The squalestatins: Inhibitors of squalene synthase. Enzyme inhibitory activities and in vivo evaluation of C3-modified analogues

Article abstract of DOI:10.1021/jm950893j

Squalestatin analogues modified at C3 were prepared and evaluated for their ability to inhibit rat liver microsomal squalene synthase in vitro. While the 4,6-dimethyloctenoate ester group at C6 was maintained, a number of modifications to the C3 carboxyli

Full text of DOI:10.1021/jm950893j

J . Med. Chem. 1996, 39, 1413-1422
1413
Th e Squ a lesta tin s: In h ibitor s of Squ a len e Syn th a se. En zym e In h ibitor y
Activities a n d in Vivo Eva lu a tion of C3-Mod ified An a logu es
Panayiotis A. Procopiou,* Brian Cox, Barrie E. Kirk, Michael G. Lester, Alun D. McCarthy,† Meenu Sareen,†
Peter J . Sharratt, Michael A. Snowden,† Stephen J . Spooner, Nigel S. Watson, and J ulia Widdowson
Departments of Medicinal Chemistry and Molecular Science, Glaxo Wellcome Research and Development, Medicines Research
Center, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom
Received December 6, 1995X
Squalestatin analogues modified at C3 were prepared and evaluated for their ability to inhibit
rat liver microsomal squalene synthase in vitro. While the 4,6-dimethyloctenoate ester group
at C6 was maintained, a number of modifications to the C3 carboxylic acid were well tolerated.
However, in the absence of the C6 ester group, similar modifications to the C3 carboxyl group
caused loss of activity. Selected compounds were evaluated for their ability to inhibit cholesterol
biosynthesis in vivo in rats 1 and 6 h postadministration. Analogues of squalestatin 1 (S1)
modified at C3 were found to possess a shorter duration of effect in vivo which is reflected in
their substantially reduced ability to lower serum cholesterol levels in marmosets. Significant
cholesterol lowering (up to 62%) for the C3 hydroxymethyl analogue 1b was observed only
when this compound was dosed three times a day for 3 days.
High levels of blood cholesterol and blood lipids are
conditions which are implicated in the onset of vessel
wall disease. Methods for effective reduction of serum
cholesterol levels are therefore of major interest. Cur-
rently, the most effective approach to lowering serum
cholesterol concentrations is by inhibiting sterol bio-
synthesis, and a number of therapeutic agents are
available which work by inhibiting 3-hydroxy-3-meth-
ylglutaryl coenzyme A (HMG-CoA) reductase, the en-
zyme which catalyzes the biosynthesis of mevalonic acid.
Mevalonic acid, however, is a common precursor of all
isoprenyl derivatives, including in animals the ubiquino-
nes, heme A, the dolichols, isopentenyl t-RNA, and
isoprenylated proteins. A more selective inhibition of
cholesterol biosynthesis may be achieved by inhibiting
steps beyond the branch in the pathway. The first
biosynthetic step which leads exclusively to sterols, the
head-to-head condensation of two farnesyl diphosphate
(FPP) molecules to give squalene via the intermediate
presqualene diphosphate (PSPP), is catalyzed by squalene
synthase1 (SQS) (farnesyldiphosphate:farnesyldiphos-
phate farnesyl transferase, EC 2.5.1.21). Agents which
inhibit this enzyme are therefore potential drugs for the
regulation of cholesterogenesis.
cholesterol levels during 7 days after a single iv dose of
1 mg/kg).6 A group at Merck has published the isolation
of zaragozic acids, the structure of zaragozic acid A7,8
being identical with that of S1. More recently a group
at Tokyo Noko UniversitysMitsubishi9 has also re-
ported the isolation of S1 from another organism,
Setosphaeria khartoumensis L1685.
As a part of our chemical program aimed at the
modification of the complex squalestatin structure and
the identification of the key structural features respon-
sible for the biological activity, we have reported on the
C1 chain-length requirements;10 on the role of the
tricarboxylic acid moiety;11 on C6 and C7 modifica-
tions;12 on the C6,C7 dideoxy,13 C3 decarboxy,14 C4
deoxy,15 monocyclic,16 acyclic,17,18 C3 hydroxymethyl19
and C3 heterocyclic20 analogues; and on modifications
at the allylic center.21 In addition a detailed review
bringing together all the published SAR of the squale-
statins and the zaragozic acids has just been com-
pleted.22 Our earlier studies have shown that the
carboxylic acid at C3 in S1 is not essential. Thus,
esterification11 of the C3 carboxylic acid to the 3-methyl
ester, decarboxylation,14 or reduction to the hydroxy-
methyl19 derivative at this position provide analogues
with potent SQS inhibitory activity. Furthermore sub-
stitution of the C3 carboxylic acid in S1 by a variety of
heterocyclic substituents can be well tolerated.20 In
addition researchers at Merck have reported23 the
preparation of the C3 methyl analogue of zaragozic acid
A which retains SQS inhibitory activity. In this paper
we report further studies at this position on squale-
statins 1a and 2a and, in particular, the effect on
biological activity of replacing the carboxylic acid moiety
at C3 by neutral and basic groups.
We have recently described the isolation2,3 and struc-
ture elucidation4 of the squalestatins, a novel group of
fungal metabolites isolated from a previously unknown
Phoma species (Coelomycetes). Squalestatins S1 (1a )
and H1 (2a ) are selective inhibitors of both rat and
Candida SQS; 50% inhibition of rat liver microsomal
SQS activity is observed in vitro at a concentration of
12 and 26 nM, respectively. Furthermore, when S1 is
administered orally to marmosets for 7 days, a 50%
reduction in serum cholesterol levels is observed at a
dose of 10 mg/kg/day.5 Moreover when S1 is adminis-
tered iv for 7 days at 1 mg/kg/day, an 86% reduction in
serum cholesterol is observed; under the same dosing
regime H1 showed a 56% reduction. Further studies
in marmosets revealed that S1 has a profound and
extended effect on lipids (50-60% decrease in serum
Ch em istr y
Esterification of 1a with excess methyl iodide in the
presence of sodium bicarbonate in DMF gave the tri-
methyl ester 3 which was selectively saponified to the
4,5-dimethyl ester 4.14 Reduction of the 3-carboxylate
function of 4 was achieved by activation as its 3-N-
† Department of Molecular Science.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
0022-2623/96/1839-1413$12.00/0 © 1996 American Chemical Society

1414 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Procopiou et al.
Sch em e 1a
provided the methoxymethyl compound 11 from which
the MEM groups were cleaved with aqueous formic acid
to give the diol 12. Selective cleavage of the methyl
esters gave the C3 methoxymethyl analogue 1d .
The preparation of compounds incorporating basic
groups at C3 utilized the 3-methyl-4,5-di-tert-butyl
triester 13 (Scheme 4).19 Protection of the C7 hydroxyl
group as the TBDMS ether gave 14 from which the
3-methyl ester was hydrolyzed selectively with base to
give the 3-carboxylic acid 15. Reduction of the carboxy-
lic acid group via the N-succinimidoyl ester provided the
hydroxymethyl analogue 16. Reaction of 16 with triflic
anhydride provided the triflate 17, displacement of
which with azide gave the C3 azidomethyl analogue 18.
Cleavage of the TBDMS group with fluoride provided
the alcohol 19 from which the aminomethyl derivative
20 was derived by reduction of the azido function with
triphenylphosphine. Cleavage of the tert-butyl ester
groups with 6.5 M HCl in dioxane provided the dicar-
boxylic acid 1e. Reductive amination of formaldehyde
with amine 1e using sodium triacetoxyborohydride as
the reducing agent afforded the corresponding dimethyl-
amino analogue 1f.
a
Conditions: (i) MeI, NaHCO3, DMF; (ii) 0.1 M NaOH, THF,
H2O; (iii) (a) N-hydroxysuccinimide, 1-cyclohexyl-3-(2-morpholi-
noethyl)carbodiimide metho-p-toluenesulfonate, (b) NaBH4, THF;
(iv) LiI, 2,4,6-collidine, 45 °C.
Sch em e 2a
With a route to C3 aminomethyl-substituted ana-
logues established, neutral and acidic derivatives of this
functionality were readily accessible. Thus, reaction of
the C3 aminomethyl dicarboxylic acid 1e with phenyl
carbamate and triethylamine in THF at reflux provided
the C3 ureidomethyl analogue 1g whereas reaction of
1e with aminoiminomethanesulfonic acid27 (21) and
triethylamine in methanol at 20 °C provided the C3
guanidinomethyl analogue 1h . The acidic trifluoro-
methanesulfonamide 1i was derived as shown in Scheme
5. Reduction of the azidomethyl derivative 18 with
triphenylphosphine gave the aminomethyl analogue 22
which on treatment with triflic anhydride and 2,4,6-
collidine provided the sulfonamido analogue 23. Re-
moval of the TBDMS group with tetrabutylammonium
fluoride gave the alcohol 24, and cleavage of the tert-
butyl esters with 6.5 M HCl in dioxane provided the
required sulfonamide 1i.
a
Conditions: (i) (a) i-BuOCOCl, N-methylmorpholine, CH2Cl2;
(b) CH2N2, Et2O; (ii) HI, CHCl3; (iii) LiI, 2,4,6-collidine, 45 °C.
hydroxysuccinimidyl ester followed by in situ reaction
with sodium borohydride24 to provide the 3-hydroxy-
methyl analogue 5. Treatment of the derived product
with lithium iodide in 2,4,6-collidine at 45 °C under a
stream of nitrogen25 resulted in selective cleavage of the
methyl esters to give the dicarboxylic acid 1b (Scheme
1).19 An alternative synthesis of this compound has
been published by the Merck group.23 The synthesis of
the C3 acetyl analogue 1c is shown in Scheme 2.
Activation of the 4,5-dimethyl ester 4 followed by
treatment with diazomethane and subsequent reduction
of the derived diazo ketone20 6 with hydrogen iodide
gave the desired C3 acetyl derivative 7 from which the
dicarboxylic acid 1c was obtained using procedures
similar to those described above.
The methoxymethyl analogue 1d was synthesized
from the trimethyl ester4 3 as outlined in Scheme 3.
