Synthesis of (E)- and (Z)-29-methylidyne-2,3-oxidosqualene derivatives as inhibitors of liver and yeast oxidosqualene cyclase

Article abstract of DOI:10.1039/b200888m

The synthesis of (E)- and (Z)-29-methylidyne-2,3-oxidosqualene derivatives is described starting from the C₂₂ and C₁₇ squalene aldehyde monobromohydrins. The conversion was achieved by means of a Wittig reaction, followed by desilylation of the terminal acetylene. For trisubstituted 1,3-enynes, preliminary alkylation with a suitable allyl bromide was performed. A new procedure for the synthesis of squalene aldehyde C₂₇, C₂₂ and C₁₇ monobromohydrins is also described. Some of the new compounds behaved as inhibitors of pig liver and yeast oxidosqualene cyclase and were time-dependent inhibitors of the animal enzyme.

Full text of DOI:10.1039/b200888m

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Synthesis of (E )- and (Z )-29-methylidyne-2,3-oxidosqualene
derivatives as inhibitors of liver and yeast oxidosqualene cyclase
Maurizio Ceruti,*†a Franca Viola,b Gianni Balliano,b Paola Milla,b Giorgio Roma,c
Giancarlo Grossic and Flavio Roccob
1
a Dipartimento Farmacochimico, Tossicologico e Biologico, Università degli Studi di Palermo,
Via Archirafi 32, 90123 Palermo, Italy
b Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino,
Via Pietro Giuria 9, 10125 Torino, Italy
c Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, Viale Benedetto XV,
3, 16132 Genova, Italy
Received (in Cambridge, UK) 23rd January 2002, Accepted 24th April 2002
First published as an Advance Article on the web 13th May 2002
The synthesis of (E)- and (Z)-29-methylidyne-2,3-oxidosqualene derivatives is described starting from the C22 and
17 squalene aldehyde monobromohydrins. The conversion was achieved by means of a Wittig reaction, followed by
C
desilylation of the terminal acetylene. For trisubstituted 1,3-enynes, preliminary alkylation with a suitable allyl
bromide was performed. A new procedure for the synthesis of squalene aldehyde C27, C22 and C17 monobromohydrins
is also described. Some of the new compounds behaved as inhibitors of pig liver and yeast oxidosqualene cyclase and
were time-dependent inhibitors of the animal enzyme.
plants, the rat, the pig and man.1–12 The predicted molecular
masses range from 80 to 90 kDa and the amino acid sequences
determined show significant homology between rat, yeast and
Introduction
2,3-Oxidosqualene cyclases (OSCs) (EC 5.4.99.7) are widely
distributed enzymes that catalyse the cyclization of (3S)-2,3-
oxidosqualene (OS) 1 to lanosterol 3 in mammals and yeasts
and to cycloartenol 4 (Scheme 1) or to a variety of tetracyclic
plant enzymes. Sequence comparison of OSCs with bacterial
squalene hopene cyclase13 show 17–27% homogeneity and
reveals the existence of a highly conserved repetitive motif
and pentacyclic triterpenes in higher plants. OSCs are
(the QW motif ) rich in aromatic amino acids.4
membrane-associated enzymes that have been purified and
cloned from different species: Candida albicans, Saccharomyces
cerevisiae, Schizosaccharomyces pombe among the fungi; higher
The cyclization of OS 1 starts with the protonation of the
epoxide by a suitable electrophilic residue of the enzyme, to give
a C-2 carbonium ion intermediate and proceeds through the
formation of a series of carbonium ion intermediates: the
monocyclic, the bicyclic, the tricyclic with a five-membered
cycle, the all six-membered tricyclic and finally the tetracyclic
intermediate 2 or protosteryl ion. This latter undergoes back-
† Present address: Dipartimento di Scienza e Tecnologia del Farmaco,
Università degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino,
Italy. E-mail: maurizio.ceruti@unito.it; fax: ϩ39 011 6707695; tel: ϩ39
011 6707694.
Scheme 1 Mechanism of cyclization of 2,3-oxidosqualene 1 to lanosterol 3 and cycloartenol 4.
DOI: 10.1039/b200888m
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This journal is © The Royal Society of Chemistry 2002
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bone rearrangement to yield either lanosterol 3 in animals and
yeasts or cycloartenol 4 in higher plants.1,2,14,15
undergo conformational equilibria caused by the mobile tails
moving around the more rigid central portion, which gives the
squalene chains the form of a dynamic precoil. Therefore,
the centre of the chain is protected from electrophilic addition
reactions.
Based on these studies, a general method for the protection
of the internal double bonds of polyenic carbonyl compounds
was developed.28 Following this procedure, C22 squalene alde-
hyde 11 was protected as the 1,3-dioxolane 14, regioselectively
reacted at the terminal double bond with NBS to form the
bromohydrin 17 and finally deprotected with bis(acetonitrile)-
palladium() dichloride to the C22 squalene aldehyde bromo-
hydrin 20. This method may be separately applied to the syn-
thesis of the three bromohydrins 19, 20 and 21 (yield of 13–15
from squalene 7: 30%). However, it requires a long separation
procedure of the three aldehydes and each protection step is
performed separately for each aldehyde.
A new procedure was therefore developed, starting from a
1 : 1 : 1 mixture of the three aldehydes 10, 11 and 12, easily
obtainable from squalene 7 (Schemes 2 and 3). The mixture was
directly protected by treatment with ethane-1,2-diol in benzene,
with toluene-p-sulfonic acid as catalyst, forming a mixture of
the dioxolanes 13, 14 and 15. The dioxolanes were separated by
reverse phase flash chromatography more easily and rapidly
than the aldehydes and in higher total yield (yield of 13–15
from squalene 7: 33%). Each dioxolane was then separately
treated with NBS in aqueous THF, affording the corresponding
dioxolane external bromohydrin 16, 17 or 18, which was depro-
tected to the C27, C22 or C17 squalene aldehyde bromohydrin 19,
20 or 21 with bis(acetonitrile)palladium() dichloride.
For many years we and others have been studying OSC
inhibitors. Initially, effective inhibitors were obtained by
mimicking the carbocationic intermediates formed during
cyclization of OS, designing squalene-derived structures in
which the positively charged carbocation was replaced by a
nitrogen.1,16–19 Other research groups have developed different
series of cyclised aza derivatives,20 and sulfur-containing OS
derivatives have been developed by the Oehlschlager group and
by ourselves.21–23 Another strategy to achieve new OSC inactiv-
ators is the introduction of a second epoxidic ring, replacing
a carbon–carbon double bond in the natural substrate.24
Finally, a strategy that has been successfully adopted is to
intercept the enzymatic active-site nucleophiles with a stable
allylic cation, resulting in an irreversible covalent modification
of OSC. Following this strategy, Prestwich,25,26 Corey27 and
our28,29 groups synthesized various series of 2,3-oxido-
squalenoid dienes and vinyl epoxides, some of which were
found to be selective and time-dependent inhibitors of yeast or
animal OSCs, the most potent being dienes 5 and 6.
Synthesis of 1,3-enynes
Xiao and Prestwich succeeded in preparing an irreversible
site-directed inhibitor of vertebrate OSC, (18Z)-29-methyl-
idene-2,3-oxidosqualene (29-MOS) 5. The proposed inhibition
mechanism involves the initial cyclization to the 21-methyl-
ideneprotosterol cation, which instead of undergoing backbone
rearrangement reacts with a nucleophilic site of the enzyme
resulting in irreversible inactivation of the enzyme.25 Tritiated
29-MOS was used in affinity labelling experiments to identify
the 29-MOS binding site region of rat liver OSC, which is the
DCTAEA motif, a well conserved region in all OSCs.30
Some years ago, in an attempt to achieve mechanism-based
inhibitors able to trap the C-20 carbocation, the 22,23-dihydro-
2,3-oxido-20-oxasqualene was synthesized.31 Subsequently,
using the same procedure, Corey14,15,32 synthesized the 2,3-
oxido-20-oxasqualene as a tool to provide information on the
17β-configuration of the protosteryl ion side chain.
