrate binding and activity suggest that this
step occurs at the end of a large central channel found
in the middle of one face of the SQS domain.5 The
stable reaction intermediate (PSQPP) passes deeper into
this central channel, where the second reaction step occurs
and is sequestered from water. Here, PSQPP undergoes
rearrangement, through a secondary cyclobutylcarbinyl
diphosphate (CYPP) and then tertiary cyclopropylcarbinyl
diphosphate (TPSQPP) and reduction with NADPH to
form S.
*Corresponding authors. Tel.: +44-161-247-1415; fax: +44-161-247-
6357; e-mail: j.dickinson@mmu.ac.uk
yCurrent address: Department of Chemistry, University of York,
Heslington, York YO10 5DD, UK. Tel.: +44-1904-434091; fax: +44-
1904-432516; e-mail: ijsf1@york.ac.uk
An impressive array of structurally diverse inhibitors of
SQS has been reported. Currently one of the most
0968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0968-0896(02)00090-1

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Scheme 1. The biosynthetic reaction mediated by SQS within cholesterol biosynthesis.
potent SQS inhibitors is Squalestatin S16 also known as
Zaragozic acid A7 (Fig. 1). S1 and its analogues exhibit
fascinating biological activity and much work has focused
on their total synthesis.8 Unfortunately S1 is toxic in
mammalian subjects, and work with the squalestatin class
has been discontinued by Glaxo-Wellcome.9
The X-ray structure shows that SQS is folded as a single
domain and has a large channel in the centre of one face.
The active sites of the two half-reactions are located in the
central channel. This is lined on both sides by conserved
aspartate and arginine residues. One end of this central
channel is exposed to solvent (water), while the other end
leads to a completely enclosed pocket surrounded by
hydrophobic residues, which presumably accepts the
hydrophobic farnesyl chain or mimetics thereof.
Recently Pandit et al.10 reported both the crystallisation
of human SQS and co-crystal structure of SQS with
various inhibitors (CP-424677, CP-458003 and CP-
320473; Fig. 1) enzyme bound.11À13 This remarkable
goal represents 30 years of extensive efforts to obtain
crystals of sufficient quality in order to allow X-ray
determination. However, at the onset of this work no
three-dimensional structure for SQS was available and
hence we embarked on the construction of a pharma-
cophore that represents the inhibitors and intermediates
(PSQPP, CYPP and TPSQPP) of SQS.
‘The flap’—The central channel contains an important
structural feature where one side is covered by a ‘flap’,
residues 50 and 54, which connects the A and B helices.
The side chain of Phe-54 forms one wall under the flap
and is largely a hydrophobic cavity.12 The X-ray co-
crystal structures of SQS-inhibitor complexes show that
bulky hydrophobic groups, for example naphthyl and
biphenyl moieties, fill a cavity under this flap. Unfortu-
nately the residues forming the flap and the side chain of
Tyr-73 show large conformational differences in each
enzyme–inhibitor complex.12 Hydrophobic moieties are
thought to stabilise a very flexible region. This property
of the enzyme active site makes computational model-
ling with new structures difficult. Indeed, the inherent
problems of hydrophobic binding due to rotation of the
side chains Phe-54 and Tyr-73 and backbone rotations in
the ‘flap’ residues illustrates the importance of rigidity
when designing novel inhibitors based on the structures
of the enzyme–inhibitor complexes. Moreover, it ought
to be possible to take advantage of the information
gained from the X-ray studies with the enzyme–inhibitors
complexes, that is, by taking advantage of the ‘flap’ in
order to design novel rigid bicyclic systems that include
aromatic isosteres. Future computational molecular
studies will address this issue.
Results—Pharmacophore for SQS
A pharmacophore is defined as the three-dimensional
arrangement of the groups essential for recognition and
activation.14 In drug design, it also refers to a set of
features that are common to both substrate/inter-
mediates and inhibitors of a given receptor/enzyme.15
Various methods exist for construction of the pharma-
cophore, such as the distance-geometry approach by
Figure 1. Squalestatin S1 (Zaragozic acid A).

I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
2643
computational molecular modelling.14,16 Using this
approach, it is possible to gain information about the
conformational properties of both substrate/inter-
mediates and known inhibitors of SQS. The inherent
conformational flexibility of the farnesyl chains increases
the difficulty associated with modelling the substrate/
intermediates of SQS, so one must focus attention on the
known inhibitors of SQS to give a training set for the
pharmacophore.
SQS inhibitors share common structural features
(Scheme 2): the presence of a hydrophilic di- or tri-acid
core, which share electronic and steric similarities with
the diphosphate head found in FPP and SQS inter-
mediates; and hydrophobic-lipophilic groups which
mimic the farnesyl moiety. A simple two-dimensional
generic structure that represents the SQS inhibitors is
presented in Scheme 2.
The initial generation of the pharmacophoric map using
S16,7 and viridiofungin A17 proved quite cumbersome,
mainly due to the dissimilarity and flexibility found in
the farnesyl mimetics. Other inhibitors chosen were the
J-10418 and 3-biarylquinuclidine19 classes, which proved
more useful. It was also possible to use modified S1
structures (MS1-A, MS1-B and MS1-C; Fig. 2).20
Clearly, the chosen compounds are structurally distinct
and were all selected as candidates in the training set for
pharmacophore generation. As the phenyl and biphenyl
moieties exhibit rigidity, molecular superposition of the
unsaturated systems (p) and two oxygens (directly
attached to carbon ‘in bold’ for the CP-, J-104 and S1
derivatives, and phosphorus for the biarylquinuclidines)
was possible.21
In this model, the p-systems are described by a vector
orthogonal to the plane of the p-system through its centre,
and then measured with respect to the two oxygen atoms,
that is forming several co-ordinates (Fig. 3, I). Four p-
systems (p1, p2, p3 and p4; Fig. 2) were used in the model
and the position of these with respect to the diphos-
phate head and di-acid bioisostere were determined.
Scheme 2. Two-dimensional generic structure for SQS inhibitors.
Figure 2. Structures used in the construction of the pharmacophore.

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Figure 3. Proposed hypothetical pharmacophore for SQS.
Figure 4. Overlays of the SQS intermediate structures.
Figure 5. The proposed target structures.
This enabled the construction of a three-dimensional
pharmacophore (Fig. 3, II). The molecular overlay of
the SQS intermediates, PSQPP, CYPP and TPSQPP,
simulated and overlaid using a best-fit parameter, fitted
well into this model (Fig. 4).
The three-dimensional structures were minimized at the
MM level until the lowest local energy minimum22
(LEM) was attained. The pharmacophore p–p distances
and limits are shown in Table 1. The target structures,
T-1 through to T-6, demonstrate the desired orientation
of the substituents. For the bicyclo[3.2.0]heptane series
(T-1, T-2 and T-3) the bicyclic ring system exhibits a
different conformation when R is changed from C2 to
C3 (Fig. 6).23 The endo envelope is preferred for T-1,
whereas for T-2 the exo envelope is predominant. This
difference demonstrated that a change in the orientation
The co-crystallised structures of CP-424677 (J-104), CP-
458003 and CP-320473 inhibitors from human SQS,12
also fit well into the pharmacophore.
Identification of new SQS inhibitors candidates
Rigid ring systems were introduced to increase the accu-
racy of the simulated structures.14 The use of flexible
structures might result in the introduction of error
through unknown conformational properties.14,21 Several
cyclic and bicyclic structures were investigated, as the
substituent orientation of these structures gives a small
‘cone-angle’ window (Fig. 3, III) and are considered
more ideal pharmacophore candidates (Fig. 5).
The substituents of the target structures (T-1–T-6) were
systematically varied so as to attain positional mimetics
of the farnesyl chains and diphosphate head of the SQS
intermediates. The best substituents were found to be
phenyl and biphenyl moieties, which exhibit the highest
rigidity with respect to the p-system.
Figure 6. Endo and exo envelope conformations for T-1 and T-2.

I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
Table 1. Distance coordinates for the proposed SQS pharmacophore
2645
Distance co-ordinatea
T-1
T-2
T-3
T-4
T-5
T-6
Limits
d1
d2
d3
d4
d5
d6
d7
d8
d9
6.41 (+1.78)
4.42 (À3.43)
8.67 (À0.74)
9.40 (À1.92)
6.32 (À0.95)
2.85 (À1.35)
9.02 (+1.43)
4.38
7.48 (+2.85)
5.46 (À2.39)
9.41
6.79 (+2.16)
6.29 (À1.56)
9.52
4.06 (À0.43)
5.96 (À1.89)
7.5 (À1.91)
7.73 (À3.59)
6.23 (À1.04)
4.20
3.83 (À0.66)
5.38 (À2.47)
6.83 (À2.58)
7.34 (À3.98)
7.27
3.84 (À0.36)
6.77 (À0.74)
4.26
4.24 (À0.25)
5.26 (À2.59)
7.31 (À2.10)
8.12 (3.20)
7.42
3.35 (À0.85)
7.48 (À0.03)
4.54 (+0.15)
9.51
4.49–4.63
7.85–9.39
9.41–9.59
11.32–11.54
7.27–10.76
4.20–4.89
7.51–7.59
3.24–4.39
9.26–12.13
7.19 (À4.13)
8.87 (À2.45)
7.66
4.68
7.63
2.75 (À1.45)
9.64 (+2.05)
4.31
10.94 (+3.35)
4.27
8.78 (À0.48)
7.12 (À0.39)
4.37
9.22 (À0.04)
7.98 (À1.28)
9.91
9.02 (À0.24)
The co-ordinate distances in bold lie between the calculated limits.
aNumber in parentheses correspond to the difference in the calculated limits.
of R might also yield a potential structure and hence led
to the simulation of T-3.
The cyclopentane structures (T-4, T-5 and T-6) exhibit
good potential in this pharmacophore. Abbott Labora-
tories24 recently published similar analogues to T-6, as
potent in vitro inhibitors of SQS. This lends credibility
to both the simulated pharmacophore and the other
structures simulated from it.
The synthesis of analogues similar to the proposed tar-
gets, T-2, T-3 and T-4, have been the focus of our recent
attention25 and the preliminary biological activities,
against a range of yeasts and bacteria and the enzyme
SQS from mammalian pig liver, of several T-2, T-3 and
T-4 analogues, are reported herein.