Protection of the C4 and C7 hydroxyl groups as (meth-
oxyethoxy)methyl (MEM) ethers gave the bis-MEM-
protected compound 8. Selective base-catalyzed hydrol-
ysis of the 3-methyl ester afforded the related carboxylic
acid18 9 which was reduced to the C3 hydroxymethyl
derivative 10. Alkylation of freshly prepared26 10
The C6 ester side chain was selectively removed from
compounds 1c-e, 1g, 1i by reaction with N-methylhy-
droxylamine according to our previously published
procedure13 to give derivatives 2c-e, 2g, 2i.
Resu lts a n d Discu ssion
The compounds listed in Table 1 were evaluated for
their inhibitory activity against SQS using the same

Squalestatins: Inhibitors of Squalene Synthase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1415
Sch em e 3a
a
Conditions: (i) (a) MEM-Cl, i-Pr2NEt, ClCH2CH2Cl, reflux; (b) NaH, DMF, MEM-Cl; (ii) 0.1 M NaOH, THF; (iii) (a) (COCl)2, DMF,
CH2Cl2; (b) NaBH4, THF, CH3CN; (iv) NaH, MeI, DMF; (v) HCO2H, H2O; (vi) LiI, 2,4,6-collidine, 45 °C.
Sch em e 4a
a
Conditions: (i) (a) MeOH, 12 M HCl; (b) Me2NCH(O-t-Bu)2, toluene; (ii) TBDMS-Cl, imidazole; (iii) 0.1 M NaOH, THF, H2O; (iv) (a)
N-hydroxysuccinimide, DCC, CH2Cl2; (b) NaBH4, THF; (v) (CF3SO2)2O, 2,4,6-collidine, CH2Cl2; (vi) NaN3, DMF; (vii) TBAF, THF; (viii)
Ph3P, THF, H2O; (ix) 6.5 M HCl, dioxane; (x) HCHO, AcOH, NaBH(OAc)3, CH3CN; (xi) PhOCONH2, Et3N, THF; (xii) H2NC(NH)SO3H
(21), Et3N, CH3CN.
Ta ble 1. In Vitro SQS Inhibitory Activitya
Sch em e 5a
compd
no.
IC50 compd
IC50
(nM)
formula
(nM)
no.
formula
1a
1b
1c
1d
1e
1f
1g
1h
1i
C35H46O14
12
15
11
13
4
10
3
65
60
2a
2b
2c
2d
2e
2f
2g
2h
2i
C25H30O13
C25H32O12
C26H32O12
C26H34O12
C25H33NO11
npc
26
C
35H48O13
395b
C36H48O13
500
C
C
C
36H50O13
35H49NO12
37H53NO12
>1000
>1000
npc
C36H50N2O13
36H51N3O12
C36H48F3NO14
C26H34N2O12
>1000
C
npc
npc
S
C26H32F3NO13
S
>1000
a
IC50 values were determined at least on two different occasions
with a minimum of five and a maximum of eight dose levels of
each inhibitor at least in duplicate and are expressed as mean
b
values, using S1 as a reference. Reference 19. c np ) not pre-
pared.
sponding analogues of squalestatin H1 (2a ) which
incorporate a C6 hydroxyl group have greatly reduced
SQS inhibitory activities. Furthermore, in a series of
squalestatin H1 analogues incorporating heterocyclic
groups directly attached to C3, only the analogue
incorporating a C3 tetrazol-5-yl group, a known car-
boxylic acid mimetic, retains potent SQS inhibitory
activity.20 These results, together with those previously
reported11 for the squalestatin methyl ester derivatives,
suggest that an acidic group must be present at C3 for
significant SQS inhibitory activity to be retained in
compounds lacking the 4,6-dimethyloctenoate ester
a
Conditions: (i) Ph3P, THF, H2O; (ii) (CF3SO2)2O, 2,4,6-
collidine, CH2Cl2; (iii) TBAF, THF; (iv) 6.5 M HCl, dioxane.
enzyme preparations and assays as described in our
earlier publications.2,5,28 The data in Table 1 clearly
show that potent SQS inhibitory activity is exhibited
by those analogues which retain the C6 dimethyl-
octenoate side chain, although some loss of activity is
apparent in the case of the guanidinomethyl and sul-
fonamido analogues 1h and 1i. In contrast, the corre-

1416 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Procopiou et al.
Ta ble 2. Effect of 1b on Serum Cholesterol Levels in
Marmosets (n ) 6) after a Single iv Dose
cholesterol reduction (%)
dose (mg/kg)
day 2
day 3
day 7
10
30
5 ( 5
30 ( 7
31 ( 5
47 ( 4
2 ( 4
20 ( 10
21 ( 3
62 ( 4
14 ( 6
16 ( 4
28 ( 8
32 ( 5
50
16.7a
a
Animals were dosed three times a day for 3 days.
cleared more rapidly into the bile providing thus an
explanation for their different lipid lowering proper-
ties.30
F igu r e 1. Time-course studies for inhibition of cholesterol
biosynthesis in rats (n ) 8) using 1a (1 mg/kg iv) and 1b (10
mg/kg iv). * Significantly (p < 0.05) below control.
Con clu sion
Squalestatin analogues modified at C3 were prepared
and evaluated for their ability to inhibit rat SQS in vitro.
Analogues incorporating neutral and basic groups at C3
were well tolerated while maintaining the C6 ester
group found in the naturally occurring squalestatins.
In the absence of the C6 ester group, however, similar
modifications to the C3 carboxylic acid caused substan-
tial reduction of inhibitory activity. Selected compounds
were tested in rats for their ability to inhibit cholesterol
biosynthesis 1 and 6 h postadministration. At a dose
of 1 mg/kg iv S1 inhibits cholesterol biosynthesis in rats
at least 8 h postadministration. Squalestatins 1b -f
possess inhibitory activities closely similar to S1 but
contrasting abilities to inhibit cholesterol biosynthesis
in vivo. Thus while these analogues showed complete
inhibition of hepatic cholesterol biosynthesis at 1 h
postadministration, none showed significant inhibition
6 h postadministration, even when dosed at 10 mg/kg
iv. These data together with time-course studies with
1b for inhibition of cholesterol biosynthesis clearly
indicated that these C3-modified analogues of S1 pos-
sess shorter duration of effect in vivo which is reflected
in their substantially reduced ability to lower serum
cholesterol levels in marmosets. Thus compounds 1b,d,e
were ineffective at lowering serum cholesterol levels at
a dose of 10 mg/kg iv. Signifigant cholesterol lowering
for 1b was observed only when this compound was dosed
three times a day for 3 days. The C3-decarboxylated
analogue14 also has a much reduced ability to lower
serum cholesterol levels in marmosets. It is clear that
the C3 carboxylic acid plays a minor role in securing
enzyme inhibitory activity in S1 analogues that have
been modified at C3. However this functional group
critically influences the in vivo profile of this series.
group at C6. However, as is apparent from the lack of
SQS inhibitory activity shown by 2i which contains the
acidic (trifluoromethyl)sulfonamido group at C3, the
nature of this acidic group is clearly important. We
have suggested previously6,11,19,20,21 that squalestatin S1
and H1 analogues are presqualene diphosphate and
farnesyl diphosphate mimetics, respectively, and that
this may account for the observed differences in SAR
between C3-modified analogues in each series. It is
noteworthy that not only can the carboxylic acid group
at C3 be replaced by neutral groups in squalestatins
possessing a C6 ester group to provide dianionic com-
pounds which retain potent SQS inhibitory activity, but
by incorporating a basic group at C3 as in the amino-
methyl and (dimethylamino)methyl analogues (1e and
1f, respectively), compounds with a net charge of -1
can retain potent SQS inhibitory activity.
We have shown previously5 that S1 inhibits choles-
terol biosynthesis from [1-14C]acetate when adminis-
tered to rats, with an ED50 of 0.1 mg/kg iv. Time-course
studies for inhibition of cholesterol biosynthesis in rats
using S1 (1 mg/kg iv) and 1b (10 mg/kg iv) revealed that
1b has a shorter duration of effect (Figure 1). In the
light of these data, analogues with further modifications
to the C3 carboxylic acid were tested at two time points
(1 and 6 h postadministration) at a dose of 10 mg/kg iv
using 1a (1 mg/kg iv) and 1b (10 mg/kg iv) as controls.
The squalestatins 1c-f showed maximal inhibition of
hepatic cholesterol biosynthesis in vivo in rats only at
1 h postdose; no significant inhibition was observed at
the later time point.
We have reported previously6 that a profound and
extended lipid-lowering effect was observed in marmo-
sets following a single iv dose of 1 mg/kg S1. Thus a
maximum effect was seen after 3 days, and 7 days
postdose serum cholesterol was still reduced by 50%.
In similar studies, analogues 1b,d ,e did not significantly
reduce serum cholesterol concentrations even at 10 mg/
kg iv. Squalestatin 1b was examined further in single-
dose lipid-lowering studies at doses of 30 and 50 mg/kg
iv. Lipid levels were only reduced marginally even at
the higher dose tested. When 1b was dosed at 16.7 mg/
kg iv three times a day for 3 days, lipids were signifi-
cantly reduced by 62% 3 days postadministration and
returned toward baseline 7 days postdose (Table 2).
These data clearly suggested a shorter elimination half-
life for 1b and studies in perfused marmoset liver using
[3H]-S129 and [3H]-1b established that the latter was
abstracted by the liver more quickly than 1a but was
Exp er im en ta l Section
Organic solutions were dried over MgSO4, and column
chromatography was performed on silica gel 60 (Merck, Art
no. 9385). TLC was performed on Merck Kieselgel 60 F254
glass-backed plates; compounds were detected by spraying the
plates with an ammonium molybdate-sulfuric acid solution
and baking the plates. Analytical HPLC was performed on a
Spherisorb 5 ODS-2 column (25 cm × 0.46 cm) using CH3CN-
H2O containing 0.15 mL/L concentrated H2SO4 or 1 mL/L TFA
as eluent, at a flow rate of 1.5 mL/min and detecting at 210
nm. Preparative HPLC was conducted on a Spherisorb 5
ODS-2 column (25 cm × 2 cm i.d.) using CH3CN-H2O
containing 0.15 mL/L concentrated H2SO4 as eluent, at a flow
rate of 15 mL/min and detecting at 210 nm. The appropriate
fractions from each run were combined, the CH3CN removed
in vacuo (bath temperature <40 °C), and the remainder
extracted with EtOAc. The combined extracts were washed
with brine and evaporated, the residue was dissolved in H2O/

Squalestatins: Inhibitors of Squalene Synthase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1417
dioxane and freeze-dried. IR spectra were recorded on a
Nicolet 5SXC FTIR spectrometer. NMR spectra were recorded
on Bruker AM 250 or Varian VXR 400 spectrometers using
standard pulse sequences. Chemical shifts are expressed as
parts per million downfield from internal tetramethylsilane.