The synthesis was developed of truncated OS derivatives con-
taining, at the terminal positions of the squalenoid skeleton,
an unsubstituted 1,3-enyne unit or an alk-2-enyl substituted
1,3-enyne, starting from the C22 or C27 squalene aldehyde
monobromohydrin 20 or 19. Various preparative methods for
derivatizing an aldehyde as a 1,3-enyne have been reported,34
but few procedures are suitable for the present purposes.
The transformation of the C22 and C27 squalene aldehyde
monobromohydrins 20 and 19 into the corresponding E and
Z methylidyne derivatives was achieved in two steps according
to the Corey35 and the Masamune34 procedures, which produce
(E)-1,3-enynes with high E stereoselectivity. Other workers36,37
have subsequently converted an aldehyde into an enyne follow-
ing this method, in E stereoselectivity or E stereospecificity. In
contrast, Z stereoselectivity was achieved when the enyne unit
was linked to a dioxolane system.38
[3-(Trimethylsilyl)prop-2-ynyl]triphenylphosphonium brom-
ide in anhydrous THF was treated with an excess of butyl-
lithium and reacted at Ϫ80 ЊC with the C22 or C27 squalene
aldehyde monobromohydrins 20 or 19 (Schemes 4 and 5) afford-
ing the Z and E trimethylsilyl derivatives 22a, 22b and 24a, 24b
in 30% and 34% yield, respectively (E : Z = 2 : 1). During the
Wittig reaction, the desired conversion of the bromohydrin to
the epoxide was also achieved. The two isomers were separated
by flash chromatography, eluting first the Z isomer and then the
E isomer. The final conversion into the E or Z methylidyne
derivatives of OS 23a, 23b and 25a, 25b was accomplished with
tetrabutylammonium fluoride in anhydrous THF, in 77% and
78% yield respectively.35–37
It was considered that the syntheses of a new class of
truncated and non-truncated methylidyne oxidosqualene
derivatives might afford better insight into the function and
reactivity of the nucleophiles of the active site of the enzyme
that stabilize the C-2 and C-20 cationic intermediates.
Results and discussion
Synthesis of squalene aldehyde C27, C22 and C17 monobromo-
hydrins
The synthesis of many OS derivatives starts from the squalene
aldehyde C27, C22 and C17 monobromohydrins 19, 20 and 21
(Schemes 2 and 3). Various methods of preparation have been
developed, but they often suffer from low yield and tedious
separation.16,17,28,31
The OS analogues having the methylidyne function at the
C-29 position of the squalene skeleton (29a and 29b) and the
nor analogues (27a and 27b) (Scheme 6) were synthesized,
although a survey of the literature showed no previously
reported method. [3-(Trimethylsilyl)prop-2-ynyl]triphenylphos-
phonium bromide in anhydrous THF was reacted with an
excess of butyllithium (see the Experimental section) and
subsequently with 1-bromo-3-methylbut-2-ene, followed by the
1H and 13C NMR and conformational and dynamical
theoretical studies have been carried out on squalene, 2,3-
oxidosqualene and derivatives33 in various solvents, in order to
explain the selective reactivity of the terminal double bond of
squalene towards reagents such as N-bromosuccinimide (NBS).
Squalene and derivatives in solution have been shown to
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Scheme 2 Synthesis of the C27, C22 and C17 squalene aldehydes 10, 11 and 12.
Table 1 Inhibition values (IC50/µM) of pig liver and Saccharomyces
C22 squalene aldehyde monobromohydrin 20 (Scheme 6). By
changing the reaction conditions during the one-pot reaction,
the trimethylsilyl derivatives of (18Z)-26a and (18E)-29-
methylidyne-20-nor-2,3-oxidosqualene (26b) (E : Z = 1 : 1) were
obtained in 26% yield. During the Wittig reaction, conversion
of the bromohydrin into the epoxide was also achieved. Alkyl-
ation with 5-bromo-2-methylpent-2-ene afforded the trimethyl-
silyl derivatives of (18Z)-28a and (18E)-29-methylidyne-2,3-
oxidosqualene (28b) in very low yields (5%). The final con-
version into the methylidyne derivatives of OS 27a, 27b and
29a, 29b was accomplished with tetrabutylammonium fluoride
in anhydrous THF, in 81% and 72% yield respectively.35–37 The
E : Z ratio was determined to be 1 : 1 by 1H NMR analysis.39
By comparing the synthesis of the two series of compounds,
it can be observed that when the 18,19-double bond is trisub-
stituted, the E and Z isomers are obtained in similar amounts,
while enynes are obtained with E stereoselectivity when the
terminal isoprene chain is absent. The synthetic procedure
developed for the synthesis of the C17, C22 and C27 squalene
aldehyde monobromohydrins may be useful for the develop-
ment of new OS derivatives functionalised at the C-15, C-19
and C-23 positions.
cerevisiae oxidosqualene cyclase (OSC) by methylidyne derivatives of
oxidosqualene
IC50/µMa
OSC
Pig liver
OSC
S. cerevisiae
Compound
6 (control)
23a
3.5
60
50
>100
>100
60
1.5
50
30
>100
>100
NDb
50
23b
25a
25b
26a ϩ 26b
27a ϩ 27b
29a ϩ 29b
20
30
30
a IC50, inhibitor concentration reducing enzymatic conversion by 50%.
b ND = not determined.
compounds are the non-truncated acetylenes 27a ϩ 27b and
29a ϩ 29b, which showed an IC50 of 20–30 µM on pig liver OSC
and of 30–50 µM on the yeast enzyme. Compounds 27a and
29a have the same isomeric structure as OS and they should be
cyclized by the enzyme to analogues of the 21-methyl-
idyneprotosterol cation, 30 and 31, which should react with
a nucleophilic site of the enzyme, resulting in irreversible
inactivation (Scheme 7). The resulting inhibitor is probably
converted into unstable allenic derivatives 32 or 33, which then
undergo further degradation. Possibly, the lower biological
activity of the 29-methylidyne derivatives compared to 29-
methylidene derivatives depends on preferential cyclization to
the 29-methylidyne lanosterol derivatives 34 and 35, instead of
covalent linkage to the enzyme.
Biological activity
Our and the previous findings of others showed that com-
pounds such as the methylidene derivatives 5 and 6, having
a non-truncated or a truncated squalenoid structure and a
correctly located reactive group adjacent to the C18–C19
double bond involved in the cyclization, are potent and selective
inhibitors of OSCs.25–30 The biological activity of the syn-
thesized methylidyne derivatives of OS was studied on animal
and yeast enzymes.
Concerning (18Z)-23a and (18E)-29-methylidynehexanor-
2,3-oxidosqualene (23b), the two isomers showed modest and
similar activity on the two cyclases. On the other hand, the
corresponding (18E)-29-methylidenehexanor-2,3-oxidosqua-
lene (6), previously synthesized by us,28 was a potent and time-
Table 1 reports the IC50 inhibition values obtained during
testing for OSC activity with solubilized and partially purified
pig liver OSC and a microsomal suspension of S. cerevisiae,
using compound 6 as a control compound.28 Most of the com-
pounds, as expected, are inhibitors of OSCs. The more active
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Scheme 3 Synthesis of the C27, C22 and C17 squalene aldehyde monobromohydrins 19, 20 and 21.
dependent inhibitor of yeast OSC, while the 18Z-isomer was
much less active. Compounds 25a and 25b did not inhibit the
animal and yeast cyclases even at 100 µM concentration, thus
confirming that the inhibitors require an epoxide and a reactive
function near the crucial positions involved in the cyclization.