Synthesis of T-2 analogues
The bicyclo[3.2.0]heptane ring 3 was synthesised in 71%
yield by [p2s+p2a] cycloaddition of phenyl(methyl)k-
etene 2 (generated in situ via dehydrochlorination of 2-
phenyl-propanoyl chloride 1 with triethylamine) to
[1,3]-cyclopentadiene (pathway A, Scheme 3).26
Recently, the mechanism of this reaction has received
renewed interest.27 The initial product is suggested to be
a [p4s+p2s] cycloaddition product 4 which undergoes a
[3,3]-sigmatropic rearrangement to the thermo-
dynamically more stable expected [2+2] product 3
(pathway B, Scheme 3). However, we failed to detect
this intermediate under various conditions (several
reaction solvents and low temperatures).
Scheme 3. Synthetic route to the T-2 analogues.
Table 2. Yields of the exo diol 5
Solvent (ratio)a
Yield%
THF–tBuOH–H2O (8:5:3)
63
82
74
33
MeCN–THF–tBuOH–H2O (4:2:1:1)
MeCN–tBuOH–H2O (4:1:1)
The concave bicyclo[3.2.0]heptane ring enables regio
and stereoselective functionalisation of the alkenic
bond.28,29 Using the method of Rahman,29b the exo diol 5
was synthesised by catalytic osmylation (OsO4) of 3 in the
presence of N-methyl-morpholine-N-oxide (NMNO) in a
THF–tBuOH–H2O mixture (Table 2).
(CH3)2CO–THF–tBuOH–H2O (4:2:1:1)
aAll reactions were run on the same scale with 2 mol% of OsO4 and 1
equiv NMNO in the specified solvent system.
Reaction of 5 with 1 equiv of the benzoyl chloride gave
the C3 (7) mono-acylated and C2–C3 (8) di-acylated
products, separable by flash chromatography in 46 and
23% yields, respectively. The exo configuration of the
C2 substituent in both 7 and 8 were confirmed by fine
1H coupling (J1-2=1.8 Hz) observed between the trans-
coaxial H-1 and H-2 protons. The cis arrangement of
both the C2 and C3 substituents were confirmed by
small H-2 and H-3 coupling for 8 (J2-3=4.0 Hz). The
The endo diol 6 was not observed in this reaction, pre-
sumably due to the steric constraints imposed by the
bicyclic ring, which blocks OsO4 attack from the endo
face.29 Alteration of the solvent system led to an
increase in the yield of 5 to 82%. C3-esterified deriva-
tives were synthesised by simple treatment of 5 with the
appropriate acyl chloride in the presence of pyridine
and DMAP in CH2Cl2 (Table 3).

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Table 3. Yields of two T-2 derivatives
Synthesis of T-3 analogues
Compda
R
Yieldb (%)
Previous work by Roberts et al. has shown that 7,7-di-
substitutedbicyclo[3.2.0]alkenones undergo electrophilic
bromination in a regioselective manner.28,29a It was
rationalised that the introduction of farnesyl mimetics
could be achieved using the electrophilic bromination
reaction. The synthesis of T-3 analogues was accom-
plished with several 7,7-disubstituted bicyclo[3.2.0]hept-
2-en-6-ones (3, 11, 12 and 13), using N-bromoacetamide
(NBA) and the appropriate nucleophile (H2O, ROH or
RCO2H) (Scheme 4). We found that N-bromosuccini-
mide (NBS) was a satisfactory source of electrophilic
bromine when H2O was used as the nucleophile. How-
ever, employment of other nucleophiles (ROH and
RCO2H) with NBS led to poor isolated yields of the
required products.
7 (8)
9 (10)
Ph
Ph(CH2)3
46 (23)
13 (44)
aNumber in parentheses corresponds to the di-substituted derivative.
bYields in parentheses refer to the C2–C3 di-substituted product.
selective C3 mono-acylation of 5 is particularly note-
worthy, as both C2 and C3 alcohols are seemingly
equally exposed. Molecular modelling30 of 5 at the PM3
level of theory shows that the exo envelope is the lowest
energy conformation in solution (Fig. 7).
It is clear that the C2 alcohol is slightly hindered by the
cyclobutane ring. Reaction of 5 with 4-phenyl-butanoyl
chloride under identical conditions gave predominantly
the C2-C3 di-acylated product 10 in 44% and the C3
mono-acylated product 9 in 13% yield. The favorable
formation of the di-acylated products is intriguing,
although the origin of this effect may stem from differ-
ing conformations of diol 5 and the mono-acylated
products 7 and 9, where addition of the second acyl
group is facilitated by introduction of the first group.
The corresponding products and yields are given in
Table 4. The electrophilic bromination of chloro-
bicycloheptenone 11 yielded two isomers 21 (9%) and
22 (37%). The minor isomer 21 was produced by addi-
tion of bromine from the more hindered face of the
alkene, followed by nucleophilic attack from the exo
face of the bicyclic ring (Scheme 5).28
When other nucleophiles (MeOH, MeCO2H and
PhCO2H) were employed in the reaction of 11 with
NBA, only the major exo-bromo stereoisomers (23, 24
and 25) were observed.
Scheme 4. Synthesis of T-3 analogues. (a) NBA, (CH3)2CO–H2O
(3:1); (b) NBA, CH2Cl2, RCO2H; (c) NBA, CH2Cl2, ROH; (d)
RCOCl, Et3N, DMAP.
Figure 7. Exo-face selective osmylation products.
Table 4. Synthesis of T-3 derivatives
Alkene
Nucleophile
H2O
Product
Compd
Yield %
R=Ph, X=Cl, 11
R=Ph, X=Cl, R1=H (C2 endo-Br)
R=Ph, X=Cl, R1=H (C2 exo-Br)
R=Ph, X=Cl, R1=Me
R=Ph, X=Cl, R1=COMe
R=Ph, X=Cl, R1=COPh
R=Ph, X=Cl, R1=CO-C6H4-p-C6H5
R=Ph, X=Br, R1=COPh
R=Ph, X=Me, R1=H (C2 exo-Br only)
R=Ph, X=Me, R1=Me
R=Ph, X=Me, R1=COMe
21
22
23
24
25
26
27
14
15
16
17
18
19
20
30
9
37
88
77
47
0
62
56
82
80
63
0
R=Ph, X=Cl, 11
R=Ph, X=Cl, 11
R=Ph, X=Cl, 11
R=Ph, X=Cl, 11
R=Ph, X=H, 12
R=Ph, X=Me, 3
R=Ph, X=Me, 3
R=Ph, X=Me, 3
R=Ph, X=Me, 3
R=Ph, X=Me, 3
R=Ph, X=Me, 3
R=Ph, X=Me, 3
R=p-C6H5-C6H4, X=Me, 13
MeOH
MeCO2H
PhCO2H
p-C6H5-C6H4CO2H
PhCO2H
H2O
MeOH
MeCO2H
PhCO2H
R=Ph, X=Me, R1=COPh
p-NO2–PhCO2H
p-MeO–PhCO2H
p-C6H5–C6H4CO2H
PhCO2H
R=Ph, X=Me, R1=CO(C6H4-p-NO2)
R=Ph, X=Me, R1=CO(C6H4-p-OMe)
R=Ph, X=Me, R1=CO-C6H4-p-C6H5
R=p-C6H5-C6H4, X=Me, R1=COPh
0
0
58

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2647
(77%). We also found that it was possible to acylate the
endo alcohol of 14 using benzoyl chloride under standard
conditions to give 17 in near quantitative yield.
Synthesis of T-4 analogues
We envisaged that pro-drug type compounds based on
the T-4 target might prove useful in vitro and in vivo
inhibitors of SQS (Scheme 7).
Functionalised bicyclo[3.3.0]octanes have been exten-
sively developed as key useful intermediates in synthetic
routes to the naturally occurring prostaglandins.28,31 T-
4 analogues can be easily approached using lactones 31
and 32. The large scale synthesis of 31 was performed
starting from dichloroacetyl chloride 33 (Scheme 8).32
Scheme 5. Major and minor stereoisomers formed from the endo and
exo envelope conformations of the bicyclo[3.2.0]heptane ring.
The successful incorporation of the benzoyl ester (25)
led reaction of 11 with para-phenylbenzoic acid,
although no product (26) was formed in this reaction.
Dehydrochlorination of 33 in situ using Et3N in the
presence of [1,3]-cyclopentadiene gave cycloadduct 34 in
90% yield. Dehalogenation of 34 using a zinc–copper
couple in acetic acid gave bicyclo[3.2.0]hept-en-6-one 35
in 70% yield. Compound 35 could be resolved using
fermenting Bakers yeast to give the S-alcohol 36 and
R-ketone of 35-(R).33 Reoxidation of S-alcohol 36 with
CrO3–H2SO4/acetone yields S-ketone 35-(S) in 32%
overall yield from (Æ)-35. Baeyer–Villiger oxidation of
35 gave the lactone 37 in 75% yield.
The use of other nucleophiles, such as E-geraniol, E,E-
farnesol, benzyl alcohol, 4-phenyl-butanoic acid and
decanoic acid failed to react.
The reaction of unhalogenated derivative 12 (X=H) with
NBA in the presence of benzoic acid resulted in bromina-
tion of the C7 position, yielding the di-bromo derivative
27 in 62% yield. Roughly 2.5 equiv of NBA were
required for this reaction to reach completion. Both bro-
mination reactions proceed at roughly equivalent rates, as
evidenced by the disappearance of the vinyl protons (H-
The key-step in this series was Prins reaction of 37 with
para-formaldehyde (monomerised with H2SO4 at 70 ꢀC)
in glacial acetic acid, followed by acylation of the crude
mixture of products (di-acetate, and C4/C5 mono-ace-
tates) yielding the di-acetate 38 in 63% yield. Hydrolysis
of the acetates with sodium methoxide in methanol,
1
2 and H-3) and the H-7 acidic proton by H NMR.
Treatment of compound 3 with NBA in acetone and
H2O, yielded the exo-bromo stereoisomer 14 exclu-
sively. This reaction was repeated several times, albeit
producing none of the endo-bromo stereoisomer. This is
the first case of a 7,7-disubstitutedbicyclo[3.2.0]hep-2-
en-6-one affording one isomer exclusively under these
conditions.28,29
The incorporation of methoxy 15, acetoxy 16 and ben-
zoxy 17 derivatives of 3 were relatively facile, with all
reactions proceeding in good yield. Compound 3 failed
to react with NBA in the presence of p-nitro-benzoic acid,
p-methoxy-benzoic acid or p-phenylbenzoic acid. 7-endo-
p-Biphenyl-7-exo-methylbicyclo[3.2.0]hept-2-en-6-one 13
was synthesised from the corresponding ketene 28 (gen-
erated in situ from 29 via dehydrochlorination with Et3N)
and [1,3]-cyclopentadiene (49% overall yield from com-
mercially available p-biphenylacetic acid) (Scheme 6).