Negative ion fast-atom-bombardment mass spectrometry [MS-
(FAB-ve)] was performed on a Finnigan MAT TSQ70B spec-
trometer and high-resolution negative ion liquid secondary ion
mass spectrometry [HRMS(LSI-ve)] was performed on a VG
Autospec spectrometer. Positive ammonia chemical ionisation
(CI) and positive FAB mass spectrometry was conducted on a
Hewlett-Packard Engine instrument. In order to increase
aqueous solubility of the squalestatins tested in vivo the
carboxylic acids were converted to their potassium salts.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-
(h ydr oxym eth yl)-4,6,7-tr ih ydr oxy-2,8-dioxabicyclo[3.2.1]-
octan e-4,5-dicar boxylic Acid, 6-(4,6-Dim eth yl-2-octen oate),
4,5-Dim eth yl Ester (5). The acid 4 (500 mg, 0.69 mmol) and
N-hydroxysuccinimide (88 mg, 0.76 mmol) in dry THF (7 mL)
were treated at 20 °C with 1-cyclohexyl-3-(2-morpholinoethyl)-
carbodiimide metho-p-toluenesulfonate (324 mg, 0.76 mmol).
After 2 h further quantities of N-hydroxysuccinimide (40 mg,
0.35 mmol) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiim-
ide metho-p-toluenesulfonate (147 mg, 0.35 mmol) were added.
The mixture was stirred for a further 1.5 h when sodium
borohydride (132 mg, 0.35 mmol) was added, followed after a
further hour by 10% aqueous citric acid (50 mL). The mixture
was extracted with ethyl acetate (3 × 50 mL), and the organic
phase was washed with brine (4 × 30 mL), dried, and
chromatographed on silica, eluting with EtOAc-cyclohexane
(3:7, 1:1, 11:9) to give 5 as a white solid (270 mg, 55%): NMR
(CDCl3) δ includes 1.05 (d, 3H, J ) 7 Hz, dCHCHCH3), 2.10
(s, 3H, AcO), 2.69 (dd, 1H, J ) 14 and 6 Hz, CH2Ph), 3.74 (br,
2H, CH2OH), 3.80 and 3.88 (2s, 3H each, COOCH3), 4.03 (br,
1H, 7-H), 4.64 (t, 1H, J ) 5 Hz, 3-H), 4.94 and 4.96 (2s, 1H
each, dCH2), 5.10 (d, 1H, J ) 5 Hz, CHOAc), 5.74 (d, 1H, J )
16 Hz, OCOCHdCH), 5.83 (d, 1H, J ) 2 Hz, 6-H), 6.85 (dd,
1H, J ) 16 and 9 Hz, OCOCHdCH), 7.1-7.4 (m, 5H, Ph). Anal.
(C37H52O13) C, H.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-
(h ydr oxym eth yl)-4,6,7-tr ih ydr oxy-2,8-dioxabicyclo[3.2.1]-
octan e-4,5-dicar boxylic Acid, 6-(4,6-Dim eth yl-2-octen oate)
(1b). A solution of 5 (200 mg, 0.28 mmol) in 2,4,6-trimeth-
ylpyridine (10 mL) was heated to 45 °C under a slow stream
of nitrogen in the presence of lithium iodide (759 mg, 5.67
mmol). After 7 d the solvent was evaporated under reduced
pressure, brine (50 mL) was added, and the mixture was
extracted with ether (3 × 50 mL). The combined organic
phases were dried and purified by reverse-phase HPLC
(eluting with 40-95% CH3CN-H2O containing 0.15 mL/L H2-
SO4) to give 1b (90.2 mg, 48%) as a white solid: NMR (CD3-
OD) δ includes 0.8-0.9 (m, 9H, CH3), 1.05 (d, 3H, J ) 7 Hz,
dCHCHCH3), 2.10 (s, 3H, AcO), 2.65 (dd, 1H, J ) 14 and 6
Hz, CH2Ph), 3.65 (br, 2H, CH2OH), 4.0 (br, 1H, 7-H), 4.64 (br,
1H, 3-H), 4.94 and 4.96 (2s 1H each, dCH2), 5.07 (d, 1H, J )
5 Hz, CHOAc), 5.80 (d, 1H, J ) 16 Hz, OCOCHdCH), 6.34
(br, 1H, 6-H), 6.85 (dd, 1H, J ) 16 and 9 Hz, OCOCHdCH),
7.1-7.3 (m, 5H, Ph); MS(FAB-ve) m/ z 675 (M - H)-. Anal.
(C35H48O13‚0.5H2O) C, H.
each, COOCH3), 4.05 (m, 1H, 7-H), 4.92 (s, 1H, 3-H), 4.98, 5.00
(2s, dCH2), 5.62 (d, 1H, J ) 5 Hz, CHOAc), 5.73 (m, 1H, 6-H),
5.76 (d, 1H, J ) 16 Hz, OCOCHdCH), 6.86 (dd, 1H, J ) 16
and 9 Hz, OCOCHdCH), 7.10-7.30 (m, 5H, Ph). Anal.
(C38H52O13‚0.5H2O) C, H.
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â]]-3-Acetyl-
1-[4-(a cet yloxy)-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl]-
4,6,7-tr ih yd r oxy-2,8-d ioxa bicyclo[3.2.1]octa n e-4,5-d ica r -
boxylic Acid , 6-(4,6-Dim eth yl-2-octen oa te) (1c). To a
stirred solution of 7 (760 mg, 1.06 mmol) in 2,4,6-collidine (38
mL) was added lithium iodide (1.52 g, 11.4 mmol). The
mixture was stirred and heated at 40 °C under a stream of
nitrogen for 24 h, then diluted with water (100 mL), acidified
to pH1 with hydrochloric acid, and extracted with ether (2 ×
100 mL). The organic extracts were washed with water, dried,
and evaporated. The residue was purified by reverse-phase
HPLC eluting with 60% CH3CN-H2O to give 1c (200 mg, 27%)
as a white solid: NMR (CD3OD) δ includes 1.03 (d, 3H, J )
7.5 Hz, dCHCHCH3), 2.10 (s, 3H, AcO), 2.21 (s, 3H, COCH3),
4.01 (d, 1H, J ) 2 Hz, 7-H), 5.00 (s, 1H, 3-H), 4.98, 5.01 (2s,
dCH2), 5.09 (d, 1H, J ) 5 Hz, CHOAc), 5.8 (d, J ) 16 Hz,
OCOCHdCH), 6.28 (d, 1H, J ) 2 Hz, 6-H), 6.85 (dd, 1H, J )
16 and 9 Hz, OCOCHdCH), 7.10-7.30 (m, 5H, Ph). Anal.
(C36H48O13‚H2O) C, H.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-6-h y-
d r oxy-3-(h yd r oxym e t h yl)-4,7-b is[(2-m e t h oxye t h oxy)-
m et h oxy]-2,8-d ioxa b icyclo[3.2.1]oct a n e-4,5-d ica r b oxyl-
ic Acid , 6-(4,6-Dim eth yl-2-octen oa te), 4,5-Dim eth yl Ester
(10). To a solution of dry DMF (1.2 mL) in dry CH2Cl2 (15
mL) under nitrogen at 0 °C was added dropwise via syringe
oxalyl chloride (1.46 mL, 16.7 mmol). Considerable gas
evolution occurred, and a white solid was precipitated. Stir-
ring at 0 °C was continued for 0.5 h before addition of 9 (7.5
g, 8.38 mmol) in dry THF (30 mL) and dry CH3CN (15 mL).
Stirring at 0 °C was continued for 1.5 h before addition of
sodium borohydride (636 mg, 16.8 mmol) and dry DMF (20
mL). After further stirring at 0 °C for 1.5 h the mixture was
allowed to warm to 20 °C and remain at this temperature for
18 h before dilution with water (250 mL), acidification with 2
M hydrochloric acid, and extraction with ether (2 × 250 mL).
The extracts were washed with water (2 × 100 mL), dried,
and chromatographed on silica gel eluting with EtOAc-
petroleum ether (1:1) to give 10 (2.6 g, 35%): NMR (CDCl3) δ
includes 0.80-0.90 (m, 9H, CH3), 1.04 (d, 3H, J ) 7.5 Hz,
dCHCHCH3), 2.10 (s, 3H, AcO), 3.32 (s, 6H, CH2OCH3), 3.45-
3.55 (m, 4H, CH2OCH3), 3.70 (s, 3H, COOCH3), 3.82 (s, 3H,
COOCH3), 4.05 (d, 1H, J ) 2.5 Hz, 7-H), 4.58-4.66 (m, 1H,
3-H), 5.70 (d, 1H, J ) 15 Hz, OCOCHdCH), 6.65 (d, 1H, J )
2.5 Hz, 6-H), 6.82 (dd, 1H, J ) 15 and 7 Hz, OCOCHdCH),
7.10-7.35 (m, 5H, Ph). Anal. (C45H68O17‚0.5H2O) C, H.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-6-h y-
d r oxy-4,7-b is[(2-m et h oxyet h oxy)m et h oxy]-3-(m et h oxy-
m et h yl)-2,8-d ioxa b icyclo[3.2.1]oct a n e-4,5-d ica r b oxylic
Acid , 6-(4,6-Dim et h yl-2-oct en oa t e), 4,5-Dim et h yl E st er
(11). To a stirred solution of 10 (2.6 g, 2.95 mmol) in dry DMF
(36 mL) under nitrogen at 20 °C was added sodium hydride
(60% oil dispersion; 195 mg, 4.87 mmol). After 15 min methyl
iodide (3.4 mL) was added, and stirring under nitrogen at 20
°C was continued for 6 h before dilution with water (200 mL),
acidification with 2 M hydrochloric acid, and extraction with
ether (2 × 200 mL). The organic solution was washed with
water, dried, evaporated, and purified by chromatography on
silica gel eluting with EtOAc-petroleum ether (3:2) to afford
11 (1.9 g, 72%) as a pale gum: NMR (CDCl3) δ includes 0.80-
0.90 (m, 9H, CH3), 1.03 (d, 3H, J ) 7.5Hz, dCHCHCH3), 2.10
(s, 3H, AcO), 3.31 (s, 3H, CH2OCH3), 3.35 (s, 6H, CH2OCH3),
3.69 (s, 3H, COOCH3), 3.82 (s, 3H, COOCH3), 4.08 (d, 1H, J )
2.5 Hz, 7-H), 4.60-4.70 (m, 1H, 3-H), 5.70 (d, 1H, J ) 15 Hz,
OCOCHdCH), 6.68 (d, 1H, J ) 2.5 Hz, 6-H), 6.82 (dd, 1H, J
) 15 and 7 Hz, OCOCHdCH), 7.10-7.35 (m, 5H, Ph).