Time-dependent inhibition experiments were performed by
pre-incubating the pig liver enzyme in the presence of the
inhibitors at 200 µM concentration (see the Experimental
section). Aliquots were withdrawn at time intervals, diluted
at least 40 times and added to substrate, to check the residual
enzymatic activity with respect to controls that were pre-
incubated in the absence of inhibitors for the same length of
time. The time-dependent decrease of activity did not depend
on insufficient dilution, as non-pre-incubated controls never
showed more than a 10% decrease of activity when tested in the
presence of inhibitor at the same final dilution used in the tests.
Pig liver OSC was inhibited in a time-dependent manner by
the methylidyne derivatives tested (23b and 27a ϩ 27b), the best
compounds being again the nor-methylidyne derivative 27a ϩ
27b, while the (18E)-hexanormethylidyne derivative 23b was
less active. The t/2 values obtained at 200 µM concentration
were 20 min for 27a ϩ 27b and 55 min for 23b.
The time-dependence of inhibition of S. cerevisiae OSC
was evaluated for compounds 23b and 27a ϩ 27b at con-
centrations similar to their IC50 values, but the low specific
activity found in yeast microsomes did not allow the dilution
necessary to test the residual activity after pre-incubation.
A search for a more active and suitable source of yeast
enzyme is underway. Recently it was shown that OSC activity
is almost exclusively associated with yeast lipid particles,
while the occurrence of OSC in other organelles, including
the endoplasmic reticulum, is negligible.6 Work is in progress
to prepare lipid particle fractions and to the test time-
dependence of the more interesting inhibitors on yeast enzyme
systems.
Experimental
1H NMR spectra were recorded on a Jeol EX 400 instrument
for samples in CDCl3 solution at room temperature, with
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Scheme 5 Synthesis of the 22Z and 22E isomers of 24-methylidyne-
30-nor-2,3-oxidosqualene 25a and 25b.
Scheme 4 Synthesis of the 18Z and 18E isomers of 29-methylidyne-
20,21,22,23,24,30-hexanor-2,3-oxidosqualene 23a and 23b.
separated and characterised as previously reported,31 to give a
1 : 1 mixture of the two internal monoepoxides 8 and 9 and the
external monoepoxide 1.
Me4Si (TMS) as internal standard. Coupling constants (J) are
given in Hz. IR spectra were recorded on a PE 781 spectro-
photometer. Mass spectra were obtained on a Finnigan MAT
TSQ 700 spectrometer. Microanalyses were determined on
an elemental analyser 1106 and were within 0.3% of the
theoretical values. The reactions were monitored by TLC on
F254 silica gel precoated sheets; after development, the sheets
were exposed to iodine vapour. Flash-column chromatography
was performed on 230–400 mesh silica gel. THF and diethyl
ether were dried over sodium benzophenone ketyl. All solvents
were distilled prior to flash chromatography.
C27, C22 and C17 Squalene aldehydes: (4E,8E,12E,16E)-
4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenal 10,
(4E,8E,12E )-4,9,13,17-tetramethyloctadeca-4,8,12,16-tetraenal
11 and (4E,8E )-5,9,13-trimethyltetradeca-4,8,12-trienal 12
(Scheme 2)
HIO4ؒ2H2O (1.5 equiv., 1.60 g, 7.04 mmol) was added to diethyl
ether (250 cm3) with vigorous stirring and, when dissolution
was almost complete, the 1 : 1 : 1 mixture of squalene epoxides
1, 8 and 9 (2.0 g, 4.69 mmol) in diethyl ether (5 cm3) was added.
Stirring was continued for 15 min after which the reaction
mixture was washed with saturated brine (3 × 100 cm3), dried
over anhydrous sodium sulfate, filtered and evaporated to
dryness. The resulting oil was purified by flash chromatography
(petroleum ether–diethyl ether 80 : 20) to give a 1 : 1 : 1 mixture
of the C27, C22 and C17 aldehydes 10, 11 and 12, (1.27 g, 86%) as
a colourless oil. A sample of the mixture was separated and
characterised as previously reported,16,31 to give sequentially the
C17 aldehyde 12, the C22 aldehyde 11 and finally the C27 alde-
hyde 10.
Squalene monoepoxides: (6E,10E,14E,18E )-22,23-epoxy-
2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18-pentaene 1,
(6E,10E,14E )-trans-18,19-epoxy-2,6,10,15,19,23-hexamethyl-
tetracosa-2,6,10,14,22-pentaene 8 and (6E,10E,18E )-trans-
14,15-epoxy-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,18,22-
pentaene 9 (Scheme 2)
A solution of squalene 7 (10 g, 24.3 mmol) dissolved in CH2Cl2
(250 cm3) at 0 ЊC was stirred, while m-chloroperbenzoic acid
(MCPBA) (85% purity, 1.5 equiv., 6.30 g, 36.5 mmol) was
added over a period of 30 min; it was then allowed to react for
further 30 min with continued stirring. The reaction mixture
was washed with 20% aqueous NaHCO3 (3 × 100 cm3) and
brine (2 × 100 cm3), dried over anhydrous sodium sulfate,
filtered and evaporated to dryness. The resulting oil was puri-
fied by flash chromatography (petroleum ether–diethyl ether
90 : 10) to give a 1 : 1 : 1 mixture of the three monoepoxides
(4.65 g, 45%) as a colourless oil. A sample of the mixture was
Squalene aldehyde dioxolanes: 2-[(3E,7E,11E,15E )-3,7,12,16,
20-pentamethylhenicosa-3,7,11,15,19-pentaenyl]-1,3-dioxolane
13, 2-[(3E,7E,11E )-3,8,12,16-tetramethylheptadeca-3,7,11,15-
tetraenyl]-1,3-dioxolane 14 and 2-[(3E,7E )-4,8,12-trimethyl-
trideca-3,7,11-trienyl]-1,3-dioxolane 15 (Scheme 3)
A solution of the 1 : 1 : 1 mixture of the C27, C22 and C17
squalene aldehydes 10, 11 and 12 (5 g, 15.8 mmol), ethylene
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Scheme 6 Synthesis of the 18Z and 18E isomers of 29-methylidyne-20-nor-2,3-oxidosqualene 27a and 27b and 29-methylidyne-2,3-oxidosqualene
29a and 29b.
glycol (3 equiv., 2.94 g, 47.4 mmol) and toluene-p-sulfonic acid
monohydrate as catalyst (0.1 equiv., 300 mg, 1.58 mmol) in
benzene (300 cm3) was refluxed for 4 h in a refrigerator
equipped with a Markusson connector. It was then cooled and
a small amount of solid NaHCO3 was added. The reaction
mixture was diluted with benzene (200 cm3), washed with
saturated NaHCO3 (2 × 100 cm3) and brine (1 × 100 cm3), dried
over anhydrous sodium sulfate, filtered and evaporated to
dryness. The resulting oil was purified by flash chromatography
(petroleum ether–diethyl ether 97 : 3), to give a 1 : 1 : 1 mixture
of dioxolanes 13, 14 and 15 (4.90 g, 86%) as a colourless oil. A
sample of the mixture was separated by flash chromatography
(petroleum ether–diethyl ether 99 : 1, then 98 : 2 and finally 97 :
3) to give sequentially the C27 squalene aldehyde dioxolane 13,
the C22 squalene aldehyde dioxolane 14 and finally the C17
squalene aldehyde dioxolane 15. Analytical data for C22
squalene aldehyde dioxolane 14 were identical to those
previously reported.28
161 (18), 135 (22), 93 (98) and 69 (100); (Found: C, 81.28;
H, 11.30; O, 7.44. C29H48O2 requires: C, 81.25; H, 11.29; O,
7.46%).