Scheme 7. Proposed metabolic lactone ring opening of the T-4 targets.
Compound 13 underwent selective reaction with NBA
in the presence of benzoic acid to give 30 in good yield
Scheme 8. Synthetic route toT-4 analogues. (a) MCPBA, NaHCO3,
CH2Cl2, 25 ꢀC; (b) (i). (CH2O)n, H2SO4, AcOH, 80 ꢀC; (ii). Ac2O,
DMAP, Pyr., CH2Cl2; (c) NaOMe, MeOH; (d) RCOCl, Pyr., DMAP,
CH2Cl2, 25 ꢀC; (e) Bakers Yeast, Sucrose, water—3 days; (f) Jones
reagent.
Scheme 6. Synthesis of alkene 13. (a) n-BuLi (2 equiv), THF, À78 ꢀC,
then CH3l. (b) (COCl)2, CHCl3, 0–25 ꢀC. (c) Et3N, 1,3-cyclopenta-
diene. (PBP=para-biphenyl).

2648
I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
and acidic work up afforded the known lactone 31 in
92% yield.32
steroid pathway and the latter species is more suscep-
tible to SQS inhibitors.36 This susceptibility was also
confirmed in our own assay.
The T-4 esterified compounds were attained by treat-
ment of 31 or 3234 with the appropriate acyl chloride in
a standard fashion (Table 5 and Scheme 8).
S1 exhibited no activity towards any of the bacteria
screened. S1 is either unable to traverse the bacterial cell
wall or the bacteria species screened are non-dependant
on SQS and ultimately sterol biosynthesis.
Table 5. Isolated yields of T-4 analogues
No.
R
R0
Yield%
53
The T-2 analogues (5, 7, 8, 9 and 10) exhibited little activ-
ity against the three yeasts. The best inhibitor of this class
was analogue 9, expected on hydrophobic grounds to be
an improved inhibitor. Interestingly, analogue 5 exhibits
modest inhibitory activity towards E. coli.
39a
40b
41b
58
91
The biological activities of the T-3 class were dis-
appointing. Compounds 14, 16, 17 and 22 showed little
or no activity against the yeasts. The best analogues
were those that contained both a halogen at the C7
position and a benzoxy substituent at C3 (25, 27 and
30). These were all modest inhibitors of S. cerevisiae and
S. pombe.
aSynthesised from compound 31.
bSynthesised from compound 32.
Antimicrobial activities
The test compounds were screened for anti-microbial
activity.35 Three yeasts (Candida albicans, Shizosaccharo-
myces pombe and Saccharomyces cerevisiae, Table 6)
and three bacteria (Botrytis cineria, Escherichia coli and
Staphylococcus aureus, Table 7) were chosen to evaluate
the effectiveness of these compounds.
On the whole, the T-3 class demonstrate little activity
against any of the bacteria that were screened.
Table 7. Biological activities against bacteria
Compd
Inhibition zone (mm)a,b
Each compound was evaluated at a concentration of
200 mg in triplicate, against two controls: squalestatin S1
(Table 8) and ethanol (inhibitor-free). It should be
noted that all three yeasts are highly dependant on the
B. subtilis
E. coli
S. aureus
5
7
8
9
8
7
—
7
13
8
—
9
6
7
—
0
10
14
15
16
17
21
22
23
24
25
27
30
32
39
40
41
8
—
0
—
0
0
5
—
0
—
0
0
0
—
0
—
7
0
Table 6. Biological activities against yeasts
Compd
Inhibition zone (mm)a,b
C. albicans
GDH2346
S. cerevisiae
NCYC1467
S. pombe
0
0
0
—
—
0
0
7
0
0
0
0
—
—
0
0
0
—
—
0
0
5
5
7
0
8
0
8
8
8
6
5
7
0
11
8
7
—
0
7
—
5
—
9
11
11
12
—
12
10
19
13
8
0
9
0
10
14
15
16
17
21
22
23
24
25
27
30
31
32
39
40
41
0
0
—
7
0
—
0
—
7
7
0
11
14
21
12
18
9
8
0
0
12
15
15
—
—
—
—
7
0
—
7
aDiameter of inhibition zone.
bAll discs contained 200 mg of the test compound. Student t-Test analysis
demonstrates that all inhibition zones >5 mm show significant differ-
ences (P<0.05). For the controls, see the experimental section.
—
10
12
10
10
—
11
7
Table 8. Yeast biological activities of S1
Organism
Concentration (mg)a
19
16
200
20
2
Candida albicans
Saccharomyces cerevisiae
Shizosaccharomyces pombe
27
27
37
15
15
25
5
7
19
aDiameter of inhibition zone.
bAll discs contained 200 mg of the test compound. Student t-Test
analysis demonstrates that all inhibition zones >5 mm show
significant differences (P<0.05).
aZones of inhibition (values are means of three experiments).

I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
2649
The T-4 analogues (31, 32, 39, 40 and 41) exhibited the
best activities against the yeasts screened. Compound 31,
without any hydrophobic substituents, is a good inhi-
bitor of C. albicans; the yeast that is considered the ‘most
hardy’ in the series with regards to SQS inhibitors—
compare S1 (Table 8). The presence of a p-biphenyl
moiety in 32 increases activity. Addition of a citranellyl
side chain results in loss of activity, although this might
be due to problems with cell transport and insufficient
lactone hydrolysis in C. albicans. This is confirmed with
analogue 41, where only modest activity was observed.
Compounds 32 and 39 are modest inhibitors of S. cerevi-
siae and S. pombe, which was surprising in light of the S1
results. Analogue 40 retained activity towards both S.
cerevisiae and S. pombe, demonstrating the promise of
this class. Analogue 41 also exhibits good activity
towards S. cerevisiae and S. pombe. The inhibitory activ-
ities of the T-4 class against the bacteria B. subtilis and S.
aureus were poor, although somewhat surprisingly good
activities for analogues 39, 40 and 41 were observed
against E. coli. This final result was the most intriguing,
as 39, 40 and 41 are seemingly selective for E. coli.
observed (5.4%). The p-biphenyl analogue 32 was a
surprise inhibitor, showing a reduction in squalene for-
mation of 12.3%. The presence of a citranellyl chain (40)
or an alkyl-phenyl tether (41) gave the best activities
(16.5 and 16.6%, respectively) in the T-4 series.
Conclusion
We have designed a three-dimensional pharmacophore
for SQS by simulation of the substrate/intermediates
and known inhibitors of the enzyme. Utilization of the
distance geometry approach provided by the pharma-
cophore has presented several novel targets for synthetic
elaboration (T-1–T-6). The choice of the targets was
based on rigidity and the desired orientation of the
hydrophobic substituents. This was rather fortuitous in
retrospect, although most appropriate when compared
to the structural architecture provided by the X-ray co-
crystal structures of SQS and inhibitors described
recently by Pandit et al.12 The synthesis of T-2 and T-3
analogues were approached with a view to identifying
promising activity early without following a time-con-
suming path to these rather complex targets. The anti-
microbial activities of these compounds were poor,
although somewhat surprisingly, the T-2 analogues 5, 7
and 9 exhibited activity against PL-SQS. We appreciate
that these compounds have a large degree of hydro-
phobic character and this is most likely to be attribu-
table to their poor anti-microbial activities. It seems
plausible that within the mammalian cell, the cyclobu-
tanones 5, 7 and 9 might be converted to a more active
form and future studies will address this.
Mammalian pig liver SQS (PL-SQS) activities
Several analogues that exhibited promising activity were
evaluated for inhibition against PL-SQS using an adap-
ted procedure as described by Higson et al.37 S1 was
used as a control and showed a 27.5% reduction in
squalene formation at 10 mM. The relatively low activity
is attributable to the crude liver cell extracts used in this
assay, although we believe that the use of this type of
enzyme preparation gives a clearer indication of the real
activity of the compounds in vitro and also enables the
T4-class to be ‘activated’ by hydrolysis of the lactone
ring. The T-2 analogues all exhibited some inhibitory
activity towards PL-SQS at 10 mM (Table 9).
The T-4 analogues exhibited the greatest activities
against mammalian PL-SQS. It was pleasing to see that
the presence of a p-biphenyl and citranellyl moiety or an
alkyl-phenyl tether produced the best results. Future
work will focus on the synthesis of the ring-opened
forms of analogues 40 and 41, and in order to evaluate
whether hydrolysis is occurring within the cell, feeding
studies with T-4 analogues will be conducted.
Table 9. Pig liver SQS inhibition results
Compda
Reduction in SQS Activityb
S1 (ZA-A)
5
7
27.5
7.6
8.3
14.4
5.4
12.3
8.3
1
0.276
0.302
0.526
0.196
0.447
0.302
0.600
0.604
9
31
32
39
40
41
Experimental
Nuclear Magnetic Resonance (1H and 13C) spectra were
recorded on a Jeol GSX270 (270 MHz) spectrometer.
Chemical shifts are reported in parts per million (d)
downfield from an internal tetramethylsilane reference.
Coupling constants (J values) are reported in hertz (Hz),
and spin multiplicities are indicated by the following
symbols: s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet). The relative proportion of solvents in
mixed chromatography solvents refers to the volume/
volume ratio. Triethylamine was dried over calcium
hydride and distilled before use. CH2Cl2 was distilled
from calcium hydride prior to use. All reactions were
performed in an inert atmosphere created by a slight
positive pressure of argon. GC spectra were recorded on
a Finnigan 2000 series GC coupled to a Finnigan Trace
MS, source Electron Impact (EI) 70 eV. 2-Phenylpro-
panoyl chloride, (+)-citranellyl carbonoyl chloride and
16.5
16.6
aAll test compounds at 10 mM.
bRelative activity with S1 in bold.
For example, diol 5 caused a modest 7.6% reduction in
squalene production. The presence of a benzoxy sub-
stituent in the exo position at C3 (7) marginally enhanced
this activity (8.3%). Addition of a tether between the
phenyl and the C3 position (9) resulted in a significant
increase in activity (14.4%). The T-4 analogues exhibited
activity that fits well with increasing the hydrophobicity
via use of a p-biphenyl moiety and the fact that these
compounds have a diphosphate isostere released on
hydrolysis. We expected the basic ‘core’ analogue 31 to
exhibit no activity, although a modest amount was

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I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
4-phenylbutanoyl chloride were synthesised from
commercially available 2-phenyl-propanoic acid, (+)-
citranellic acid and 4-phenyl-butyric acid, respectively.