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-
(m eth oxym eth yl)-4,6,7-tr ih ydr oxy-2,8-dioxabicyclo[3.2.1]-
octan e-4,5-dicar boxylic Acid, 6-(4,6-Dim eth yl-2-octen oate),
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â]]-3-Acetyl-
1-[4-(a cet yloxy)-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl]-
4,6,7-tr ih yd r oxy-2,8-d ioxa bicyclo[3.2.1]octa n e-4,5-d ica r -
boxylic Acid , 6-(4,6-Dim eth yl-2-octen oa te), 4,5-Dim eth yl
Ester (7). To a stirred solution of 620 (1.0 g, 1.35 mmol) in
CHCl3 (3 mL) at 0 °C was added dropwise 66% hydriodic acid
(1 mL). After the addition, the mixture was kept at 0 °C for
3 min, then diluted with water (50 mL), and extracted with
ether (50 mL). The organic phase was washed successively
with water, aqueous sodium thiosulfate, and brine, then dried,
and evaporated. The residue was purified by chromatography
on silica gel, eluting with EtOAc-petroleum ether (2:3) to give
7 (820 mg, 85%) as a pale foam: NMR (CDCl3) δ includes 1.02
(d, 3H, J ) 7.5 Hz, dCHCHCH3), 2.10 (s, 3H, AcO), 2.28 (s,
COCH3), 3.25 (d, 1H, J ) 2.5 Hz, 7-OH), 3.80 and 3.95 (2s, 3H

1418 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Procopiou et al.
4,5-Dim eth yl Ester (12). A solution of 11 (345 mg, 0.39
mmol) in formic acid-water (3:1) was heated at 65 °C for 4 h.
The solvents were removed in vacuo, and the residue was
dissolved in toluene (50 mL) and re-evaporated to give 12 (270
mg, 97%) as a pale foam: NMR δ (CDCl3) includes 0.80-0.90
(m, 9H, CH3), 1.02 (d, 3H, J ) 7.5 Hz, dCHCHCH3), 2.08 (s,
3H, AcO), 3.30 (s, 3H, CH2OCH3), 3.80 (s, 3H, COOCH3), 3.87
(s, 3H, COOCH3), 4.00 (s, 1H, 7-H), 4.65-4.75 (m, 1H, 3-H),
4.95 and 4.99 (2s, 1H each, dCH2), 5.10 (d, 1H, J ) 5 Hz,
CHOAc), 5.75 (d, 1H, J ) 15 Hz, OCOCHdCH), 5.85 (s, 1H,
6-H), 6.85 (dd, 1H, J ) 15 and 7 Hz, OCOCHdCH), 7.10-
7.35 (m, 5H, Ph).
at 65 °C under nitrogen for 16.5 h and then partitioned
between EtOAc (200 mL) and 2 M hydrochloric acid (200 mL).
The aqueous phase was extracted with EtOAc (200 mL). The
combined organic extracts were washed with 2 M hydrochloric
acid (100 mL), water, and brine (2 × 100 mL each), dried, and
evaporated to an oil. This was chromatographed on silica gel
eluting with EtOAc-cyclohexane (1:4). The required fractions
were combined and evaporated to give 14 (9.01 g, 76%) as a
colorless gum: NMR (CDCl3) δ includes 1.02 (d, 3H, J ) 6
Hz, dCHCHCH3), 1.4 and 1.65 (2s, 9H each, t-BuO), 2.1 (s,
3H, AcO), 3.73 (s, COOCH3), 4.02 (s, 1H, 4-OH), 4.12 (d, 1H,
J ) 2 Hz, 7-H), 4.98 and 5.00 (2s, 1H each, dCH2), 5.12 (d,
1H, J ) 5 Hz, CHOAc), 5.28 (s, 1H, 3-H), 5.8 (d, 1H, J ) 16
Hz, OCOCHdCH), 6.38 (d, 1H, J ) 2 Hz, 6-H), 6.93 (dd, 1H,
J ) 16 and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph).
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-7-
[[d im et h yl(1,1-d im et h ylet h yl)silyl]oxy]-4,6-d ih yd r oxy-
2,8-d ioxa b icyclo[3.2.1]oct a n e-3,4,5-t r ica r b oxylic Acid ,
6-(4,6-Dim eth yl-2-octen oa te), 4,5-Bis(1,1-d im eth yleth yl)
Ester (15). A solution of 14 (9.01 g, 9.7 mmol) in THF (450
mL) was treated with 0.1 M sodium hydroxide (116 mL) with
stirring at room temperature. After 0.5 h the solution was
evaporated to low volume and then partitioned between EtOAc
(250 mL) and 2 M hydrochloric acid (500 mL). The aqueous
phase was extracted with further EtOAc (2 × 250 mL). The
combined extracts were washed with water and brine (2 × 250
mL each), dried, and evaporated to give 15 (8.7 g, 98%) as a
white foam: IR νmax (CHBr3) 3444, 1776, 1730 cm-1; NMR
(CDCl3) δ includes 1.02 (d, 3H, J ) 6 Hz, dCHCHCH3), 1.4
and 1.65 (2s, 9H each, t-BuO), 2.1 (s, 3H, AcO), 4.13 (d, 1H, J
) 2 Hz, 7-H), 5.02 and 5.05 (2s, 1H each, dCH2), 5.11 (d, 1H,
J ) 5 Hz, CHOAc), 5.22 (s, 1H, 3-H), 5.78 (d, 1H, J ) 16 Hz,
OCOCHdCH), 6.34 (d, 1H, J ) 2 Hz, 6-H), 6.95 (dd, 1H, J )
16 and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph); MS(FAB+ve)
m/ z 917 (M + H)+; HRMS(FAB+ve) found 917.5082, calcd for
C49H77O14Si 917.5073.
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â]]-1-[4-(Acety-
loxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-7-[[d im eth yl-
(1,1-d im et h ylet h yl)silyl]oxy]-4,6-d ih yd r oxy-3-h yd r oxy-
m et h yl-2,8-d ioxa b icyclo[3.2.1]oct a n e-4,5-d ica r b oxylic
Acid , 6-(4,6-Dim eth yl-2-octen oa te), 4,5-Bis(1,1-d im eth yl-
eth yl) Ester (16). A solution of 15 (5.89 g, 6.4 mmol),
N-hydroxysuccinimide (0.8 g, 6.95 mmol), and N,N′-dicyclo-
hexylcarbodiimide (1.46 g, 7.08 mmol) in THF (60 mL) was
stirred at room temperature for 17 h. The resulting suspen-
sion was filtered, and the filtrate was evaporated to a white
foam. This was dissolved in DMF (60 mL), stirred at room
temperature, and treated with sodium borohydride (242 mg,
6.4 mmol). After 55 min the suspension was filtered. The
filtrate was partitioned between EtOAc (500 mL) and 2 M
hydrochloric acid (500 mL). The organic phase was washed
with water (500 mL), saturated aqueous sodium bicarbonate,
and brine (2 × 500 mL each), dried, and evaporated. The
residue was chromatographed on silica gel eluting with
EtOAc-cyclohexane (1:3) to give 16 (2.55 g, 44%) as a white
foam: NMR (CDCl3) δ includes 1.02 (d, 3H, J ) 6 Hz,
dCHCHCH3), 1.4 and 1.61 (2s, 9H each, t-BuO), 2.1 (s, 3H,
AcO), 3.58 and 3.76 (m, 2H, CH2OH), 3.86 (s, 1H, 4-OH), 4.11
(d, 1H, J ) 2 Hz, 7-H), 4.65 (dd, 1H, J ) 4 and 6 Hz, 3-H),
4.98 and 5.0 (2s, dCH2), 5.12 (d, 1H, J ) 5 Hz, CHOAc), 5.8
(d, 1H, J ) 16 Hz, OCOCHdCH), 6.32 (d, 1H, J ) 2 Hz, 6-H),
6.92 (dd, 1H, J ) 16 and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H,
Ph).
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-
(m eth oxym eth yl)-4,6,7-tr ih ydr oxy-2,8-dioxabicyclo[3.2.1]-
octan e-4,5-dicar boxylic Acid, 6-(4,6-Dim eth yl-2-octen oate)
(1d ). To a stirred solution of 12 (230 mg, 0.32 mmol) in dry
collidine (12 mL) under nitrogen was added lithium iodide (430
mg, 3.21 mmol). The mixture was heated under nitrogen at
45 °C for 18 h; then diluted with ether (75 mL); washed
consecutively with 2 M hydrochloric acid (2 × 100 mL), water
(20 mL), and saturated aqueous sodium thiosulfate (5 mL);
then dried; evaporated; and purified by reverse phase column
chromatography on silica gel (Whatman partisil prep P40; 100
mL) eluting with CH3CN-H2O (2:3) containing H2SO4 (0.15
mL/L) to afford 1d (124 mg, 56%) as a colorless foam: NMR
(CD3OD) δ includes 0.80-0.90 (m, 9H, CH3), 1.02 (d, 3H, J )
7.5 Hz, dCHCHCH3), 2.10 (s, 3H, AcO), 3.45-3.55 (m, 2H,
CH2OCH3), 3.99 (d, 1H, J ) 2 Hz, 7-H), 4.75-4.82 (m, 1H,
3-H), 4.95 and 4.99 (2s, 1H each, dCH2), 5.06 (d, 1H, J ) 5
Hz, CHOAc), 5.80 (d, 1H, J ) 15 Hz, OCOCHdCH), 6.30 (d,
1H, J ) 2 Hz, 6-H), 6.85 (dd, 1H, J ) 15 and 7 Hz,
OCOCHdCH), 7.10-7.35 (m, 5H, Ph). Anal. (C36H50O13
0.5H2O) C, H.