C17 squalene aldehyde dioxolane 15: νmax(film)/cmϪ1 2940,
2870, 1450, 1385 and 1140; δH(200 MHz, CDCl3) 1.57–1.76 (14
H, m, allylic CH3 and CH2-dioxolane), 1.94–2.14 (10 H, m,
allylic CH2), 3.80–4.00 (4 H, m, OCH2CH2O), 4.85 (1 H, t,
J 4.8, dioxolane CH) and 5.05–5.18 (3 H, m, vinylic CH);
m/z(EI) 292 (8%), 249 (5), 223 (7), 204 (10), 161 (12), 142 (40),
121 (18) and 99 (100); m/z(CI, isobutane) 293 (100%); (Found:
C, 78.01; H, 11.05; O, 10.93. C19H32O2 requires: C, 78.03;
H, 11.03; O, 10.94%).
Squalene aldehyde dioxolane bromohydrins: (6E,10E,14E,18E )-
3-bromo-21-(1,3-dioxolan-2-yl)-2,6,10,15,19-pentamethyl-
henicosa-6,10,14,18-tetraen-2-ol 16, (6E,10E,14E )-3-bromo-17-
(1,3-dioxolan-2-yl)-2,6,10,15-tetramethylheptadeca-6,10,14-
trien-2-ol 17 and (6E,10E )-3-bromo-13-(1,3-dioxolan-2-yl)-
2,6,10-trimethyltrideca-6,10-dien-2-ol 18 (Scheme 3)
C27 squalene aldehyde dioxolane 13: νmax(film)/cmϪ1 2940,
2865, 1450, 1380 and 1140; δH(200 MHz, CDCl3) 1.60–1.75
(20 H, m, allylic CH3 and CH2-dioxolane), 1.97–2.15 (18 H,
m, allylic CH2), 3.82–3.98 (4 H, m, OCH2CH2O), 4.86 (1 H,
t, J 4.8, dioxolane CH) and 5.00–5.18 (5 H, m, vinylic CH);
m/z(EI) 428 (2%), 371 (0.7), 359 (1.6), 291 (2), 229 (4.2), 203 (6),
The 1 : 1 : 1 mixture of dioxolanes 13, 14 and 15 (1.0 g,
2.77 mmol) was dissolved in THF (80 cm3) in a two-necked
flask and stirred under nitrogen at 0 ЊC. Water was added
until the solution became lightly opalescent. NBS (1.1 equiv.,
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Scheme 7 Hypothetical inhibition mechanism of oxidosqualene cyclase by (18Z)-29-methylidyne-2,3-oxidosqualene 29a and its 20-nor derivative
27a.
542 mg, 3.05 mmol) was added in small portions with vigorous
stirring over 15 min, with, at intervals, addition of a few
drops of water to keep the reaction mixture lightly opalescent.
The mixture was allowed to stand for 15 min at 0 ЊC, again
with addition of a few drops of water at intervals when it
began to clear. The reaction mixture was quenched with cold
10% NaHCO3 (80 cm3), extracted with diethyl ether (3 ×
80 cm3), washed with 10% NaHCO3 (1 × 80 cm3) and brine
(1 × 80 cm3), dried with anhydrous sodium sulfate, filtered
and evaporated in vacuo. The resulting oil was purified by
RP18 reverse phase flash chromatography (acetonitrile–water
80 : 20, then 90 : 10, finally pure acetonitrile) to give sequen-
tially the C17 squalene aldehyde dioxolane bromohydrin 18
(185 mg), the C22 squalene aldehyde dioxolane bromohydrin 17
(215 mg) and finally the C27 squalene aldehyde dioxolane
bromohydrin 16 (242 mg), in 51% total yield, as light yellow
oils.
C22 squalene aldehyde bromohydrin: (4E,8E,12E )-16-bromo-17-
hydroxy-4,9,13,17-tetramethyloctadeca-4,8,12-trienal 20
(Scheme 3)
Dioxolane bromohydrin 17 (400 mg, 0.87 mmol) was dissolved
in acetone (200 cm3) under dry nitrogen, with stirring. Bis-
(acetonitrile)palladium() dichloride [PdCl2(CH3CN)2] (0.2
equiv., 44 mg, 0.17 mmol) was added and the mixture allowed
to stand for 8 h under nitrogen, with stirring. Controls on silica
gel TLC revealed that in most eluants, the dioxolane 17 and the
aldehyde 20 had about the same Rf, while dichloromethane–
ethyl acetate 95 : 5 differentiated the two compounds. The reac-
tion mixture was quenched with cold 10% NaHCO3 (100 cm3)
and extracted with diethyl ether (2 × 100 cm3). The combined
extracts were washed with 10% NaHCO3 (1 × 50 cm3) and brine
(1 × 50 cm3), dried with anhydrous sodium sulfate, filtered
and evaporated in vacuo. The resulting oil was purified by flash
chromatography (petroleum ether–diethyl ether, 95 : 5 to
remove impurities, then 92 : 8, finally 90 : 10) to give 259 mg
(72%) of C22 squalene aldehyde bromohydrin 20 as a colourless
oil.28 νmax(film)/cmϪ1 3500–3400, 2965, 2920, 2860, 1725, 1450,
1385 and 1110; δH(200 MHz, CDCl3) 1.28 and 1.32 [6 H, 2 s,
(CH3)2COH], 1.50–1.70 (11 H, m, allylic CH3 and CH2CHBr),
1.88–2.16 (12 H, m, allylic CH2), 2.35–2.40 (2 H, m, CH2CHO),
3.84 (1 H, m, CHBr), 5.00–5.23 (3 H, m, vinylic CH) and 9.78
(1 H, m, CHO); m/z(EI) 414 (0.5%), 412 (0.5), 332 (3), 316 (1),
247 (1), 153 (6), 135 (15), 111 (16), 93 (38), 81 (90) and 43 (100);
(Found: C, 63.88; H, 9.02; Br, 19.31; O, 7.75. Calc. for
C22H37BrO2: C, 63.91; H, 9.02; Br, 19.33; O, 7.74%).
Analytical data for C22 aldehyde dioxolane bromohydrin 17
were identical to those previously reported.28
C27 squalene aldehyde dioxolane bromohydrin 16: νmax(CCl4)/
cmϪ1 3570, 2980, 2930, 2855, 1550, 1450, 1385 and 1140; δH(200
MHz, CDCl3) 1.28 and 1.32 [6 H, 2 s, (CH3)2COH], 1.50–1.72
(16 H, m, allylic CH3, CH2CHBr and CH2-dioxolane), 1.96–
2.13 (16 H, m, allylic CH2), 3.78–3.95 (5 H, m, OCH2CH2O and
CHBr), 4.85 (1 H, t, J 4.8, dioxolane CH) and 5.02–5.20 (4 H,
m, vinylic CH); m/z(EI) 526 (0.7%), 524 (0.7), 462 (0.6), 444 (1),
427 (1.2), 365 (1.1), 291 (3.5), 255 (1.5), 161 (22), 135 (35) and
93 (100); m/z(CI, isobutane) 527 (72%) and 525 (100); (Found:
C, 66.30; H, 9.38; Br, 15.20; O, 9.11. C29H49BrO3 requires: C,
66.27; H, 9.40; Br, 15.20; O, 9.13%).