Benzoyl chloride and compound 32 were purchased
from Lancaster Synthesis and used as received. 7-exo-
Chloro-7-endo-phenylbicyclo[3.2.0]hept-2-en-6-one (11)
and 7-endo-phenylbicyclo[3.2.0]hept-2-en-6-one (12) were
synthesised using known literature procedures29b (Æ)-
Bicyclo[3.2.0]hept-2-en-6-one (35) was prepared as pre-
viously described.28 Synthesis of both enantiomers of
bicyclo[3.2.0]hept-2-en-6-one [35-(R) and 35-(S)] was
performed on a 20g scale.33 The synthesis of compounds
37, 38 and 31 were conducted on a 10g scale as previously
described.32 High resolution mass spectra were run by the
EPSRC high-resolution mass spectrometer facility at the
University of Swansea (UK).
2957, 2868, 1770, 1602, 1493, 1445, 1292, 1241, 1058,
1
941, 862, 700, 534. d H (CDCl3) 7.21–7.35 (5H, m, Ph-
H), 3.64–3.82 (3H, m, C2-H, C3-H and C5-H), 2.87–
2.89 (1H, d, J=7.3, C1-H), 2.20–2.29 (2H, b, C2-OH
and C3-OH), 2.12–2.19 (1H, m, C4-H endo), 1.84–1.96
(1H, m, C4-H exo), 1.62 (3H, s, C7(C10-H3)). d 13C
215.2 (C6), 139.7 (C7(C10ipso), 128.7 (2C), 127.0 (2C),
125.9, 74.7 (C2), 73.9 (C3), 66.5 (C7), 57.0 (C5), 49.4
(C1), 32.8 (C4), 28.3 (C7(C10)). LRMS (EI) m/z 232
(M+), 215.2 (M+ÀOH)+, 198 (M+À2OH)+, 172, 158,
147 (PhCH3CCOCH)+ 100%, 132 (PhCH3CCO)+, 129,
115, 104.1 (PhCCH3)+, 99.1 (C5H7O2)+ 91, 77 (Ph)+, 65
(C5H5)+. Found: C, 72.65; H, 7.09 expected for
C14H16O3 C, 72.39; H, 6.94.
(Æ)-2-exo-Hydroxy-3-exo-benzoxy-7-endo-phenyl-7-exo-
methylbicyclo[3.2.0]hept-2-en-6-one (7). Compound 5
(0.10 g, 0.44 mmol) was added to a stirred mixture of
DMAP (5 mg, Cat.) in pyridine (2 mL). The mixture was
stirred for 30 min and then benzoyl chloride (67 mg,
0.48 mmol, 1.1 equiv) in pyridine (0.5 mL) was added
dropwise over 10 min. The mixture was placed under an
atmosphere of argon and stirred for 18 h. The mixture
was quenched with water (2 mL) and extracted with
ether (3ꢁ5 mL). The combined organic extracts were
washed with 10% aqueous hydrochloric acid (3ꢁ5 mL)
and water (1ꢁ5 mL), dried (MgSO4) and concentrated
in vacuo to give an oil. Purification by flash chromato-
graphy using EtOAc/hexane (1:10, v/v) gave impure 8 as
a colourless oil (32 mg, 16.3%, 23.4% based on recov-
ered 5, repeated chromatography failed to give 8 in higher
purity) followed by 7 as a white solid (46 mg, 31.2%,
45.5% based on recovered 5, mp 113–114 ꢀC). Data for
compound 7: nmax (KBr)/cmÀ1 3555, 3026, 2967, 2872,
1949, 1769, 1743, 1602, 1585, 1444, 1103, 702. d 1H
(CDCl3) 7.94–7.97 (2H, d, J=7.4, C3(C40-H and C80-
H), 7.53–7.58 (1H, dd, J=7.4 and 7.4, C3(C60-H), 7.26–
7.37 (7H, m, C3(C50-H and C70-H) and C7(Ph-H), 4.90–
4.97 (1H, m, C3-H), 4.05 (1H, dd, J=1.8 and 4.1 Hz,
C2-H), 3.91–3.98 (1H, d, J=8.1, C5-H), 2.99–3.02 (1H,
dd, J=8.1 and 1.8, C1-H), 2.40–2.47 (1H, m, C4-H
exo), 2.16–2.24 (1H, m, C4-H endo), 2.02 (1H, s, C2-
OH), 1.68 (3H, s, C7(C10-H3)). LRMS (EI) m/z 215
(M+-OCOPh)+ 21%, 196 (M+ÀOCOPhÀOH)+, 132.0
(PhCMeCO)+, 129.1, 122, 104.8 (PhCMe)+ 100%,
91.0, 76.9 (Ph), 55, 51. Found: C, 74.80; H, 6.48, expec-
ted for C21H20O4, C, 74.98; H, 5.99. Representative data
for compound 8 (Selected): d 1H (CDCl3) 8.02–8.07
(2H, m, C3(C40-H and C80-H)), 7.82–7.85 (2H, dd,
J=8.1 and 8.1, C2(C40-H and C80-H)), 7.26–7.62 (11H,
m, C7(Ph-H), C3(C50-H, C60-H and C70-H) and C2(C50-
H, C60-H and C70-H), 5.36–5.38 (1H, dd, J=4.0 and
1.8, C2-H), 5.03–5.08 (1H, m, C3-H), 3.95–4.02 (1H, dd,
J=8.1 and 8.1, C5-H), 3.16–3.19 (1H, dd, J=8.1 and
1.8, C1-H), 2.61–2.68 (1H, d, J=19.4, C4-H endo),
2.27–2.36 (1H, m, C4-H exo), 1.70 (3H, s, C7(C10-H3).
(Æ)-7-endo-Phenyl-7-exo-methylbicyclo[3.2.0]hept-2-en-6-
one (3). 2-Phenylpropanoyl chloride 1 (3.01 g, 17.9
mmol) was added dropwise to a magnetically stirred
solution of [1,3]-cyclopentadiene (2.6 g, 39.3 mmol, 2.2
equiv) and Et3N (2.7 g, 26.8 mmol, 1.5 equiv) in CHCl3
(100 mL) at 0 ꢀC over 30 min. The mixture was allowed
to warm to 25 ꢀC and then stirred for 3 h. CHCl3 was
removed in vacuo to ca. 10 mL, then hexane (50 mL)
was added and the white precipitate was filtered through
a pad of Celite, which was washed with 3ꢁ10 mL por-
tions of hexane. Concentration of the filtrate in vacuo
afforded a dark coloured oil. Purification by flash chro-
matography using ether/hexane (1:6, v/v) afforded the
known38 title compound as a viscous lime green oil (2.52g,
71.2%). nmax (neat) cmÀ1 3059, 2960, 2922, 1771, 1603,
1371, 1203, 702. d 1H (CDCl3) 7.15–7.36 (5H, m, Ph-H),
5.55–5.70 (1H, m, C2-H), 5.47–5.52 (1H, m, C3-H),
3.99–4.06 (1H, m, C5-H), 3.55–3.59 (1H, m, C1-H),
2.63–2.73 (1H, m, C4-H endo), 2.41–2.54 (1H, m, C4-H
exo), 1.67 (3H, s, C7(C10)-H3). d 13C (CDCl3) 211.1
(C6), 140.7 (C10 ipso), 133.2, 128.2 (2C), 127.4 (2C), 126.2,
126.1, 71.5 (C7), 57.2 (C5), 51.6 (C1), 34.2 (C4), 27.1
(C7(C10)). LRMS (EI) m/z 198 (M+) 6%, 183 (M+
ÀCH3)+, 170 (M+-CO)+, 155, 132 (Ph(CH3)CCO)+,
104 (Ph(CH3)C)+, 91, 77 (Ph)+, 66 (C5H6)+, 51 (100%).
(Æ)-2-exo-Hydroxy-3-exo-hydroxy-7-endo-phenyl-7-exo-
methylbicyclo[3.2.0]hept-2-en-6-one (5). Solvent system
A: Tetrahydrofuran (8 mL), tert-butyl alcohol (5 mL)
and water (3 mL). To a stirred mixture of N-methyl-
morpholine-N-oxide (0.60 g, 5.11 mmol), osmium tetra-
oxide (25 mg, 0.1 mmol, 2 mol%) in solvent system A,
was added compound 3 (1 g, 5.1 mmol) in small portions
over a few min. The mixture was left to stir at room
temperature under an atmosphere of argon and mon-
itored by TLC. After 18 h the alkene was consumed
completely (TLC). The mixture was diluted with CH2
Cl2 (50 mL) and washed with 20% aqueous sodium sul-
phite solution (3ꢁ20 mL), 10% aqueous hydrochloric
acid (2ꢁ20mL) and finally water (2ꢁ20mL). The aqu-
eous phases were back extracted with CH2Cl2 (2ꢁ20mL).
The combined organic extracts were dried (MgSO4) and
concentrated in vacuo to give a yellow oil. Purification
by flash chromatography using EtOAc/ hexane (1:1, v/v)
gave the title compound as a white solid (0.73 g, 62.6%,
Rf=0.37, EtOAc, mp 98–99 ꢀC). nmax (KBr)/cmÀ1 3349,
(Æ)-2-exo-Hydroxy-3-exo-(30 -phenylpropyl)-carboxy-7-
endo-phenyl-7-exo-methylbicyclo[3.2.0]hept-2-en-6-one (9)
and (Æ)-2-exo-(30-phenylpropyl)-carboxy-3-exo-(30-phenyl-
propyl)-carboxy-7-endo-phenyl-7-exo-methyl bicyclo[3.2.0]
hept-2-en-6-one (10). Following the procedure for com-
pound 7: Compound 5 (104 mg, 0.45 mmol) was reacted

I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
2651
with 4-phenyl butanoyl chloride (89.9 mg, 0.49 mmol) in
pyridine (2 mL). Purification by flash chromatography
using EtOAc/hexane (1:4, v/v) gave 10 as a colourless oil
(87.7 mg, 37.2%, 43.9% based on recovered 5) followed
by 9 as a viscous yellow oil (19.2 mg, 11.3%, 13.4%
based on recovered 5). The last component eluted was 5
J=1.0, 7.8, C1-H), 2.43–2.56 (1H, m, C4-H exo), 2.28–
2.34 (1H, d, J=14.8, C4-H endo), 1.62 (1H, s, C3-OH).
d
13C (CDCl3) 200.8 (C6), 136.9 (C7 (C10 ipso)), 129.0,
128.9, 128.8, 127.6, 127.4, 80.9 (C3), 80.5 (C7), 59.8
1
(C2), 57.0 (C5), 53.1 (C1), 35.1 (C4). d H–1H COSY
(CDCl3) 4.32 (C2-H and C3-H) and 2.43 (C4-H exo),
4.32 (C2-H and C3-H) and 4.05 (C1-H), 4.05 (C1-H)
and 3.93 (C5-H). LRMS (CI) m/z 334 (M 81Br, +NH4)
7%, 332 (M 79Br, +NH4) 5.5%, 314 (2.5%), 312 (2%),
232, 218, 202 (100%), 185, 118, 108, 91, 55. HRMS
(CI): m/z exact mass calculated for C13H16NO279BrCl
332.0052; found 332.0050.