‚
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-4,6,7-
tr ih yd r oxy-2,8-d ioxa bicyclo[3.2.1]octa n e-3,4,5-tr ica r box-
ylic Acid , 6-(4,6-Dim et h yl-2-oct en oa t e), 4,5-Bis(1,1-d i-
m eth yleth yl) Ester , 3-Meth yl Ester (13). A stirred suspen-
sion of 1a (15.6 g, 19.4 mmol) in methanol (1 L) was treated
dropwise with concentrated hydrochloric acid (13 mL). The
resulting clear solution was stirred at room temperature for
24 h. It was then treated with solid sodium bicarbonate (13.2
g), and most of the solvent was evaporated under reduced
pressure. The residue was acidified with 2 M hydrochloric acid
(500 mL) and extracted with EtOAc (1 L × 3). The organic
extract was washed with water (1 L), dried, filtered, and
evaporated. The residue was dissolved in dry toluene (130
mL), heated to 80 °C under nitrogen, and then treated
dropwise with N,N-dimethylformamide di-tert-butyl acetal (38
mL, 0.15 mol) over 30 min. The reaction mixture was stirred
at 80 °C for 3.25 h and then allowed to cool. It was diluted
with ether (700 mL) and washed with brine (600 mL). The
organic layer was dried, and the solvent was evaporated under
reduced pressure. The residue subjected to flash column
chromatography on silica gel eluting with EtOAc-cyclohexane
(1:5, 1:1). The appropriate fractions were combined, and the
solvent was evaporated to give 13 (5.93 g, 37%) as a foam: IR
νmax 3580, 3450, 1729, 1250, 1023 cm-1; NMR (CDCl3) δ
includes 0.8-0.9 (m, 9H, 3CH3), 1.05 (d, 3H, J ) 6.2 Hz,
dCHCHCH3), 1.48 and 1.6 (2s, 9H each, t-BuO), 2.1 (s, 3H,
AcO), 2.71 (dd, 1H, J ) 14 and 5 Hz, CH2Ph), 2.96 (d, 1H, J )
2 Hz, 7-OH), 3.73 (s, 3H, COOCH3), 4.1 (s, 1H, 4-OH), 4.05 (t,
1H, J ) 2 Hz, 7-H), 4.97 (s, 2H, dCH2), 5.1 (d, 1H, J ) 5 Hz,
CHOAc), 5.26 (s, 1H, 3-H), 5.77 (d, 1H,
J ) 16 Hz,
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-7-
[[d im eth yl(1,1-d im eth yleth yl)silyl]oxy]-4,6-d ih yd r oxy-3-
[[[(tr iflu or om eth yl)su lfon yl]oxy]m eth yl]-2,8-dioxabicyclo-
[3.2.1]octa n e-4,5-tr ica r boxylic Acid , 6-(4,6-Dim eth yl-2-
OCOCHdCH), 5.97 (d, 1H, J ) 2 Hz, 6-H), 6.91 (dd, 1H, J )
16 and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph). Anal.
(C44H64O14) C, H.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-7-
[[d im et h yl(1,1-d im et h ylet h yl)silyl]oxy]-4,6-d ih yd r oxy-
2,8-d ioxa b icyclo[3.2.1]oct a n e-3,4,5-t r ica r b oxylic Acid ,
6-(4,6-Dim eth yl-2-octen oa te), 4,5-Bis(1,1-d im eth yleth yl)
Ester , 3-Meth yl Ester (14). A solution of 13 (10.38 g, 12.7
mmol), tert-butyldimethylsilyl chloride (19.6 g, 130 mmol), and
imidazole (17.7 g, 260 mmol) in dry DMF (26 mL) was stirred
oct en oa t e), 4,5-Bis(1,1-d im et h ylet h yl) E st er (17).
A
solution of 16 (1.19 g, 1.32 mmol) in dry CH2Cl2 (20 mL) was
stirred under nitrogen in an ice bath and treated with 2,4,6-
collidine (0.225 mL, 1.7 mmol) and then trifluoromethane-
sulfonic anhydride (0.28 mL, 1.7 mmol). After 45 min the
yellow solution was diluted with CH2Cl2 (150 mL) and washed
with 2 M hydrochloric acid (2 × 50 mL), water (100 mL),

Squalestatins: Inhibitors of Squalene Synthase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1419
saturated aqueous sodium bicarbonate (2 × 100 mL), and brine
(2 × 100 mL), dried, and evaporated to give 17 (1.296 g, 95%)
as a foam: IR νmax (CHBr3) 1729 cm-1; NMR (CDCl3) δ includes
1.02 (d, 3H, J ) 6 Hz, dCHCHCH3), 1.41 and 1.65 (2s, 9H
each, t-BuO), 2.1 (s, 3H, AcO), 3.88 (s, 1H, 4-OH), 4.13 (br s,
1H, 7-H), 4.32 (dd, 1H, J ) 2.5 and 11 Hz, CH2OSO2), 4.57
(dd, 1H, J ) 7.5 and 11 Hz, 3-H), 4.95-5.05 (m, 3H, dCH2
and one of CH2OSO2), 5.11 (d, 1H, J ) 5 Hz, CHOAc), 5.80 (d,
1H, J ) 16 Hz, OCOCHdCH), 6.30 (br s, 1H, 6-H), 6.95 (dd,
1H, J ) 9 and 16 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph). Anal.
(C50H77F3O15SSi) C, H, S.
4.97 (2s, 1H each, dCH2), 5.09 (d, 1H, J ) 5 Hz, CHOAc), 5.78
(d, 1H, J ) 16 Hz, OCOCHdCH), 5.95 (d, 1H, J ) 2 Hz, 6-H),
6.90 (dd, 1H, J ) 16 and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H,
Ph).
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-ph en ylh exyl]-3-(am i-
n om eth yl)-4,6,7-tr ih ydr oxy-2,8-dioxabicyclo[3.2.1]octan e-
4,5-d ica r boxylic Acid , 6-(4,6-Dim eth yl-2-octen oa te) (1e).
A solution of 20 (170 mg, 0.22 mmol) in 6.5 M hydrogen
chloride in dioxane (10 mL) was kept at room temperature for
8.25 h and then evaporated to a pale yellow solid. This was
redissolved in 6.5 M hydrogen chloride in dioxane (5 mL), and
the solution was kept at room temperature for 4 h and then
evaporated. The residue was dissolved in ether, and the
solution was evaporated to a pale yellow solid which was
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-3-(a zi-
d om eth yl)-7-[[d im eth yl(1,1-d im eth yleth yl)silyl]oxy]-4,6-
d ih yd r oxy-2,8-d ioxa bicyclo[3.2.1]octa n e-4,5-d ica r boxy-
lic Acid , 6-(4,6-Dim eth yl-2-octen oa te), 4,5-Bis(1,1-d im eth -
yleth yl) Ester (18). A solution of 17 (584 mg, 0.56 mmol) in
dry DMF (6 mL) was stirred at 20 °C and treated with sodium
azide (112 mg, 1.72 mmol). After 4 h the mixture was
partitioned between EtOAc (100 mL) and 2 M hydrochloric
acid (50 mL). The organic phase was washed with 2 M
hydrochloric acid (50 mL), then water, and brine (2 × 50 mL
each), dried, and evaporated to give 18 (475 mg, 91%) as a
colorless gum: IR νmax (CHBr3) 2104, 1728 cm-1; NMR (CDCl3)
δ includes 1.02 (d, 3H, J ) 6 Hz, dCHCHCH3), 1.42 and 1.64
(2s, 9H each, t-BuO), 2.09 (s, 3H, AcO), 2.91 and 3.52 (2dd,
1H each, J ) 2.5, 13 and 7.5, 13 Hz, CH2N3), 3.81 (s, 1H, 4-OH),
4.12 (br s, 1H, 7-H), 4.78 (dd, 1H, J ) 2.5 and 7.5 Hz, 3-H),
4.97 and 5.0 (2s, 1H each, dCH2), 5.11 (d, 1H, J ) 5 Hz,
CHOAc), 5.8 (d, 1H, J ) 16 Hz, OCOCHdCH), 6.32 (br s, 1H,
6-H), 6.94 (dd, 1H, J ) 9 and 16 Hz, OCOCHdCH), 7.1-7.35
(m, 5H, Ph). Anal. (C49H77N3O12Si‚0.75C4H8O2) C, H, N.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-3-(a zi-
dom eth yl)-4,6,7-tr ih ydr oxy-2,8-dioxabicyclo[3.2.1]octan e-
4,5-d ica r boxylic Acid , 6-(4,6-Dim eth yl-2-octen oa te), 4,5-
Bis(1,1-d im eth yleth yl) Ester (19). A solution of 18 (453 mg,
0.49 mmol) in THF (10 mL) was stirred at room temperature
and treated with tetrabutylammonium fluoride (1 M, 0.5 mL).