C27 squalene aldehyde bromohydrin: (4E,8E,12E,16E )-20-
C17 squalene aldehyde dioxolane bromohydrin 18: νmax(CCl4)/
cmϪ1 3570, 2970, 2930, 2855, 1550, 1450, 1390 and 1140; δH(200
MHz, CDCl3) 1.28 and 1.32 [6 H, 2 s, (CH3)2COH], 1.52–1.75
(10 H, m, allylic CH3, CH2CHBr and CH2-dioxolane), 1.97–
2.15 (8 H, m, allylic CH2), 3.84–3.96 (5 H, m, OCH2CH2O and
CHBr), 4.85 (1 H, t, J 4.8, dioxolane CH) and 5.02–5.18 (2 H,
m, vinylic CH); m/z(EI) 390 (2%), 388 (2), 375 (1), 373 (1), 309
(6), 291 (8), 229 (4), 223 (6), 181 (6), 142 (42) and 99 (100);
m/z(CI, isobutane) 391 (95%) and 389 (100); (Found: C, 58.64;
H, 8.56; Br, 20.50; O, 12.32. C19H33BrO3 requires: C, 58.61; H,
8.54; Br, 20.52; O, 12.33%).
bromo-21-hydroxy-4,8,13,17,21-pentamethyldocosa-4,8,12,16-
tetraenal 19 (Scheme 3)
Compound 19 was obtained and purified using the method
described for 20, starting from the dioxolane 16, in 70% yield.
νmax(film)/cmϪ1 3500–3400, 2960, 2920, 2860, 1725, 1450,
1390 and 1110; δH(200 MHz, CDCl3) 1.28 and 1.32 [6 H, 2 s,
(CH3)2COH], 1.48–1.70 (14 H, m, allylic CH3 and CH2CHBr),
1.87–2.16 (16 H, m, allylic CH2), 2.34–2.40 (2 H, m, CH2CHO),
3.84 (1 H, m, CHBr), 5.00–5.24 (4 H, m, vinylic CH) and 9.78
(1 H, m, CHO); m/z(EI) 482 (0.3%), 480 (0.3), 383 (2.5), 301
J. Chem. Soc., Perkin Trans. 1, 2002, 1477–1486
1483

View Article Online
(1.8), 273 (2), 229 (2.2), 217 (8), 203 (6), 135 (36), 93 (50) and 81
(100); m/z(CI, isobutane) 483 (36%), 481 (38), 465 (95) and 463
(100); (Found: C, 67.31; H, 9.45; Br, 16.61; O, 6.66. C27H45BrO2
requires: C, 67.34; H, 9.42; Br, 16.59; O, 6.65%).
argon. Tetrabutylammonium fluoride (1.0 M solution in THF,
1.15 equiv., 135 µl, 0.135 mmol) was added and the mixture was
stirred for 15 min at 0 ЊC and concentrated in vacuo. It was then
poured into cold 10% NH4Cl-diethyl ether (1 : 1, 50 cm3)
and extracted with diethyl ether (3 × 30 cm3). The combined
extracts were washed with saturated brine (2 × 30 cm3), dried
with anhydrous sodium sulfate, filtered and evaporated in
vacuo. The resulting oil was purified by flash chromatography
(petroleum ether–diethyl ether, 99.8 : 0.2 to remove impurities,
then 95 : 5) to give 32 mg (77%) of the (18Z)-methylidyne
derivative 23a, as a colourless oil. νmax(CCl4)/cmϪ1 3310, 2965,
2930, 2860, 1450, 1380 and 1250; δH(200 MHz, CDCl3) 1.26 and
1.31 (6 H, 2 s, epoxidic CH3), 1.58–1.72 (11 H, m, allylic CH3
C17 squalene aldehyde bromohydrin: (4E,8E )-12-bromo-13-
hydroxy-5,9,13-trimethyltetradeca-4,8-dienal 21 (Scheme 3)
Compound 21 was obtained and purified using the method
described for 20, starting from the dioxolane 18, in 73% yield.
νmax(film)/cmϪ1 3500–3400, 2970, 2920, 2850, 1725, 1445, 1385
and 1115; δH(200 MHz, CDCl3) 1.28 and 1.32 [6 H, 2 s,
(CH3)2COH], 1.50–1.72 (8 H, m, allylic CH3 and CH2CHBr),
1.88–2.20 (8 H, m, allylic CH2), 2.35–2.41 (2 H, m, CH2CHO),
3.85 (1 H, m, CHBr), 5.02–5.20 (2 H, m, vinylic CH) and 9.77
(1 H, m, CHO); m/z(EI) 346 (3%), 344 (3), 328 (3), 326 (3), 302
(1), 300 (1), 264 (5), 243 (4), 229 (2), 203 (1), 135 (32), 107 (15),
93 (35) and 81 (100); (Found: C, 59.10; H, 8.47; Br, 23.09;
O, 9.25. C17H29BrO2 requires: C, 59.13; H, 8.47; Br, 23.14; O,
9.27%).
and epoxidic CH2), 1.92–2.22 (14 H, m, allylic CH2), 2.71 (1 H,
᎐
t, J 6.3, epoxidic CH), 3.08 (1 H, d, J 2.3, C᎐CH, Z isomer),
᎐
5.06–5.21 (3 H, m, vinylic CH), 5.44 (1 H, br d, Z CH᎐CH–
᎐
᎐
᎐
C᎐CH) and 5.97 (1 H, dt, J 11.2 and J 6.9, Z CH᎐CH–C᎐CH);
᎐
᎐
᎐
m/z(EI) 354 (0.2%), 325 (1.2), 297 (2), 279 (3), 203 (6), 149 (44),
135 (50), 81 (100); m/z(CI, isobutane) 355 (100%); (Found: C,
84.72; H, 10.80; O, 4.48. C25H38O requires: C, 84.69; H, 10.80;
O, 4.51%).
(3Z,7E,11E,15E )-19,20-Epoxy-7,12,16,20-tetramethyl-1-
(trimethylsilyl)henicosa-3,7,11,15-tetraen-1-yne 22a and
(3E,7E,11E,15E )-19,20-epoxy-7,12,16,20-tetramethyl-1-
(trimethylsilyl)henicosa-3,7,11,15-tetraen-1-yne 22b (Scheme 4)
(18E )-29-Methylidyne-20,21,22,23,24,30-hexanor-2,3-oxido-
squalene: (3E,7E,11E,15E )-19,20-epoxy-7,12,16,20-tetra-
methylhenicosa-3,7,11,15-tetraen-1-yne 23b (Scheme 4)
In a three-necked flask containing anhydrous THF (25 cm3),
[3-(trimethylsilyl)prop-2-ynyl]triphenylphosphonium bromide
(1.2 equiv., 465 mg, 1.02 mmol) was suspended at Ϫ80 ЊC,
under a flux of dry nitrogen, with stirring. Butyllithium (1.6 M
solution in hexane, 3 equiv., 1.6 cm3, 2.55 mmol) was added,
during which the reaction mixture turned red. It was left for
15 min at Ϫ40 ЊC and then cooled to Ϫ80 ЊC. C22 squalene
aldehyde monobromohydrin 20 (1 equiv., 351 mg, 0.85 mmol)
in anhydrous THF (2 cm3) was added after 5 min and allowed
to react for 1 h at Ϫ80 ЊC, during which time the colour turned
orange. It was then gradually allowed to reach room tempera-
ture, poured into cold 10% NH4Cl–diethyl ether (1 : 1, 50 cm3)
and extracted with diethyl ether (3 × 30 cm3). The combined
extracts were washed with saturated brine (2 × 30 cm3), dried
with anhydrous sodium sulfate, filtered and evaporated in vacuo
at ϩ35 ЊC. The resulting oil was purified by flash chroma-
tography (petroleum ether to remove impurities, then petroleum
ether–diethyl ether, 99 : 1, 98 : 2 and finally 95 : 5) to give 36 mg
of the Z isomer of silyl acetylene 22a and 73 mg of the E isomer
22b, as colourless oils, in 30% total yield (E : Z = 2 : 1).