1
as a white solid (16 mg). Data for compound 9: d H
(CDCl3) 7.13–7.42 (10H, m, Ph-H), 4.63–4.70 (1H, m,
C3-H), 3.82–3.89 (2H, m, C2-H and C5-H), 2.89–2.92
(1H, dd, J=1.8, 6.2, C1-H), 2.57–2.63 (2H, t, J=7.4, C3
(C30-H2)), 2.23–2.40 (3H, m, C3 (C50-H2) and C4-H
exo), 1.99–2.04 (1H, d, J=12.1, C4-H endo), 1.89–1.98
(2H, m, C3(C40-H2)), 1.63 (3H, s, C7 (C10-H3)). d
1H–1H COSY (CDCl3) 4.63 (C3-H) and 3.82 (C2-H),
3.82 (C2-H and C5-H) and 2.89 (C1-H), 3.82 (C5-H)
and 2.23 (C4-H exo), 4.88 (C3-H) and 2.23 (C4-H exo).
Found: C, 75.74; H, 7.16, expected for C21H20O4, C,
76.17; H, 6.92. Representative data for compound 10: 0d
1H (CDCl3) 7.07–7.41 (15H, m, C7 (Ph–H), C2 (C5 -
Ph–H) and C3 (C50-Ph–H)), 4.98–5.00 (1H, dd, J=1.8,
4.1, C2-H), 4.70–4.78 (1H, m, C3-H), 3.79–3.86 (1H, d,
J=7.7, C5-H), 2.87–2.91 (1H, dd, J=1.8, 7.7, C1-H),
2.62–2.65 (2H, t, J=7.4, C2 (C30-H2)), 2.49–2.55 (2H, t,
J=7.5, C3 (C30-H2)), 2.27–2.38 (3H, m, C4-H exo and
C2 (C40-H2)), 2.13–2.18 (2H, t, J=7.5, C3 (C40-H2)),
1.75–2.02 (5H, m, C4-H endo, C2 (C50-H2) and C3 (C50-
H3)), 1.63 (3H, s, C7 (C10-H3)). d 13C (CDCl3) 211.1
(C6), 172.3 (C2 (C20)), 171.9 (C3 (C20)), 141.3 (C ipso),
138.7 (C ipso), 128.9, 128.4, 128.4, 128.35, 127.4 (2C),
126.1 (2C), 125.9, 125.7, 74.2 (C2), 73.3 (C3), 67.5 (4)
(C7), 56.1 (C5), 47.5 (C1), 35.1 (C2 (C30), 34.9 C3 (C30),
33.6 (C2 (C40), 33.3 (C4), 30.6 (C3 (C40)), 28.2 (C2
(C40)), 26.4 (C3 (C40)), 26.2 (C8).
(Æ)-2-exo-Bromo-3-endo-methoxy-7-endo-phenyl-7-exo-
chlorobicyclo[3.2.0]hept-6-one (23). To a stirred mixture
containing compound 11 (0.56 g, 2.56 mmol) in metha-
nol (10 mL) was added NBA (0.53 g, 3.84 mmol, 1.5
equiv). Reaction time: 16 h (TLC) and standard work
up gave an oil. Purification by flash chromatography
using ether/hexane (1:4, v/v) gave the title compound as
a colourless oil (0.74 g, 87.8%). nmax (neat)/cmÀ1 3064,
2989, 2931, 2829, 1794, 1600, 1581, 1450, 1088, 911,
1
732, 699, 658, 613. d H (CDCl3) 7.26–7.60 (5H, m, Ph-
H), 4.30 (1H, s, C3-H), 4.27–4.28 (1H, d, J=4.8, C1-H),
3.90 (1H, s, C2-H), 3.86–3.89 (1H, d, J=4.8, C5-H),
2.52 (3H, s, C3 (C20-H3)), 2.25–2.49 (2H, m, C4-H2). d
13C (CDCl3) 200.5 (C6), 136.9 (C7 (C10 ipso)), 129.4,
129.4, 128.8, 128.1, 127.6, 89.3 (C3), 80.5 (C7), 59.9
(C1), 57.5 (C2), 55.9 (C5), 49.4 (C3 (C20)), 33.1 (C4).
Found: C, 50.52; H, 3.97, expected for C14H14BrClO2
C, 51.01; H, 4.28.
(Æ)-2-exo-Bromo-3-endo-acetoxy-7-endo-phenyl-7-exo-
chlorobicyclo[3.2.0]hept-6-one (24). To a stirred mixture
containing compound 11 (1.0 g, 4.6 mmol) in acetic acid
(10 mL) and CH2Cl2 (10 mL) was added NBA (0.94 g,
6.81 mmol, 1.5 equiv) over 30 min. Reaction time: 16 h
(TLC). Purification by flash chromatography using
EtOAc/hexane (1:9, v/v) gave the title compound as a
light green crystalline solid (1.26 g, 77.3%, mp 38 ꢀC,
Rf=0.12, EtOAc/hexane (1:9, v/v)). nmax (neat)/cmÀ1
3064, 2977, 2869, 1795, 1746, 1600, 1583, 1448, 1371,
(Æ)-2-endo-Bromo-3-exo-hydroxy-7-endo-phenyl-7-exo-
chlorobicyclo[3.2.0]hept-6-one (21) and (Æ)-2-exo-bromo-
3-endo-hydroxy-7-endo-phenyl-7-exo-chlorobicyclo[3.2.0]-
hept-6-one (22). To a stirred mixture containing 11
(2.46 g, 11.3 mmol) in acetone (30 mL) and water
(10 mL) was added NBA (2.33 g, 16.9 mmol, 1.5 equiv)
over 30 min. After 16 h compound 11 was completely
consumed (TLC) and standard work up gave a yellow
oil. Purification by flash chromatography using ether/
hexane (1:4, v/v) gave the known25 bromohydrin 22 as a
fluffy white solid (1.33 g, 37.3%, mp 102–103 ꢀC) fol-
lowed by 21 as a white solid (0.33 g, 9.3%, mp 107–
108 ꢀC). Data for compound 21: nmax (KBr)/cmÀ1 3552,
1
1221, 1026, 910, 733, 699. d H (CDCl3) 7.70–7.75 (2H,
m, C7 (C20-H and C60-H)), 7.41–7.59 (3H, m, C7 (C30-
H, C40-H and C50-H)), 5.33–5.34 (1H, d, J=4.0, C3-H),
4.53–4.59 (1H, dd, J=4.4, 7.7, C5-H), 4.45 (1H, s, C2-
H), 4.12–4.15 (1H, d, J=7.7, C1-H), 2.66–2.77 (1H, m,
C4-H exo), 2.44–2.50 (1H, d, J=15.0, C4-H endo), 1.68
(3H, s, C3 (C30-H3)). d 13C (CDCl3) 201.0 (C6), 168.8
(C3 (C20)), 135.9 (C7 (C10 ipso)), 129.3, 129.1, 128.9
(2C), 127.4, 82.2 (C3), 79.5 (C7), 60.6 (C5), 57.4 (C2),
1
3079, 3022, 2961, 1786, 1487, 1256, 1145, 1029. d H
(CDCl3) 7.51–7.55 (2H, m, C7 (C20-H and C60-H)),
7.33–7.36 (3H, m, C7 (C30-H, C40-H and C50-H)), 4.20–
4.27 (2H, m, C2-H and C3-H), 4.02–4.08 (1H, m, C1-
H), 3.66–3.72 (1H, m, C5-H), 2.51–2.59 (1H, m, C4-H
exo), 2.16 (1H, s, C3-OH), 1.80 (1H, m, C4-H endo).
LRMS (CI) m/z 334 (M 81Br, +NH4) 2%, 332 (M 79Br,
+NH4) 1.5%, 314 (5%), 312 (5%), 260, 232, 216, 202
(100%), 185, 118, 108, 91. HRMS (CI): m/z exact mass
calculated for C13H16NO279BrCl 332.0052; Found
332.0056. Data for compound 22: nmax (KBr)/cmÀ1
3539, 3088, 3025, 2981, 2958, 1786, 1488, 1393, 1258,
0
1
49.7 (C1), 32.4 (C4), 20.5 (C3 (C3 )). d H–1H COSY
(CDCl3) 5.33 (C3-H) and 2.66 (C4-H exo), 4.53 (C5-H)
and 4.12 (C1-H), 2.66 (C4-H exo) and 2.44 (C4-H endo).
LRMS (CI) m/z 376 (M 81Br, +NH4) 23%, 374 (M
79Br, +NH4) 17%, 342, 340, 298, 296, 260, 218 (100%),
200, 183, 155. HRMS (CI): m/z exact mass calculated
for C15H18NO379BrCl 374.0158; Found 374.0154.
Found: C, 50.23; H, 3.86; Br, 22.13, expected for C15
H14BrClO3 C, 50.38; H, 3.95; Br, 22.34.