After 70 min the solution was evaporated to dryness. The
residue was partitioned between EtOAc (100 mL) and water
(50 mL). The organic phase was washed with water (50 mL)
and brine (2 × 50 mL), dried, and evaporated to a colorless
gum. This was chromatographed on silica gel eluting with
EtOAc-cyclohexane (1:5). The required fractions were com-
bined and evaporated to give 19 (363 mg, 91%) as a colorless
gum: IR νmax (CHBr3) 2106, 1727 cm-1; NMR (CDCl3) δ
includes 0.8-0.9 (m, 9H, CH3), 1.05 (d, 3H, J ) 6 Hz,
dCHCHCH3), 1.5 and 1.58 (2s, 9H each, t-BuO), 2.1 (s, 3H,
AcO), 2.98 (d, 1H, J ) 2 Hz, 7-OH), 3.12 and 3.55 (2dd, 1H
each, J ) 5, 12.5 and 7.5, 12.5 Hz, CH2N3), 3.85 (s, 1H, 4-OH),
4.06 (d, 1H, J ) 2 Hz, 7-H), 4.65 (dd, 1H, J ) 5 and 7.5 Hz,
3-H), 4.95 and 4.98 (2s, 1H each, dCH2), 5.09 (d, 1H, J ) 5
Hz, CHOAc), 5.78 (d, 1H, J 16 Hz, OCOCHdCH), 5.92 (d, 1H,
J 2 Hz, 6-H), 6.91 (dd, 1H, J ) 16 and 9 Hz, OCOCHdCH),
7.1-7.3 (m, 5H, Ph); MS(FAB+ve) m/ z 814 (M + H)+; HRMS-
purified by preparative HPLC eluting with 70% CH3
CN-H2O
containing 0.1% TFA. The required fractions were combined
and concentrated by rotary evaporation. A white solid pre-
cipitated out which was collected by filtration, washed with
water, and dried under vacuum to provide 1e (61 mg, 38%):
NMR (DMSO-d6) δ includes 0.75-0.88 (m, 9H, CH3), 0.98 (d,
3H, J ) 6 Hz, dCHCHCH3), 2.1 (s, 3H, AcO), 2.85 (m, 2H,
CH2NH2), 3.82 (dd, 1H, J ) 2 and 5 Hz, 7-H), 4.58 (dd, 1H, J
) 4 and 6 Hz, 3-H), 4.9 (br s, 2H, dCH2), 4.98 (d, 1H, J ) 5
Hz, CHOAc), 5.75 (d, 1H, J ) 16 Hz, OCOCHdCH), 6.12 (d,
1H, J ) 2 Hz, 6-H), 6.72 (dd, 1H, J ) 9 and 16 Hz,
OCOCHdCH), 7.12-7.3 (m, 5H, Ph); MS(CI) m/ z 676 (M +
H)
+. Anal. (C35H49NO12‚0.1C2HF3O2‚1.2H2O) C, H, N.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-
[(d im e t h yla m in o)m e t h yl]-4,6,7-t r ih yd r oxy-2,8-d ioxa -
b icyclo[3.2.1]oct a n e-4,5-d ica r b oxylic Acid , 6-(4,6-Di-
m eth yl-2-octen oa te) (1f). A suspension of 1e (250 mg, 0.37
mmol) in CH3CN (12 mL) was stirred at 20 °C under nitrogen
and treated with glacial acetic acid (21 µL, 0.37 mmol),
aqueous 40% formaldehyde (280 µL, 3.7 mmol), and sodium
triacetoxyborohydride (235 mg, 1.1 mmol) to give a clear
solution. After 2 h the solution was evaporated to dryness.
The residue was stirred with EtOAc (10 mL) and filtered. The
filtrate was evaporated to a yellow foam which was purified
by preparative HPLC eluting with 70% CH3CN/H2O containing
0.1% TFA to give 1f (189 mg, 56%) as a white solid: NMR
(DMSO-d6) δ includes 0.75-0.9 (m, 9H, CH3), 0.99 (d, 3H, J )
6 Hz, dCHCHCH3), 2.1 (s, 3H, AcO), 2.81 (s, 6H, (CH3)2N),
3.99 (br d, 1H, J ) 3 Hz, 7-H), 4.7 (d, 1H, J ) 9 Hz, 3-H), 4.92
(br s, 2H, dCH2), 4.97 (d, 1H, J ) 5 Hz, CHOAc), 5.8 (d, 1H,
J ) 16 Hz, OCOCHdCH), 6.2 (br s, 6-H), 6.77 (dd, 1H, J ) 16
and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph). Anal. (C37H53
NO12‚1.5C2HF3O2‚2H2O) C, H, N, F.
-
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-
[[(a m in oca r b on yl)a m in o]m et h yl]-4,6,7-t r ih yd r oxy-2,8-
d ioxa bicyclo[3.2.1]octa n e-4,5-d ica r boxylic Acid , 6-(4,6-
Dim eth yl-2-octen oa te) (1g). A solution of 1e (93 mg, 0.14
mmol), phenyl carbamate (123 mg, 0.9 mmol), and triethyl-
amine (0.086 mL, 0.6 mmol) in THF (5 mL) was refluxed for
27 h and then evaporated to dryness. The residue was
partitioned between EtOAc (25 mL) and 2 M hydrochloric acid
(25 mL). The organic phase was washed with further acid (10
mL), water, and brine (2 × 25 mL each), dried, and evaporated
to give a white solid which was purified by HPLC eluting with
60% CH3CN-H2O containing 0.15 mL/L H2SO4 to give 1g as
a white solid (38 mg, 38%): NMR (DMSO-d6) 0.75-0.9 (m,
9H, CH3), 0.98 (d, 3H, J ) 7 Hz, dCHCHCH3), 2.09 (s, 3H,
AcO), 2.95 (m, 2H, CH2N), 3.85 (dd, 1H, J ) 2 and 5 Hz, 7-H),
4.38 (dd, 1H, J ) 5 and 7 Hz, 3-H), 4.91 (br s, 2H, dCH2),
4.96 (d, 1H, J ) 5 Hz, CHOAc), 5.77 (d, 1H, J ) 16 Hz,
OCOCHdCH), 6.21 (br s, 1H, 6-H), 6.73 (dd, 1H, J ) 16 and
8 Hz, OCOCHdCH), 7.12-7.32 (m, 5H, Ph); MS(FAB+ve) m/ z
719 (M + H)+. Anal. (C36H50N2O13‚0.5H2O) C, H, N.
(FAB+ve) found
814.4490.
814.4457, calcd
for
C43H64N3O12
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-ph en ylh exyl)-3-(am i-
n om eth yl)-4,6,7-tr ih ydr oxy-2,8-dioxabicyclo[3.2.1]octan e-
4,5-d ica r boxylic Acid , 6-(4,6-Dim eth yl-2-octen oa te), 4,5-
Bis(1,1-d im eth yleth yl) Ester (20). A solution of 19 (283 mg,
0.35 mmol) in THF (3 mL) and triphenylphosphine (100 mg,
0.38 mmol) was stirred and treated with water (0.6 mL).
Stirring was continued for 23 h at 44 °C, and then the solution
was evaporated. The residue was dissolved in EtOAc (100
mL), and the solution was washed with saturated aqueous
sodium bicarbonate (2 × 25 mL) and brine (2 × 25 mL), then
dried, and evaporated to a foam which was chromatographed
on silica eluting with MeOH-CHCl3 (1:19, 1:9). The required
fractions were combined and evaporated to give 20 (180 mg,
66%) as a colorless gum: NMR (CDCl3) δ includes 0.8-0.9 (m,
9H, CH3), 1.04 (d, 3H, J ) 6 Hz, dCHCHCH3), 1.50 and 1.56
(2s, 9H each, t-BuO), 2.1 (s, 3H, AcO), 2.92 (m, 2H, CH2NH2),
4.02 (d, 1H, J ) 2 Hz, 7-H), 4.4 (t, 1H, J 4 Hz, 3-H), 4.96 and
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-
[[(a m in oim in om et h yl)a m in o]m et h yl]-4,6,7-t r ih yd r oxy-
2,8-dioxabicyclo[3.2.1]octan e-4,5-dicar boxylic Acid, 6-(4,6-
Dim eth yl-2-octen oate), Hydr och lor ide Salt (1h ). A solution
of 1e (61.3 mg, 0.09 mmol) in MeOH (1 mL) was treated with

1420 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Procopiou et al.
triethylamine (0.051 mL, 0.36 mmol), followed by aminoimi-
nomethanesulfonic acid 21 (17 mg, 0.13 mmol), and the
mixture was stirred at 20 °C for 18 h. The solvent was
removed under reduced pressure, and the residue was parti-
tioned between EtOAc and 2 M hydrochloric acid and brine,
dried, and evaporated to dryness to give 1h (62 mg, 91%) as a
glass: NMR (DMSO-d6) 0.75-0.9 (m, 9H, CH3), 0.99 (d, 3H, J
) 7 Hz, dCHCHCH3), 2.10 (s, 3H, AcO), 3.18 (m, 2H, NCH2),
3.89 (br d, 1H, J ) 6 Hz, 7-H), 4.52 (br t, 1H, J ) 4 Hz, 3-H),
4.90 (s, 2H, dCH2), 4.96 (d, 1H, J ) 5 Hz, CHOAc), 5.79 (d,
1H, J ) 15 Hz, OCOCHdCH), 6.04 (d, 1H, J ) 6 Hz, 7-OH),
6.08 (br s, 1H, 6-H), 6.75 (dd, 1H, J )15 and 8 Hz,
3H, J ) 7 Hz, dCHCHCH3), 1.48 and 1.58 (2s, 9H each,
t-BuO), 2.11 (s, 3H, AcO), 2.88 (br, 1H, 7-OH), 3.25-3.40 (m,
2H, CH2NH), 3.83 (s, 1H, 4-OH), 4.01 (br s, 1H, 7-H), 4.60 (dd,
1H, J ) 12 and 6 Hz, 3-H), 4.98 and 5.00 (2s, 1H each, dCH2),
5.09 (d, 1H, J ) 5 Hz, CHOAc), 5.77 (d, 1H, J ) 16 Hz,
OCOCHdCH), 5.88 (m, 2H, 6-H, NH), 6.92 (dd, 1H, J ) 16
and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph). Anal. (C44H64F3-
NO14S) C, H, N, S.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-ph en ylh exyl)-3-[[[(tr i-
flu or om eth yl)su lfon yl]a m in o]m eth yl]-4,6,7-tr ih yd r oxy-
2,8-dioxabicyclo[3.2.1]octan e-4,5-dicar boxylic Acid, 6-(4,6-
Dim eth yl-2-octen oa te) (1i). A solution of the diester 24 (51
mg, 0.06 mmol) in 6.5 M hydrogen chloride in dioxane (3 mL)
was left to stand at 20 °C for 8 h and then evaporated to
dryness. The residue was purified by HPLC 65% CH3CN-
H2O containing 0.15 mL/L H2SO4 to give 1i (20 mg, 41%) as a
white solid: NMR (CD3OD) δ 1.02 (d, 3H, J ) 7 Hz,
dCHCHCH3), 2.10 (s, 3H, AcO), 2.69 (dd, 1H, J ) 14 and 6
Hz, CH2Ph), 3.1-3.5 (m, 2H, CH2NH), 4.00 (d, 1H, J ) 2 Hz,
7-H), 4.65 (dd, 1H, J ) 6 and 2 Hz, 3-H), 4.93 and 5.00 (2s,
1H each, dCH2), 5.05 (d, 1H, J ) 5 Hz, CHOAc), 5.80 (d, 1H,
J ) 16 Hz, OCOCHdCH), 6.30 (d, 1H, J ) 2 Hz, 6-H), 6.85
(dd, 1H, J ) 16 and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph).