E-isomer 23b was obtained starting from the silyl derivative
22b, using the method described for compound 23a, in 75%
yield. νmax(CCl4)/cmϪ1 3310, 2970, 2930, 2860, 1450, 1380 and
1250; δH(200 MHz, CDCl3) 1.26 and 1.31 (6 H, 2 s, epoxidic
CH3), 1.58–1.72 (11 H, m, allylic CH3 and epoxidic CH2),
1.92–2.22 (14 H, m, allylic CH2), 2.71 (1 H, t, J 6.3, epoxidic
᎐
CH), 2.78 (1 H, d, J 2.2, C᎐CH, E isomer), 5.06–5.21 (3 H,
᎐
᎐
m, vinylic CH), 5.48 (1 H, br d, E CH᎐CH–C᎐CH) and 6.23
(1 H, dt, J 16.1 and J 6.9, E CH᎐CH–C᎐CH); m/z(EI) 354
᎐
᎐
᎐
᎐
᎐
(0.2%), 325 (1.2), 297 (2), 279 (3.2), 203 (6), 149 (40), 135 (55),
81 (100); m/z(CI, isobutane) 355 (100%); (Found: C, 84.66;
H, 10.82; O, 4.49. C25H38O requires: C, 84.69; H, 10.80; O,
4.51%).
(3Z,7E,11E,15E,19E )-23,24-Epoxy-7,11,16,20-tetramethyl-1-
(trimethylsilyl)pentacosa-3,7,11,15,19-pentaen-1-yne 24a and
(3E,7E,11E,15E,19E )-23,24-epoxy-7,11,16,20-tetramethyl-1-
(trimethylsilyl)pentacosa-3,7,11,15,19-pentaen-1-yne 24b
(Scheme 5)
22a (Z): νmax(KBr pellet)/cmϪ1 2970, 2920, 2850, 1450, 1380
and 1245; δH(200 MHz, CDCl3) 0.19 [9 H, s, (CH3)3Si], 1.26 and
1.31 (6 H, 2 s, epoxidic CH3), 1.56–1.70 (11 H, m, allylic CH3
and epoxidic CH2), 1.90–2.20 (14 H, m, allylic CH2), 2.71 (1 H,
Compounds 24a and 24b were obtained in 34% total yield
(E : Z = 2 : 1) and separated using the method described for the
silyl derivatives 22a and 22b, using the C27 squalene aldehyde
bromohydrin 19 instead of the C22 squalene aldehyde bromo-
hydrin 20.
t, J 6.3, epoxidic CH), 5.05–5.20 (3 H, m, vinylic CH), 5.47
24a (Z): νmax(KBr pellet)/cmϪ1 2970, 2920, 2850, 1450, 1380
and 1245; δH(200 MHz, CDCl3) 0.19 [9 H, s, (CH3)3Si], 1.26 and
1.31 (6 H, 2 s, epoxidic CH3), 1.56–1.70 (14 H, m, allylic CH3
and epoxidic CH2), 1.94–2.22 (18 H, m, allylic CH2), 2.71 (1 H,
᎐
(1 H, br d, Z CH᎐CH–C᎐C) and 5.92 (1 H, dt, J 10.7 and J 6.8,
᎐
᎐
᎐
Z CH᎐CH–C᎐C); m/z(CI, isobutane) 427 (100%); (Found: C,
᎐
᎐
78.82; H, 10.85; O, 3.76; Si, 6.55. C28H46OSi requires: C, 78.80;
H, 10.86; O, 3.75; Si, 6.58%).
22b (E): νmax(KBr pellet)/cmϪ1 2970, 2920, 2850, 1450, 1380
and 1245; δH(200 MHz, CDCl3) 0.19 [9 H, s, (CH3)3Si], 1.26 and
1.31 (6 H, 2 s, epoxidic CH3), 1.56–1.70 (11 H, m, allylic CH3
and epoxidic CH2), 1.90–2.20 (14 H, m, allylic CH2), 2.71 (1 H,
t, J 6.3, epoxidic CH), 5.00–5.16 (4 H, m, vinylic CH), 5.47
᎐
(1 H, br d, Z CH᎐CH–C᎐C) and 5.92 (1 H, dt, J 10.7 and J 6.8,
᎐
᎐
᎐
Z CH–CH–C᎐C); m/z(EI) 494 (1%), 479 (0.5), 451 (0.7), 421
᎐
(0.8), 341 (3), 273 (6), 267 (2), 245 (2), 225 (3), 204 (12), 199
(18), 189 (33), 135 (35), 93 (47), 81 (80) and 73 (100); (Found:
C, 80.07; H, 11.02; O, 3.25; Si, 5.68. C33H54OSi requires: C,
80.09; H, 11.00; O, 3.23; Si, 5.68%).
t, J 6.3, epoxidic CH), 5.05–5.20 (3 H, m, vinylic CH), 5.51
᎐
(1 H, br d, E CH᎐CH–C᎐C) and 6.20 (1 H, dt, J 15.9 and J 6.8,
᎐
᎐
᎐
E CH᎐CH–C᎐C); m/z(CI, isobutane) 427 (100%); (Found: C,
᎐
᎐
24b (E): νmax(KBr pellet)/cmϪ1 2965, 2920, 2850, 1450, 1385
and 1245; δH(200 MHz, CDCl3) 0.19 [9 H, s, (CH3)3Si], 1.26 and
1.31 (6 H, 2 s, epoxidic CH3), 1.56–1.70 (14 H, m, allylic CH3
and epoxidic CH2), 1.94–2.22 (18 H, m, allylic CH2), 2.71 (1 H,
78.81; H, 10.88; O, 3.74; Si, 6.55. C28H46OSi requires: C, 78.80;
H, 10.86; O, 3.75; Si, 6.58%).
(18Z )-29-Methylidyne-20,21,22,23,24,30-hexanor-2,3-oxido-
squalene: (3Z,7E,11E,15E )-19,20-epoxy-7,12,16,20-tetra-
methylhenicosa-3,7,11,15-tetraen-1-yne 23a (Scheme 4)
t, J 6.3, epoxidic CH), 5.00–5.16 (4 H, m, vinylic CH), 5.51
᎐
(1 H, br d, E CH᎐CH–C᎐C) and 6.21 (1 H, dt, J 15.9 and J 6.8,
᎐
᎐
᎐
E CH᎐CH–C᎐C); m/z(EI) 494 (1.2%), 479 (0.6), 451 (0.6), 421
᎐
᎐
(Z)-Silyl acetylene 22a (1 equiv., 50 mg, 0.117 mmol) was dis-
solved in anhydrous THF (3 cm3) at 0 ЊC under a flux of dry
(0.8), 341 (4), 273 (5), 267 (2), 225 (2), 204 (10), 199 (18), 189
(35), 93 (47), 81 (90) and 73 (100); (Found: C, 80.11; H, 11.02;
1484
J. Chem. Soc., Perkin Trans. 1, 2002, 1477–1486

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O, 3.22; Si, 5.69. C33H54OSi requires: C, 80.09; H, 11.00; O,
3.23; Si, 5.68%).