1
1169, 1033, 990, 832, 700, 660, 629. d H (CDCl3) 7.52–
7.57 (2H, m, C7 (C20-H and C60-H)), 7.34–7.42 (3H, m,
C7 (C30-H, C40-H and C50-H)), 4.32–4.41 (2H, m, C2-H
and C3-H), 4.05 (1H, m, C5-H), 3.93–3.95 (1H, dd,
(Æ)-2-exo-Bromo-3-endo-benzoxy-7-endo-phenyl-7-exo-
chlorobicyclo[3.2.0]hept-6-one (25). To a stirred mixture

2652
I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
containing compound 11 (1.01 g, 4.6 mmol) and benzoic
acid (4.47 g, 36.61 mmol, 8 equiv) in CH2Cl2 (30 mL)
was added NBA (0.94 g, 6.8 mmol, 1.5 equiv) over
30 min. Reaction time: 16 h (TLC) and standard work
up gave a yellow solid. Recrystallisation of the yellow
solid from ether gave the title compound as white crys-
tals (0.90 g, 47.1%, mp 148–149 ꢀC). nmax (KBr)/cmÀ1
3070, 3030, 2996, 2949, 1948, 1852, 1781, 1722, 1599,
1493, 1261, 1098, 991, 826. d 1H (CDCl3) 7.51–7.58 (3H,
m, C3 (C40-H), C3 (C60-H) and C3 (C80-H)), 7.33–7.44
(4H, m, C7 (C20-H)), C7 (C60-H), C3 (C50-H and C70-
H)), 6.95–7.01 (1H, dd, J=7.2 and 7.3, C7 (C40-H)),
6.82–6.87 (2H, dd, J=7.5, 7.5, C7 (C30-H and C50-H)),
5.31–5.32 (1H, d, J=4.4, C3-H), 4.48–4.55 (2H, m, C5-
H and C2-H), 3.02–3.07 (1H, m, C1-H), 2.64–2.74 (1H,
m, C4-H exo), 2.50–2.55 (1H, d, J=14.7, C4-H endo). d
13C (CDCl3) 201.0 (C6), 165.1 (C3 (C20)), 135.2 (C ipso),
134.6 (C ipso), 133.3, 129.9 (2C), 128.8 (2C), 128.6 (2C),
128.1 (2C), 127.3, 83.5 (C3), 79.8 (C7), 61.1 (C2), 57.3
(C5), 49.6 (C1), 32.1 (C4). Found: C, 57.33; H, 3.74,
expected for C20H16BrClO3 C, 57.24; H, 3.84.
(EI) m/z 294 (M+ 79Br) and 296 (M+ 81Br) 13%, 276
(M+ 79Br-H2O)+ and 278 (M+ 81BrÀH2O)+, 215
(M+ÀBr)+, 197 (M+ÀBrÀH2O)+, 155, 132 (Ph(CH3)
CCO)+, 104 (Ph(CH3)C)+, 91, 77 (Ph)+, 55 (100%).
LRMS (CI) m/z 314 (M 81Br, +NH4) 84%, 312 (M
79Br, +NH4) 85%, 296 (M+ 81Br) 10%, 294 (M+ 79Br)
10.5%, 232 (100%), 216, 197, 169, 160, 132, 55. HRMS
(CI) m/z (+NH4 enhanced) exact mass calculated for
C14H19N79O2Br 312.0599; Found 312.0595. Found: C,
56.64; H, 5.37; Br, 26.72, expected for C14H15BrO2, C,
56.97; H, 5.12; Br, 27.07.
(Æ)-2-exo-Bromo-3-endo-methoxy-7-endo-phenyl-7-exo-
methylbicyclo[3.2.0] hept-6-one (15). To a stirred mix-
ture containing compound 3 (206 mg, 1.04 mmol) in dry
methanol (10 mL) was added NBA (0.196 g, 1.41 mmol,
1.4 equiv). Reaction time: 8 h (TLC) and standard work
up gave an oil. Purification by flash chromatography
using ether/hexane (1:4, v/v) gave the title compound as a
colourless oil (0.26g, 81.7%, mp 56–57 ꢀC, Rf=0.44, 3:7
ether/hexane v/v), which crystallised on standing. nmax
(KBr)/cmÀ1 3525, 3061, 3019, 2923, 2829, 1951, 1879,
1
(Æ)-2-exo-Bromo-3-endo-benzoxy-7-endo-phenyl-7-exo-
bromobicyclo[3.2.0] hept-6-one (27). To a stirred mix-
ture containing compound 12 (0.40 g, 2.2 mmol) and
benzoic acid (1.59 g, 13.0 mmol, 6 equiv) in CH2Cl2
(20 mL) was added NBA (0.45 g, 3.3 mmol, 1.5 equiv)
over 30 min. Further NBA (1 equiv) was added in one
portion after 8 h. Reaction time: 16 h (TLC) and stan-
dard work up gave a yellow solid. Recrystallization of
the yellow solid from ether gave the title compound as a
white fluffy solid (0.52 g, 62.3%, mp 157–158 ꢀC). nmax
(KBr)/cmÀ1 3069, 3028, 2944, 1950, 1848, 1782, 1723,
1600, 1497, 1260, 998, 719. d 1H (CDCl3) 7.54–7.66 (3H,
m, C3 (C40-H), C3 (C60-H) and C3 (C80-H)), 7.26–7.46
(4H, m, C7 (C20-H), C7 (C60-H), C7 (C30-H) and C7
(C50-H)), 6.94–6.99 (1H, dd, J=7.5 and 7.5, C7 (C40-
H)), 6.81–6.87 (2H, dd, J=7.5, 7.5, C3 (C50-H) and C3
(C70-H)), 5.34–5.35 (1H, d, J=4.4, C3-H), 4.52–4.56
(2H, m, C5-H and C2-H), 2.98–3.02 (1H, m, C1-H),
2.66–2.77 (1H, m, C4-H exo), 2.51–2.56 (1H, d, J=14.7,
C4-H endo). d 13C (CDCl3) 200.9 (C6), 165.4 (C3 (C20)),
135.6 (C ipso), 133.6 (C ipso), 130.3, 128.9, 128.8, 128.7
(2C), 128.6 (2C), 128.3, 127.6, 127.5, 83.9 (C3), 71.7
(C7), 60.9 (C2), 57.9 (C5), 49.4 (C1), 31.9 (C4). Found:
C, 51.24; H, 3.31; Br, 34.19 expected for C20H16Br2O3
C, 51.75; H, 3.47; Br, 34.43.
1772, 1600, 1493, 1250, 1089, 761, 695. d H (CDCl3)
7.17–7.40 (5H, m, Ph–H), 4.36 (1H, s, C3-H), 3.91–3.97
(1H, m, C5-H), 3.86–3.88 (1H, d, J=4.0, C2-H), 3.33–
3.36 (1H, d, J=7.7, C1-H), 2.49 (3H, s, C3 (C20-H3)),
2.33–2.42 (1H, m, C4-H exo), 2.21–2.26 (1H, d, J=13.9,
C4-H endo), 1.61 (3H, s, C7 (C10-H3)). d 13C (CDCl3)
211.6 (C6), 141.7 (C ipso), 128.4 (2C), 126.2 (2C), 125.8,
89.8 (C3), 70.0 (C7), 58.0 (C2), 55.9 (C5), 53.6 (C1), 50.7
(C3 (C20)), 32.8 (C4), 30.6 (C7 (C10)). LRMS (EI) m/z
308.1 (M+ 79Br), 310.1 (M+ 81Br) 10%, 277.8 (M+
ÀOMe)+, 229.1 (M+À Br)+, 197 (M+À BrÀOMe)+,
131.8 (PhCMeCO)+, 104.0 (PhCMe)+, 91.0, 79.0 (79Br),
77.0 (Ph)+, 65 (C5H5)+, 55 (100%). LRMS (CI) m/z 330
(M 81Br, +NH4) 100%, 328 (M 79Br, +NH4) 98%,
216, 197, 169, 132, 99. HRMS (CI) (+NH4 enhanced)
m/z exact mass calculated for C15H21N79O2Br 326.0755;
Found 326.0758.
79
79
(Æ)-2-exo-Bromo-3-endo-acetoxy-7-endo-phenyl-7-exo-
methylbicyclo[3.2.0]hept-6-one (16). To a stirred mixture
containing compound 3 (640 mg, 3.2 mmol) in CH2Cl2
(10 mL) and acetic acid (3 mL) was added NBA
(669 mg, 4.9 mmol, 1.5 equiv). Reaction time: 4 h (TLC)
and standard work up gave a colourless oil. Purification
by flash chromatography using ether/hexane (1:4, v/v)
gave the title compound as a colourless oil (0.86 g,
79.2%). nmax (neat)/cmÀ1 3057, 3023, 2967, 1959, 1839,
1776, 1743, 1601, 1494, 1444, 1371, 1224, 1027, 764. d
1H (CDCl3) 7.17–7.42 (5H, m, Ph–H), 5.18–5.20 (1H, d,
J=4.1, C3-H), 4.34 (1H, s, C2-H), 4.02–4.07 (1H, d,
J=8.0, C1-H), 3.42–3.50 (1H, m, C5-H), 2.47–2.57 (1H,
m, C4-H exo), 2.02–2.08 (1H, d, J=11.4, C4-H endo),
1.63 (3H, s, C7 (C10-H3)), 1.51 (3H, s, C3 (C30-H3)). d
13C (CDCl3) 211.8 (C6), 169.2 (C3 (C10)), 141.0 (C7 (C10
ipso)), 128.6 (2C), 126.5 (2C), 125.5, 82.5 (C3), 69.8
(C7), 58.6 (C2), 53.6 (C5), 51.1 (C5), 31.8 (C4), 31.0 (C3
(C30)), 20.4 (C7 (C10)). LRMS (EI) m/z 198
(M+ÀBrÀOCOCH3)+ 8%, 182, 154.1, 141, 128, 115,
104 (PhCH3C)+, 91, 77 (Ph)+, 65 (C5H5)+, 55 (100%),
43 (COCH3)+. Found: C, 57.42; H, 5.45, expected for
C16H17BrO3 C, 56.99; H, 5.08.