Anal. (C36H48F3NO14S) C, H, N, S.
OCOCHdCH), 7.15 (m, 5H, Ph). Anal. (C36H51N3O12
0.5HCl‚H2O) C, H, N, Cl.
‚
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl)-3-
(am in om eth yl)-7-[[dim eth yl(1,1-dim eth yleth yl)silyl]oxy]-
4 ,6 -d i h y d r o x y -2 ,8 -d i o x a b i c y c l o [3 .2 .1 ]o c t a n e -4 ,5 -
d ica r boxylic Acid , 6-(4,6-Dim eth yl-2-octen oa te), 4,5-Bis-
(1,1-d im eth yleth yl) Ester (22). A stirred solution of the
azide 18 (655 mg, 0.71 mmol) in THF (7 mL) was treated with
triphenylphosphine (202 mg, 0.77 mmol) and water (1.2 mL)
at 45 °C. Aqueous sodium bicarbonate was added (25 mL),
and the mixture was extracted with EtOAc (3 × 50 mL). The
organic phase was washed with sodium bicarbonate (50 mL)
and brine (2 × 50 mL), dried, and chromatographed eluting
with MeOH-CHCl3 (1:49) to give 22 (518 mg, 81%) as a white
foam: νmax (CHBr3) 3400, 3250, 1728 cm-1; NMR (CDCl3) 0.99
(d, 3H, J ) 7 Hz, dCHCHCH3), 1.36 and 1.57 (2s, 9H each,
t-BuO), 2.07 (s, 3H, AcO), 2.84 (br t, 2H, CH2N), 4.07 (d, 1H,
J ) 2 Hz, 7-H), 4.45 (t, 1H, J ) 4 Hz, 3-H), 4.93 and 4.97 (2s,
dCH2), 5.08 (d, 1H, J ) 5 Hz, CHOAc), 5.75 (d, 1H, J ) 16
Hz, OCOCHdCH), 6.29 (d, 1H, J ) 2 Hz, 6-H), 6.90 (dd, 1H,
J ) 16 and 9 Hz, OCOCHdCH), 7.1-7.3 (m, 5H, Ph); MS-
(FAB+ve) m/ z 902 (M + H)+; HRMS(FAB+ve) found 902.5482,
calcd for C49H80NO12Si 902.5449.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Ace t yloxy)-5-m e t h yl-3-m e t h yle n e -6-p h e n ylh e xyl)-7-
[[d im eth yl(1,1-d im eth yleth yl)silyl]oxy]-4,6-d ih yd r oxy-3-
[ [ ( t r i f l u o r o m e t h y l ) s u l f o n y l ] a m i n o m e t h y l ] -2 ,8 -
d ioxa bicyclo[3.2.1]octa n e-4,5-d ica r boxylic Acid , 6-(4,6-
Dim eth yl-2-octen oa te), 4,5-Bis(1,1-d im eth yleth yl) Ester
(23). A solution of the amine 22 (164 mg, 0.18 mmol) in CH2-
Cl2 (4 mL) was treated with 2,4,6-collidine (0.234 mL, 1.8
mmol) and then trifluoromethanesulfonic anhydride (0.2 mL,
1.2 mmol) with stirring in an ice bath under nitrogen. After
20 min the solution was diluted with EtOAc (100 mL), washed
with 2 M hydrochloric acid (100, 50 mL) and brine (100, 50
mL), dried, and chromatographed eluting with EtOAc-cyclo-
hexane (1:6). The required fractions were combined and
evaporated to a colourless gum (87 mg, 46%): νmax (CHBr3)
1727, 1200, 1187 cm-1; NMR (CDCl3) δ includes 1.02 (d, 3H,
J ) 7 Hz, dCHCHCH3), 1.39 and 1.63 (2s, 9H each, t-BuO),
2.11 (s, 3H, AcO), 3.11-3.36 (m, 2H, CH2NH), 3.80 (s, 1H,
4-OH), 4.11 (d, 1H, J ) 2 Hz, 7-H), 4.68 (dd, 1H, J ) 4 and 8
Hz, 3-H), 4.99 and 5.00 (2s, 1H each, dCH2), 5.12 (d, 1H, J )
5 Hz, CHOAc), 5.80 (m, 2H, NH and OCOCHdCH), 6.29 (d,
1H, J ) 2 Hz, 6-H), 6.92 (dd, 1H, J ) 16 and 9 Hz,
OCOCHdCH), 7.1-7.3 (m, 5H, Ph); MS(CI) 1052 (M + NH4)+;
HRMS(FAB+ve) found 1034.4932, calcd for C50H79F3NO14SSi
1034.4942.
[1S-[1r(4R*,5S*),3r,4â,5r,6r,7â]]-1-[4-(Acetyloxy)-5-m eth -
yl-3-m et h ylen e-6-p h en ylh exyl]-3-(a m in om et h yl)-4,6,7-
t r ih yd r oxy-2,8-d ioxa b icyclo[3.2.1]oct a n e-4,5-d ica r b ox-
ylic Acid (2e). A solution of 1e (100 mg, 0.15 mmol) in DMF
(0.5 mL) was treated with N-methylhydroxylamine hydrochlo-
ride (28 mg, 0.33 mmol) and triethylamine (110 µL, 0.76
mmol). The mixture was stirred for 7 h at 20 °C, and then
the solvent was removed by rotary evaporation. The resulting
white solid was purified by preparative HPLC eluting with
35% CH3CN-H2O containing 0.1% TFA to give 2e as a white
solid (57 mg, 57%): NMR (DMSO-d6) δ includes 0.78 (d, 3H,
J ) 6 Hz, CHCH3), 2.1 (s, 3H, AcO), 3.9 (br d, 1H, J ) 4 Hz,
7-H), 4.48 (br d, 1H, J ) 6 Hz, 3-H), 4.85 and 4.91 (2s, 3H,
dCH2 and 6-H), 4.99 (d, 1H, J ) 5 Hz, CHOAc), 7.1-7.3 (m,
5H, Ph); MS(FAB+ve) m/ z 524 (M + H)+. Anal. (C25H33
NO11‚C2HF3O2‚1.5 H2O) C, H, N.
-
[1S -[1r(4R *,5S *),3r,4â,5r,6r,7â]]-3-Ac e t y l-1-[(4-
a cet yloxy)-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl]-4,6,7-
t r ih yd r oxy-2,8-d ioxa b icyclo[3.2.1]oct a n e-4,5-d ica r b ox-
ylic Acid (2c). 2c was similarly prepared from 1c (50 mg,
0.072 mmol) to give after HPLC purification (30-90% CH3-
CN-H2O containing 0.1% TFA) a white solid (16.6 mg, 43%):
NMR (CD3OD) δ includes 0.88 (d, 3H, J ) 6.3 Hz, dCHCHCH3),
2.10 (s, 3H, AcO), 2.20 (s, 3H, COCH3), 4.04 (d, 1H, J ) 2 Hz,
7-H), 4.96 (s, 1H, 3-H), 4.99 and 5.02 (2s, 1H each, dCH2),
5.10 (m, 2H, CHOAc and 6-H), 7.1-7.3 (m, 5H, Ph); MS(FAB-
ve) m/ z 535 (M - H)-; MS(TSP+ve) m/ z 554 (M + NH4)+.
Anal. (C26H32O12‚2H2O) C, H.
[1S-[1r(4R*,5S*),3r,4â,5r,6r,7â]]-1-[4-(Acetyloxy)-5-m eth -
yl-3-m eth ylen e-6-p h en ylh exyl]-3-(m eth oxym eth yl)-4,6,7-
tr ih yd r oxy-2,8-d ioxa bicyclo[3.2.1]octa n e-4,5-d ica r boxy-
lic Acid (2d ). Similarly, 2d was prepared from 1d (41 mg,
0.06 mmol) to give after HPLC purification (35% CH3CN-H2O
containing 0.1% TFA) a white solid (13.9 mg, 43%): NMR
(D2O) δ includes 0.89 (d, 3H, J ) 7 Hz, dCHCHCH3), 2.18 (s,
3H, AcO), 3.32 (s, 3H, CH2OCH3), 3.4-3.6 (m, 2H, CH2OCH3),
4.08 (d, 1H, J ) 2 Hz, 7-H), 4.55-4.6 (m, 1H, 3-H), 4.93 (d,
1H, J ) 5 Hz, CHOAc), 5.00 and 5.04 (2s, 1H each, dCH2),
5.18 (d, 1H, J ) 2 Hz, 6-H), 7.2-7.4 (m, 5H, Ph); MS(FAB-ve)
m/ z 537 (M - H)-. Anal. (C26H34O12‚2H2O) C, H.
[1S-[1r(4R*,5S*),3r,4â,5r,6r,7â]]-1-[4-(Acetyloxy)-5-m eth -
yl-3-m e t h yle n e -6-p h e n ylh e xyl]-3-[[(a m in oca r b on yl)-
a m in o]m eth yl]-4,6,7-tr ih yd r oxy-2,8-d ioxa bicyclo[3.2.1]-
octa n e-4,5-d ica r boxylic Acid (2g). 2g was similarly pre-
pared from 1g (25 mg, 0.03 mmol) to give after HPLC
purification (35% CH3CN-H2O containing 0.1% TFA) a white
solid (2 mg, 12%): NMR (CD3OD) δ includes 0.85 (d, 3H, J )
6 Hz, CHCH3), 2.1 (s, 3H, AcO), 2.7 (m, 2H, CH2N), 4.02 (d,
1H, J ) 2 Hz, 7-H), 4.98 (s, 2H dCH2), 5.07 (d, 1H, J ) 5 Hz,
CHOAc), 5.1 (br s, 1H, 6-H), 7.1-7.3 (m, 5H, Ph); MS(LSI-ve)
m/ z 565 (M - H)-.