3.23; Si, 5.66. C33H54OSi requires: C, 80.09; H, 11.00; O, 3.23;
Si, 5.68%).
(22Z )-24-Methylidyne-30-nor-2,3-oxidosqualene: (3Z,7E,11E,
15E,19E )-23,24-epoxy 7,11,16,20-tetramethylpentacosa-
3,7,11,15,19-pentaen-1-yne 25a (Scheme 5)
(18Z )- and (18E )-29-Methylidyne-20-nor-2,3-oxidosqualene:
(5Z,9E,13E,17E )-21,22-epoxy-5-ethynyl-2,9,14,18,22-
pentamethyltricosa-2,5,9,13,17-pentaene 27a and
(5E,9E,13E,17E )-21,22-epoxy-5-ethynyl-2,9,14,18,22-
pentamethyltricosa-2,5,9,13,17-pentaene 27b (Scheme 6)
Compound 25a was obtained in 78% yield, using the method
described for 23a, starting from the silyl derivative 24a.
νmax(KBr pellet)/cmϪ1 3310, 2970, 2930, 2855, 1445, 1380 and
1250; δH(200 MHz, CDCl3) 1.26 and 1.31 (6 H, 2 s, epoxidic
CH3), 1.58–1.73 (14 H, m, allylic CH3 and epoxidic CH2), 1.92–
2.20 (18 H, m, allylic CH2), 2.71 (1 H, t, J 6.3, epoxidic CH),
Compounds 27a and 27b were obtained in 81% yield (E : Z =
1 : 1), starting from the silyl derivatives 26a and 26b, using the
method described for compound 23a. νmax(CCl4)/cmϪ1 3310,
2965, 2930, 2850, 1450 and 1380; δH(200 MHz, CDCl3) 1.26 and
1.31 (6 H, 2 s, epoxidic CH3), 1.56–1.73 (17 H, m, allylic CH3
and epoxidic CH2), 1.91–2.20 (16 H, m, allylic CH2), 2.71 (1 H,
᎐
3.09 (1 H, d, J 2.3, C᎐CH, Z isomer), 5.06–5.20 (4 H, m, vinylic
᎐
᎐
CH), 5.45 (1 H, br d, Z CH᎐CH–C᎐CH) and 5.98 (1 H, dt,
J 11.2 and J 6.9, Z CH᎐CH–C᎐CH); m/z(CI, isobutane) 423
᎐
᎐
᎐
᎐
t, J 6.3, epoxidic CH), 2.72 (1 H × 1/2, br s, C᎐CH, E isomer),
᎐
᎐
᎐
᎐
(100%); (Found: C, 85.27; H, 10.95; O, 3.81. C30H46O requires:
C, 85.25; H, 10.97; O, 3.79%).
2.97 (1 H × 1/2, br s, C᎐CH, Z isomer), 5.20 (4 H, m, vinylic
CH) and 5.35–5.42 (1 H, m, CH᎐C–C᎐C); m/z(EI) 422 (0.3%),
᎐
᎐
᎐
᎐
353 (0.4), 279 (1), 267 (2), 245 (4), 217 (5), 203 (6), 135 (42)
and 81 (100); m/z(CI, isobutane) 423 (100%); (Found: C, 85.28;
H, 10.99; O, 3.77. C30H46O requires: C, 85.25; H, 10.97; O,
3.79%).
(22E )-24-Methylidyne-30-nor-2,3-oxidosqualene: (3E,7E,11E,
15E,19E )-23,24-epoxy-7,11,16,20-tetramethylpentacosa-
3,7,11,15,19-pentaen-1-yne 25b (Scheme 5)
Compound 25b was obtained in 75% yield, using the method
described for 23a, starting from the silyl derivative 24b.
νmax(KBr pellet)/cmϪ1 3310, 2970, 2930, 2850, 1445, 1385 and
1250; δH(200 MHz, CDCl3) 1.26 and 1.31 (6 H, 2 s, epoxidic
CH3), 1.58–1.73 (14 H, m, allylic CH3 and epoxidic CH2), 1.92–
(6Z,10E,14E,18E )-22,23-Epoxy-2,10,15,19,23-pentamethyl-6-
(trimethylsilylethynyl)tetracosa-2,6,10,14,18-pentaene 28a and
(6E,10E,14E,18E )-22,23-epoxy-2,10,15,19,23-pentamethyl-6-
(trimethylsilylethynyl)tetracosa-2,6,10,14,18-pentaene 28b
(Scheme 6)
2.20 (18 H, m, allylic CH2), 2.71 (1 H, t, J 6.3, epoxidic CH),
᎐
2.79 (1 H, d, J 2.2, C᎐CH, E isomer), 5.06–5.20 (4 H, m, vinylic
᎐
Compounds 28a and 28b were obtained in E : Z = 1 : 1 ratio,
using the method described for the silyl derivatives 26a and 26b,
using 5-bromo-2-methylpent-2-ene instead of 1-bromo-3-
methylbut-2-ene. In this case, they were obtained in very low
yield (5%), together with the non-alkylated silyl derivatives 22a
and 22b, in 21% yield. The alkylated silyl derivatives 28a and
28b were obtained first, separated from the non-alkylated silyl
derivatives 22a and 22b by flash chromatography (petroleum
ether to remove impurities, then petroleum ether–diethyl ether
99.5 : 0.5).
᎐
CH), 5.48 (1 H, br d, E CH᎐CH–C᎐CH) and 6.23 (1 H, dt,
J 16.1 and J 6.9, E CH᎐CH–C᎐CH); m/z(CI, isobutane) 423
᎐
᎐
᎐
᎐
᎐
(100%); (Found: C, 85.28; H, 10.99; O, 3.79. C30H46O requires:
C, 85.25; H, 10.97; O, 3.79%).
(5Z,9E,13E,17E )-21,22-Epoxy-2,9,14,18,22-pentamethyl-5-
(trimethylsilylethynyl)tricosa-2,5,9,13,17-pentaene 26a and
(5E,9E,13E,17E )-21,22-epoxy-2,9,14,18,22-pentamethyl-5-
(trimethylsilylethynyl)tricosa-2,5,9,13,17-pentaene 26b
(Scheme 6)
28a and 28b: νmax(KBr pellet)/cmϪ1 2965, 2930, 2850, 1450,
1380 and 1245; δH(200 MHz, CDCl3) 0.18 [9 H, s, (CH3)3Si],
1.26 and 1.31 (6 H, 2 s, epoxidic CH3), 1.56–1.72 (17 H, m,
allylic CH3 and epoxidic CH2), 1.93–2.21 (18 H, m, allylic CH2),
In a three-necked flask containing anhydrous THF (15 cm3),
[3-(trimethylsilyl)prop-2-ynyl]triphenylphosphonium bromide
(1.2 equiv., 274 mg, 0.60 mmol) was suspended at Ϫ80 ЊC,
under a flux of dry nitrogen, with stirring. Butyllithium (1.6 M
solution in hexane, 4 equiv., 1.3 cm3, 2.0 mmol) was added,
during which the reaction mixture turned red. It was left for
15 min at Ϫ40 ЊC and then 1-bromo-3-methylbut-2-ene
(1.5 equiv., 112 mg, 0.75 mmol) was added, during which the
solution turned orange. The solution was then cooled to Ϫ80 ЊC
and additional butyllithium (1.6 M solution in hexane, 2 equiv.,
0.6 cm3, 1.0 mmol) was added, during which the solution turned
dark red. After 15 min of stirring at Ϫ80 ЊC, the C22 squalene
aldehyde monobromohydrin 20 (1 equiv., 207 mg, 0.50 mmol)
in anhydrous THF (1 cm3) was added and allowed to react
for 1 h at Ϫ80 ЊC, during which time the colour turned pink. It
was then gradually allowed to reach room temperature, poured
into cold 10% NH4Cl–diethyl ether (1 : 1, 50 cm3) and extracted
with diethyl ether (3 × 30 cm3). The combined extracts were
washed with saturated brine (2 × 30 cm3), dried with anhydrous
sodium sulfate, filtered and evaporated in vacuo at ϩ35 ЊC. The
resulting oil was purified by flash chromatography (petroleum
ether to remove impurities, then petroleum ether–diethyl ether,
99.5 : 0.5) to give 64 mg (26%) of the two silyl acetylenes 26a
and 26b (E : Z = 1 : 1), as a colourless oil. νmax(CCl4)/cmϪ1 2965,
2930, 2850, 1450, 1380 and 1250; δH(200 MHz, CDCl3) 0.18
[9 H, s, (CH3)3Si], 1.26 and 1.31 (6 H, 2 s, epoxidic CH3), 1.58–
1.75 (17 H, m, allylic CH3 and epoxidic CH2), 1.90–2.22 (16 H,
2.71 (1 H, t, J 6.3, epoxidic CH), 5.06–5.22 (4 H, m, vinylic CH)
᎐
and 5.33–5.41 (1 H, m, CH᎐C–C᎐C); m/z(CI, isobutane) 509
᎐
᎐
(100%); (Found: C, 80.25; H, 11.11; O, 3.12; Si, 5.50. C34H56OSi
requires: C, 80.25; H, 11.09; O, 3.14; Si, 5.52%).