(Æ)-2-exo-Bromo-3-endo-hydroxy-7-endo-phenyl-7-exo-
methylbicyclo[3.2.0] hept-6-one (14). To a stirred mix-
ture containing compound 3 (0.52 g, 2.63 mmol) in ace-
tone (15 mL) and water (5 mL) was added NBA (0.54 g,
3.95 mmol, 1.5 equiv) over 30 min. Reaction time: 16 h
(TLC) and standard work up gave a white solid. Pur-
ification by flash chromatography using ether/hexane
(1:3, v/v) gave the title compound as white crystals (0.43
g, 55.9%, mp 98–99 ꢀC). nmax (KBr)/cmÀ1 3504, 3023,
2933, 2861, 1955, 1889, 1772, 1600, 1446, 1109, 1033,
1
948, 700. d H (CDCl3) 7.18–7.40 (5H, m, Ph-H), 4.33–
4.35 (1H, m, C3-H), 3.92–4.00 (2H, m, C2-H and C5-
H), 3.31–3.34 (1H, d, J=7.7, C1-H), 2.35–2.46 (1H, m,
C4-H exo), 2.13–2.18 (1H, d, J=14.3, C4-H endo), 1.63
(3H, s, C7 (C10-H3)), 1.33–1.35 (1H, s, C3-OH). LRMS

I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
2653
(Æ)-2-exo-Bromo-3-endo-benzoxy-7-endo-phenyl-7-exo
methylbicyclo[3.2.0]hept-6-one (17). To a stirred mixture
containing compound 3 (606 mg, 3.06 mmol) and ben-
zoic acid (2.24 g, 18.4 mmol, 6 equiv) in CH2Cl2 (30 mL)
was added NBA (0.55 g, 3.98 mmol, 1.3 equiv). Reac-
tion time: 24 h (TLC) and standard work up gave a
white solid. Recrystallization from ether gave the title
compound as white crystals (0.77 g, 62.7%, mp 147–
148 ꢀC). nmax (KBr)/cmÀ1 3059, 3030, 2931, 2898, 2867,
1952, 1842, 1773, 1720, 1602, 1492, 1320, 1178, 1096,
precipitate was filtered and the filtrate concentrated in
vacuo to give the title compound as a light brown
oil (0.80 g, 100%), which was used without further
purification for the reaction with [1,3]-cyclopentadiene.
(Æ)-7-endo-para-Biphenyl-7-exo-methylbicyclo[3.2.0] hept-
2-en-6-one (13). Following a similar procedure for the
synthesis of compound 3: 2-para-Biphenylpropanoyl
chloride 29 (0.8 g, 3.3 mmol) was reacted with [1,3]-
cyclopentadiene (0.86 g, 13 mmol, 4 equiv) and Et3N
(0.6 g, 5.9 mmol, 1.8 equiv) in CHCl3 (30 mL). The usual
work up gave a dark brown oil. Purification by flash
chromatography using ether/hexane (1:6, v/v) gave the
title compound (0.59 g, 65.2%, mp 35–36 ꢀC) as light
1
989, 887, 761, 706, 626. d H (CDCl3) 6.76–7.61 (10H,
br m, 2ꢁPh-H), 5.31–5.33 (1H, d, J=4.4, C3-H), 4.56
(1H, s, C2-H), 4.14–4.17 (1H, dd, J=4.2 and 7.7, C5-
H), 3.45–3.48 (1H, d, J=7.7, C1-H), 2.60–2.70 (1H, m,
C4-H exo), 2.41–2.46 (1H, d, J=14.7, C4-H endo), 1.62
(3H, s, C7 (C10-H3)). d 13C (CDCl3) 211.8 (C6), 165.3
(C3 (C10)), 139.8 (C ipso), 133.1 (C ipso), 130.1 (2C), 128.3
(2C), 127.9 (2C), 126.3 (2C), 125.6 (2C), 83.9 (C3), 70.1
(C7), 59.1 (C2), 53.5 (C5), 51.1 (C1), 31.8 (C4), 31.1 (C7
(C10)). LRMS (EI) m/z 279.9 (M+ 81BrÀPhCO)+, 277.9
1
green crystals. d H (CDCl3) 7.26–7.58 (9H, m, ar–H),
5.68–5.72 (1H, m, C2-H), 5.52–5.56 (1H, m, C3-H), 4.00–
4.06 (1H, m, C5-H), 3.56–3.61 (1H, m, C1-H), 2.65–2.75
(1H, m, C4-H endo), 2.39–2.52 (1H, m, C4-H exo), 1.74
(3H, s, C7(C10-H3). d 13C (CDCl3) 213.2 (C6), 140.9
(C7(C10 ipso)), 139.8, 139.3, 133.4, 131.3, 128.7 (2C), 127.1
(2C), 126.9, 126.8, 126.6, 57.3 (C1), 51.6 (C5), 34.2 (C4),
26.9 (C7 (C10)). LRMS (EI) m/z 274 (M+) 6%, 208
(M+ÀC5H6)+, 165, 153 (C6H5ÀC6H4)+, 151, 131, 115,
89, 66 (C5H6)+ 100%, 51 (C4H3)+. Found: C, 87.02; H,
6.76, expected for C20H18O; C, 86.92; H, 7.26.
79
(M+ 79BrÀPhCO)+ 2%, 197 (M+À BrÀPhCO)+, 132
(PhCH3CCO)+, 105 (PhCO)+, 104 (PhCH3C)+, 91, 79.1
(79Br)+, 77 (Ph)+, 65.1 (C5H5)+, 55 (100%). LRMS
(CI) m/z 418 (M 81Br, +NH4) 100%, 416 (M 79Br,
+NH4) 98%, 338, 279, 277, 216, 199, 169, 132, 105.
HRMS (CI): m/z exact mass calculated for C21
H23N79BrO3 416.0861; Found 416.0866. Found: C,
63.43; H, 5.24, expected for C21H19BrO3 C, 63.17; H,
4.80.
(Æ)-2-exo-Bromo-3-endo-benzoxy-7-endo-para-biphenyl-
7-exo-methylbicyclo[3.2.0]hept-6-one (20). To a stirred
mixture containing compound 13 (128 mg, 0.47 mmol)
and benzoic acid (0.46 g, 3.7 mmol, 8 equiv) in CH2Cl2
(30 mL) was added NBA (96.7 mg, 0.7 mmol, 1.5 equiv).
Reaction time: 24 h (TLC) and standard work up gave
an oil. Purification by flash chromatography using
ether/hexane (1:4, v/v) gave the title compound as a vis-
cous light green oil which crystallised on standing
2-para-Biphenylpropanoyl chloride (29). To a stirred
solution of diisopropylamine (1.03 g, 10.3 mmol, 2.2
equiv) in dry THF (20 mL) at À84 ꢀC was added n-butyl
lithium (4 mL, 9.9 mmol, 2.1 equiv, 1.6 M solution in
hexanes) over 20 min under an atmosphere of argon.
The mixture was stirred for a further 30 min and then
allowed to warm to À30 ꢀC over 1 h. The colourless
LDA solution was cooled to À84 ꢀC and 2-para-biphe-
nylpropanoic acid (1.0 g, 7.7 mmol, 1 equiv) in dry THF
(5 mL) was added dropwise over 30 min. The mixture
was allowed to stir for 2 h at À84 ꢀC and then methyl
iodide (0.66 g, 5.2mmol, 1.1 equiv) in dry THF (2 mL)
was added dropwise over 30 min. Stirring was continued
for 2 h at À84ꢀC and then the mixture was allowed to
warm to 25 ꢀC over 3 h. The reaction was quenched with
satd NH4Cl (100 mL) and extracted with EtOAc (3
ꢁ100 mL). The combined organic extracts were washed
with satd brine (2ꢁ100 mL) and water (2ꢁ100 mL),
dried (MgSO4) and concentrated in vacuo to give a vis-
cous brown oil. The oil was taken up in ether and hex-
ane (1:1, 20 mL), and after standing for several hours
the acid precipitated to give the title compound as a fine
brown powder (0.81 g, 76.4%, mp 137–139 ꢀC). nmax
(KBr)/cmÀ1 3330, 2951, 1699, 1599, 1488, 1282, 1231,
1077, 1005, 953, 765, 722, 692. 2-para-Biphenylpropa-
noic acid (0.67 g, 2.9 mmol) in dry CHCl3 (10 mL) was
stirred at 0 ꢀC for 30 min, then oxalyl chloride (0.75 g,
5.9 mmol, 2 equiv) in dry CHCl3 (5 mL) was added
dropwise over 15 min, followed by addition of 10 mL of
N,N0-dimethylformamide (cat.) and stirred at 0 ꢀC for
1 h and then allowed to warm to 25 ꢀC and stirring
continued overnight. Concentration in vacuo afforded a
brown solid, which was taken up in ether (10 mL). The
ꢀ
1
(172 mg, 77.0%, mp 65–66 C). d H (CDCl3) 7.47–7.60
(2H, m, Ph–H), 7.21–7.41 (8H, m, Ph–H), 6.98–7.02
(2H, m, Ph–H), 5.32–5.34 (1H, d, J=4.4, C3-H), 4.66
(1H, s, C2-H), 4.14–4.20 (1H, dd, J=4.4, 7.7, C5-H),
3.49–3.52 (1H, d, J=7.7, C1-H), 2.62–2.73 (1H, m, C4-
H exo),02.44–2.50 (1H, d, J=14.7, C4 endo), 1.66 (3H, s,
C7 (C1 -H3)). FAB LRMS m/z 476.2 (M+ 81Br) and
474.2 (M+ 79Br), 355.1 (M+-PhCO2)+, 273.2, 245.2,
231.2, 208.2, 181.2, 153.1 (C6H5–C6H4)+ 100%, 105.1.
FAB HRMS: m/z exact mass calculated for C27
H23BrO3Na 497.0728; found 497.0732. Found: C, 67.94;
H, 4.89; Br, 16.65 expected for C27H23BrO3 C, 68.22; H,
4.88; Br, 16.81.
General procedure for the synthesis of the T-4 di-esters
To the alcohol 31 (100 mg, 0.28 mmol) in pyridine
(5 mL) was added the acid chloride (0.31 mmol, 1.1
equiv) in CH2Cl2 (2 mL) over 15 min, followed by addi-
tion of DMAP (3.5 mg, 10 mol%). The mixture was
stirred at 25 ꢀC until all 31 was consumed (TLC). The
mixture was diluted with water (5 mL) and extracted
with EtOAc (3ꢁ10 mL). The combined extracts were
washed successively with 5% aqueous hydrochloric acid
(3ꢁ10 mL) and water (1ꢁ10 mL), dried (MgSO4) and
concentrated in vacuo to give the product as an oil or
solid. Purification by flash chromatography gave the
products as white solids or viscous oils. For alcohol 32,

2654
I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
2.2 equiv of the acid chloride was used. All reactions
were run on a 0.28–0.30 mmol scale.