(18Z )- and (18E )-29-Methylidyne-2,3-oxidosqualene:
(6Z,10E,14E,18E )-22,23-epoxy-6-ethynyl-2,10,15,19,23-
pentamethyltetracosa-2,6,10,14,18-pentaene 29a and
(6E,10E,14E,18E )-22,23-epoxy-6-ethynyl-2,10,15,19,23-
pentamethyltetracosa-2,6,10,14,18-pentaene 29b (Scheme 6)
Compounds 29a and 29b were obtained in 72% yield (E : Z =
1 : 1), starting from the silyl derivatives 28a and 28b, using the
method described for compound 23a. νmax(film)/cmϪ1 3310,
2970, 2930, 2850, 1450 and 1380; δH(200 MHz, CDCl3) 1.26 and
1.31 (6 H, 2 s, epoxidic CH3), 1.56–1.75 (17 H, m, allylic CH3
and epoxidic CH2), 1.92–2.22 (18 H, m, allylic CH2), 2.71 (1 H,
᎐
t, J 6.3, epoxidic CH), 2.72 (1 H × 1/2, br s, C᎐CH, E isomer),
᎐
᎐
2.98 (1 H × 1/2, br s, C᎐CH, Z isomer), 5.05–5.20 (4 H,
m, vinylic CH) and 5.35–5.42 (1 H, m, CH᎐C–C᎐C);
᎐
᎐
᎐
᎐
m/z(CI, isobutane) 437 (100%); (Found: C, 85.22; H, 11.08; O,
3.64. C31H48O requires: C, 85.26; H, 11.08; O, 3.66%).
Enzyme assays
m, allylic CH2), 2.71 (1 H, t, J 6.3, epoxidic CH), 5.05–5.22
Solubilisation and purification of OSCs. Partially purified
OSCs from pig liver microsomes and yeast microsomes were
obtained as previously described.16
᎐
(4 H, m, vinylic CH) and 5.34–5.41 (1 H, m, CH᎐C–C᎐C);
᎐
᎐
m/z(CI, isobutane) 495 (100%); (Found: C, 80.10; H, 10.98; O,
J. Chem. Soc., Perkin Trans. 1, 2002, 1477–1486
1485

View Article Online
11 W. R. Moore and G. L. Schatzman, J. Biol. Chem., 1992, 267, 22003.
12 C. J. Buntel and J. H. Griffin, J. Am. Chem. Soc., 1992, 114, 9711.
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J. Med. Chem., 1992, 35, 3050.
Assay of OSC activity and kinetic determination. The enzyme
activity of OSC was determined by incubating the partially
purified pig enzyme for 30 min at 45 ЊC and the solubilized
yeast enzyme for 30 min at 35 ЊC, with [3-3H]-3-(R,S)-2,3-
oxidosqualene (50000 cpm), as previously described.19 IC50
values (the concentration of inhibitor that reduces the enzym-
atic conversion of OS to lanosterol by 50%) were determined
at 25 µM substrate concentration in the presence of different
concentrations of inhibitors, using (18E)-29-methylidene-
hexanor-2,3-oxidosqualene 6 as a control compound.28
18 L. Cattel and M. Ceruti, in Biochemistry and Function of Sterols, eds.
E. J. Parish and W. D. Nes, American Oil Chemists’ Society,
Champaign, Il, 1997, p. 1.
19 F. Viola, M. Ceruti, L. Cattel, P. Milla, K. Poralla and G. Balliano,
Lipids, 2000, 35, 297.
20 M. Taton, P. Benveniste, A. Rahier, W. S. Johnson, H. Liu and
A. R. Sudhakar, Biochemistry, 1992, 31, 7892.
21 I. Abe, W. Liu, A. C. Oehlschlager and G. D. Prestwich, J. Am.
Chem. Soc., 1996, 118, 9180.
22 D. Stach, Y. F. Zheng, A. L. Perez, A. C. Oehlschlager, I. Abe,
G. D. Prestwich and P. G. Hartman, J. Med. Chem., 1997, 40, 201.
23 M. Ceruti, G. Balliano, F. Rocco, P. Milla, S. Arpicco, L. Cattel and
F. Viola, Lipids, 2001, 36, 629.
24 J. Abad, M. Guardiola, J. Casas, F. Sanchez-Baeza and
A. Messeguer, J. Org. Chem., 1996, 61, 7603.
Time-dependent inactivation of the OSC. Time-dependent
inactivation was determined at 37 ЊC by adding the inhibitors
to the enzyme solution in the absence of substrate. Aliquots
were withdrawn at time intervals from 5 min to 40 min and
diluted 40-fold for pig enzyme or 10-fold for yeast enzyme,
by transfer to test tubes containing cold and labelled substrate
OS (25 µM) and Tween-80 (0.5 mg mlϪ1) in Na–K phosphate
buffer. Residual activity was determined by incubating pig or
yeast enzyme under the same conditions. The t/2 values were
calculated by plotting the log of the residual activity against
time.19
25 X. Xiao and G. D. Prestwich, J. Am. Chem. Soc., 1991, 113, 9673.
26 B. A. Madden and G. D. Prestwich, Bioorg. Med. Chem. Lett., 1997,
7, 309.
27 E. J. Corey, H. Cheng, C. H. Baker, S. P. T. Matsuda, D. Li and
X. Song, J. Am. Chem. Soc., 1997, 119, 1289.
28 M. Ceruti, F. Rocco, F. Viola, G. Balliano, P. Milla, S. Arpicco and
L. Cattel, J. Med. Chem., 1998, 41, 540.
Acknowledgements
This work was supported by the Ministero dell’Istruzione,
Università e Ricerca (MIUR) (40% and 60%). Thanks are due
to Mr Daniele Zonari.
29 F. Viola, G. Balliano, P. Milla, L. Cattel, F. Rocco and M. Ceruti,
Bioorg. Med. Chem., 2000, 8, 223.
30 I. Abe, M. Bai, X.-Y. Xiao and G. D. Prestwich, Biochem. Biophys.
Res. Commun., 1992, 187, 32.
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