3:2 v/v). d 1H (CDCl3) 7.16–8.08 (14H, m, Ph–H), 5.35–
5.40 (1H, m, C6a-H), 5.07–5.12 (1H, m, C5-H), 4.11–
4.16 (2H, dd, J=1.1, 7.7, C4 (C10-H2), 2.89–2.99 (1H,
m, C4-H), 2.76–2.83 (1H, m, C3a-H), 2.61–2.68 (2H, m,
C3-H2), 2.33–2.51 (6H, m, C6-H2, C4 (C40-H2) and C4
(C60-H2)), 1.91–2.05 (2H, m, C4 (C50-H2)). FAB LRMS:
m/z 512.3 (M+Na)+ 48%, 499.3 (M+1)+ 17%, 353.2,
335.2 (MÀPh(CH2)3CO2)+, 198.1 (C6H4C6H5CO2+1)+,
196.1 (C6H4C6H5CO2À1)+, 181.1 (C6H4C6H5CO)+
100%, 165.1 (C6H4C6H5C)+, 121.1 (PhCO2)+, 119.1.
FAB HRMS: m/z exact mass calculated for C31H30
NaO6 521.1940; found, 521.1940.
(3aꢀ,4ꢀ,5ꢁ,6aꢀ)-(À)-Hexahydro-4-(10 -phenylcarboxy-
methyl)-2-oxo-2H-cyclopenta[b]furan-5-yl-1-phenyl-5-
carboxylate (39). Purification by flash chromatography
using EtOAc–hexane (1:1, v/v) gave the title compound
as a white solid, which was recrystallised from EtOAc
and hexane (117 mg, 53.1%, mp 100–102 ꢀC). d 1H
(CDCl3) 7.98–8.03 (4H, m, C4 (C50-H and C90-H) and
C5 (C40-H and C80-H)), 7.53–7.62 (2H, m, C4 (C70-H)
and C5 (C60-H)), 7.41–7.50 (4H, m, C4 (C60-H and C80-
H) and C5 (C50-H and C70-H)), 5.45–5.50 (1H, m, C5-
H), 5.11–5.16 (1H, dddd, J=2.1, 2.2, 5.9, 5.9, C6a-H),
4.39–4.42 (2H, d, J=7.0, C4 (C10-H2)), 2.88–3.02 (2H,
m, C3a-H and C4-H), 2.61–2.70 (3H, m, C6-H endo and
C3-H2), 2.36–2.43 (1H, m, C6-H exo). d 13C (CDCl3)
176.1 (C2), 166.5 (C5 (C20), 165.9 (C4 (C30), (1ꢁC ipso
not observed), 133.3 (C ipso), 129.6 (2C), 129.5 (2C),
128.5 (3C), 128.4, 83.9 (C5), 64.5 (C6a), 51.7 (C4 (C10),
40.7 (C3a), 38.3 (C3), 35.7 (C6). FAB LRMS: m/z 380
(M+), 303 (M+ÀPh)+, 226 (M+À2Ph)+, 132 (Ph
(Me)CCO)+, 104 (Ph(Me)C)+, 77 (Ph)+. FAB HRMS:
m/z exact mass calculated for C22H20O6 380.4011;
found, 380.4016.
Antimicrobial procedures
The antimicrobial activity of each compound was
assessed using an established standard disc-plate
method.35 Sabourands agar (Oxoid Products Ltd) was
prepared according to the manufacturers instructions,
then dispensed in 20 mL amounts into glass universal
bottles and these were autoclaved at 121 ꢀC and 15 lb
for 15 min. The molten agar was poured into agar plates
(Sterilin). Each strain was inoculated into 10 mL of
Sabourands broth (prepared according to manu-
facturers instructions) and incubated at the appropriate
temperatures (25 ꢀC for S. pombe and S. cerevisiae,
37 ꢀC for C. albicans for 12 h. After incubation the
resultant broth culture was diluted in sterile saline to
produce a 1/100 dilution. Aseptically, a 0.1 mL aliquot
of this dilution was pipetted onto the agar plate and
carefully spread around the agar surface. A sterile 4 mm
paper disc was placed on the centre of agar plate and
then the test compound (20 mL of a 10, 1, 0.1 or 0.01 mg/
mL in absolute ethanol or water with final concentra-
tions of 200, 20, 2, 0.2 mg, respectively) was inoculated
onto the sterile paper disc. These plates were incubated
at the appropriate temperatures 25 ꢀC for S. pombe and
S. cerevisiae, 37 ꢀC for C. albicans for 24 h. To investi-
gate the antimicrobial activity of the test compounds
against bacteria, the previous procedure was used,
however, nutrient agar (LAB M) and nutrient broth
(LAB M) were the preferred growth media. Incubation
of the bacterial species was conducted at 37 ꢀC for 24 h.
An antibiotic assay ‘ring’, which contains several known
potent anti-bacterial agents (Novobiocin, Penicillin G,
Streptomycin, Tetracyline, Chloroamphenicol, Ery-
thromycin, Fusidic acid and Methicillin), was used as
the control in these assays (individual discs contain 1
unit). Zones of inhibition (ꢂ5–8 mm) were observed for
these compounds.
(3aꢀ,4ꢀ,5ꢁ,6aꢀ)-(À)-Hexahydro-4-(50,90-dimethyl-hept-
80-en-10-carboxymethyl)-2-oxo-2H-cyclopenta[b]furan-5-
yl-1,10-biphenyl-5-carboxylate (40).
Purification by flash chromatography using EtOAc/
hexane (1:1, v/v) gave the title compound as a light yel-
1
low viscous oil (83.1 mg, 58.1%). d H (CDCl3) 7.36–
8.08 (9H, br m, Ph–H), 5.39–5.41 (1H, m, C6a-H), 5.05–
5.13 (2H, m, C5-H and C4 (C80-H)), 4.14–4.16 (2H, d,
J=6.2, C4 (C10-H2)), 2.91–3.01 (1H, m, C4-H), 2.80–
2.86 (1H, m, C3a-H), 2.31–2.64 (4H, m, C3-H2 and C6-
H2), 1.97–2.21 (3H, m, C4 (C50-H) and C4 (C40-H2)),
1.60 (3H, s, C4 (C100-H3)), 1.57 (3H, s, C4 (C110-H3)),
1.30–1.50 (4H, m, C4 (C60-H2) and C4 (C70-H2)), 0.93–
1.01 (3H, d, J=8.1, C4 (C120-H3)). Found: C, 73.18%;
H, 7.15%, C28H24O6 requires C, 73.79%; H, 7.19%.
(3aꢀ,4ꢀ,5ꢁ,6aꢀ) - (À) - Hexahydro - 4 - (30 - phenylpropyl
carboxymethyl)-2-oxo-2H-cyclopenta[b]furan-5-yl-1,10 -
biphenyl-5-carboxylate (41).
Pig liver SQS bioassay (HPLC-based)37
Pig liver was used as the mammalian source of SQS,
transported at 0 ꢀC and used within 24 h. Liver samples
were perfused with HEPES buffer pH 7.5 to rinse the
liver of excess blood whilst cooling the liver sample. The
liver was minced and two volumes of pH 7.5HEPES
lysis buffer (containing 1 mM PMSF, 3 mM DTT and
5.5 mM MgCl2) were added. PMSF was included to
decrease the rate of proteolysis in the samples. Metallo-
proteinase inhibitors, including EDTA, are metal che-
lators. Such compounds could not be used in this
Purification by flash chromatography using EtOAc/
hexane (3:2, v/v) gave the title compound as a white
solid, which was recrystallised from EtOAc (28 mg,
90.8%. Mp 84–86 ꢀC, Rf=0.24, ethyl acetate/hexane,

I. J. S. Fairlamb et al. / Bioorg. Med. Chem. 10 (2002) 2641–2656
2655
analysis, since the SQS requires Mg2+ for its activity.
Metalloproteinases remained active during these analyses.
The minced sample was homogenised for 10 min, then
divided into three aliquots and stored overnight at
À80 ꢀC. The frozen liver samples were slowly thawed,
on ice, to limit any further protease activity. Stock
solutions of KF, NADPH, NB-598 and test compounds
were prepared. All enzyme inhibitors were dissolved in
DMSO and added to enzyme preparations to give final
concentrations of 3.2 nM (NB-598) and 10 mM (SQS test
compounds and S1), respectively. Inhibition assays of
each test compound were run in duplicate. Homogenate
samples were divided into aliquots of 2 mL and placed
into glass vials. The stock solutions of KF (KF is an
inhibitor of phosphatase enzymes and added to limit
farnesyl pyrophosphate degradation) and NADPH were
added to give final concentrations of 11 and 1 mM,
respectively. NB-598 is a potent squalene epoxidase
inhibitor and was included so as to prevent squalene
consumption during the assay. All potential inhibitors
were compared to S1 and a control containing no inhi-
bitor. Assay initiation was performed by addition of
farnesyl pyrophosphate (5 mM). The SQS enzyme solu-
tions were then shaken for 1 h at 37 ꢀC. The enzyme
reaction was stopped by addition of 10% KOH (1 mL)
to each sample vial and thoroughly mixed by vortexing.
Saponification: Failure to saponify liver samples resul-
ted in low recoveries of squalene. The addition of a base
dissolves rather than precipitates membrane proteins,
thereby facilitating squalene release. Methanol (1 mL)
was added to each sample to aid saponification, fol-
lowed by heating to 60 ꢀC for 90 min. Extraction of
squalene and other non-polar metabolites: Heptane
(15 mL) was added to the saponified liver homogenate
sample (4 mL) and vortexed for 1 min, shaken for 5 min
and then vortexed for a further 2 min. This ensured that
the majority of squalene in the sample was transferred
to the organic phase. Heptane removal and sample pre-
treatment for HPLC: Prior to HPLC analysis heptane
was removed from each sample at 50 ꢀC under a steady
stream of nitrogen and then replaced with propan-2-ol
(2 mL); a solvent previously used in our research for the
dissolution of squalene.37 The sample was then filtered
to remove any particulate matter prior to HPLC analy-
sis. A standard squalene sample was run by HPLC.
Squalene (20 mM) on an ODS HPLC column at 30 ꢀC
had a retention time of 32 min. Duplicate extracted
samples were analysed without SQS inhibitor (peak
area; 57,977, Æ2.85%). The S1 control inhibitor caused
a reduction in the squalene concentration (peak area;
41,994, Æ1.92%) equating to a 27.5% decrease in
squalene. The inhibition results were compared directly
to both control experiments.
mass spectrometry facility at the University of Swansea
(UK) for accurate mass determinations.
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Acknowledgements
We would like to thank the Manchester Metropolitan
University (MMU) for funding in the form of a PhD
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for guidance regarding the anti-microbial studies. Pro-
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