[1S -[1r(4R *,5S *),3r,4â,5r,6r(2E ,4R *,6R *),7â]]-1-[4-
(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-ph en ylh exyl)-3-[[[(tr i-
flu or om eth yl)su lfon yl]a m in o]m eth yl]-4,6,7-tr ih yd r oxy-
2,8-dioxabicyclo[3.2.1]octan e-4,5-dicar boxylic Acid, 6-(4,6-
Dim eth yl-2-octen oa te), 4,5-Bis(1,1-d im eth yleth yl) Ester
(24). A solution of the silyl ether 23 (80 mg, 0.08 mmol) in
THF (2 mL) was stirred at 20 °C and treated with a THF
solution of Bu4NF (1 M, 0.077 mL). After 30 and 60 min
further quantities of Bu4NF (2 × 0.077 mL) were added. After
3 h the reaction mixture was evaporated to dryness, and the
residue was dissolved in EtOAc. The solution was washed
with water and brine, dried, and chromatographed eluting
with EtOAc-cyclohexane (1:4). The required fractions were
combined and evaporated to a white foam (58 mg, 79%): νmax
(CHBr3) 1726, 1186 cm-1; NMR (CDCl3) δ includes 1.03 (d,

Squalestatins: Inhibitors of Squalene Synthase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1421
(4) Sidebottom, P. J .; Highcock, R. M.; Lane, S. J .; Procopiou, P. A.;
Watson, N. S. The Squalestatins, Novel Inhibitors of Squalene
Synthase Produced by a Species of Phoma II. Structure Elucida-
tion. J . Antibiot. 1992, 45, 648-658.
[1S-[1r(4R*,5S*),3r,4â,5r,6r,7â]]-1-[4-(Acetyloxy)-5-m eth -
yl-3-m eth ylen e-6-p h en ylh exyl)-3-[[[(tr iflu or om eth yl)su l-
fon yl]a m in o]m eth yl]-4,6,7-tr ih yd r oxy-2,8-d ioxa bicyclo-
[3.2.1]octa n e-4,5-d ica r boxylic Acid (2i). Similarly, 2i was
prepared from 1i (59 mg, 0.073 mmol) to give after HPLC
purification (48% CH3CN-H2O containing 0.1% TFA) a white
solid (25 mg, 52%): NMR (CD3OD) includes 0.83 (d, 3H, J )
7 Hz, CHCH3), 2.70 (dd, 1H, J ) 14 and 6 Hz, CH2Ph), 3.1-
3.45 (m, 2H, CH2NH), 4.01 (d, 1H, J ) 2 Hz, 7-H), 4.55 (d,
1H, J ) 10 Hz, 3-H), 4.95 and 4.99 (2s, 1H each, dCH2), 5.07
(d, 1H, J ) 5 Hz, CHOAc), 5.29 (d, 1H, J ) 2 Hz, 6-H), 7.1-
7.3 (m, 5H, Ph). Anal. (C26H32F3NO13S‚1.5H2O) C, H, N.
An im a ls. The rats used in these studies were juvenile,
male CD (Charles River) weighing ∼150 g, fed on standard
laboratory chow, and allowed access to water ad libitum. The
marmosets used were adults between 15 and 28 months of age,
and fed a standard primate diet supplemented with fruit.
Ra t SQS Assa y. The enzyme assay procedures measured
the conversion of [2-14C]FPP to [14C]squalene. The assay
methodology is described in detail in our earlier publication.28
Mea su r em en t of Ch olester ol Biosyn th esis in Vivo in
Ra ts. Squalestatins were dosed iv to rats (n ) 8 per group)
followed immediately by intraperitoneal administration of
[1-14C]acetate (250 µCi/kg). One group of rats were killed after
1 h, and another after 6 h, the livers removed, and a sample
of 0.5 g was saponified in alcoholic KOH at 80 °C for 1 h. After
extraction [14C]cholesterol was separated by HPLC using a
Spherisorb ODS-2 column eluted with methanol-2-propanol
(4:1). The [1-14C]cholesterol was quantified using the proce-
dure described previously.5
Tim e Cou r se Stu d ies in Ra ts. Compounds were admin-
istered iv at time 0, and the rats sacrificed 1, 2, 4, and 7 h
later. One hour prior to sacrifice [1-14C]acetate (250 µCi/kg)
was administered ip. Rats were killed by CO2 asphyxiation
and the liver dissected free. A 0.5 g sample of liver was
saponified and the [1-14C]cholesterol separated and quantified
as above.
Effect on Ser u m Ch olester ol Levels in Ma r m osets.
Marmosets of mixed sex (n ) 6) were allocated to treatment
groups on the basis of their fasting serum cholesterol levels
such that the mean and distribution of serum cholesterol levels
were similar for each group. The assay is described in detail
in our earlier publication.6 Squalestatins were converted to
their potassium salts and dissolved in water before adminis-
tration. One control group of animals (n ) 6) were dosed with
vehicle, and another group (n ) 6) were dosed with S1 (1 mg/
kg). Serum cholesterol levels were determined at days 0, 2,
3, and 7 postdosing. All results are expressed as the mean of
the change in serum cholesterol levels from predose values
((SEM).
(5) Baxter, A.; Fitzgerald, B. J .; Hutson, J . L.; McCarthy, A. D.;
Motteram, J . M.; Ross, B. C.; Sapra, M.; Snowden, M. A.; Watson,
N. S.; Williams, R. J .; Wright, C. Squalestatin 1, a Potent
Inhibitor of Squalene Synthase, Which Lowers Serum Choles-
terol in Vivo. J . Biol. Chem. 1992, 267, 11705-11708.
(6) Procopiou, P. A.; Bailey, E. J .; Bamford, M. J .; Craven, A. P.;
Dymock, B. W.; Houston, J . G.; Hutson, J . L.; Kirk, B. E.;
McCarthy, A. D.; Sareen, M.; Scicinski, J . J .; Sharratt, P. J .;
Snowden, M. A.; Watson, N. S.; Williams, R. J . The Squale-
statins: Novel Inhibitors of Squalene Synthase. Enzyme Inhibi-
tory Activities and in Vivo Evaluation of C1-Modified Analogues.
J . Med. Chem. 1994, 37, 3274-3281.
(7) Bergstrom, J . D.; Kurtz, M. M.; Rew, D. J .; Amend, A. M.;
Karkas, J . D.; Bostedor, R. G.; Bansal, V. S.; Dufresne, C.;
VanMiddlesworth, F. L.; Hensens, O. D.; Liesch, J . M.; Zink, D.
L.; Wilson, K. E.; Onishi, J .; Milligan, J . A.; Bills, G.; Kaplan,
L.; Nallin Omstead, M.; J enkins, R. G.; Huang, L.; Meinz, M.
S.; Quinn, L.; Burg, R. W.; Kong, Y. L.; Mochales, S.; Mojena,
M.; Martin, I.; Pelaez, F.; Diez, M. T.; Alberts, A. W. Zaragozic
Acids: A Family of Fungal Metabolites that are Picomolar
Competitive Inhibitors of Squalene Synthase. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 80-84.
(8) Hensens, O. D.; Dufresne, C.; Liesch, J . M.; Zink, D. L.; Reamer,
R. A.; VanMiddlesworth, F. The Zaragozic Acids: Structure
Elucidation of a New Class of Squalene Synthase Inhibitors.
Tetrahedron Lett. 1993, 34, 399-402.
(9) Hasumi, K.; Tachikawa, K.; Sakai, K.; Marakawa, S.; Yoshikawa,
N.; Kumazawa, S.; Endo, A. Competitive Inhibition of Squalene
Synthase by Squalestatin 1. J . Antibiot. 1993, 46, 689-691.
(10) Procopiou, P. A.; Bailey, E. J .; Hutson, J . L.; Kirk, B. E.; Sharratt,
P. J .; Spooner, S. J .; Watson, N. S. The Squalestatins: Novel
Inhibitors of Squalene Synthase. The Optimal C1 Chain-length
Requirements. BioMed. Chem. Lett. 1993, 3, 2527-2532.
(11) Watson, N. S.; Bell, R.; Chan, C.; Cox, B.; Hutson, J . L.; Keeling,
S. E.; Kirk, B. E.; Procopiou, P. A.; Steeples, I. P.; Widdowson,
J . The Squalestatins: Potent Inhibitors of Squalene Synthase.
The Role of the Tricarboxylic Acid Moiety. BioMed. Chem. Lett.
1993, 3, 2541-2546.
(12) Giblin, G. M. P.; Bell, R.; Hancock, A. P.; Hartley, C. D.; Inglis,
G. G. A.; Payne, J . J .; Procopiou, P. A.; Shingler, A. H.; Smith,
C.; Spooner, S. J . Semisynthetic Squalestatins: Squalene Syn-
thase Inhibition and Antifungal Activity. The SAR of C6 and
C7 Modifications. BioMed. Chem. Lett. 1993, 3, 2605-2610.
(13) Lester, M. G.; Giblin, G. M. P.; Inglis, G. G. A.; Procopiou, P A.;
Ross, B. C.; Watson, N. S. Structurally simplified Squale-
statins: A Convenient Route to a 6,7-Unsubstituted Derivative.
Tetrahedron Lett. 1993, 34, 4357-4360.
(14) Chan, C.; Inglis, G. G. A.; Procopiou, P. A.; Ross, B. C.; Srikantha,
A. R. P.; Watson, N. S. The Squalestatins: C-3 Decarboxylation
Studies and Rearrangement to the 6,8-Dioxabicyclo[3.2.1]octane
Ring System. Tetrahedron Lett. 1993, 34, 6143-6146.
(15) Chan, C.; Andreotti, D.; Dymock, B. W.; Keeling, S. E.; Procopiou,
P. A.; Ross, B. C.; Sareen, M.; Sharratt, P. J .; Snowden, M. A.;
Watson, N. S. The Squalestatins: Decarboxy and 4-Deoxy
Analogues as Potent Squalene Synthase Inhibitors. J . Med.
Chem. 1996, 39, 207-216.
(16) Sharratt, P. J .; Hutson, J . L.; Inglis, G. G. A.; Lester, M. G.;
Procopiou, P. A.; Watson, N. S. Structurally Simplified Squale-
statins: Monocyclic 1,3-Dioxane Analogues. BioMed. Chem. Lett.
1994, 4, 661-666.
(17) Andreotti, D.; Procopiou, P. A.; Watson, N. S. The Squale-
statins: Cleavage of the Bicyclic Core via the Novel 2,8,9-
Trioxabicyclo[3.3.1]nonane Ring System. Tetrahedron Lett. 1994,
35, 1789-1792.
(18) Procopiou, P. A.; Bailey, E. J .; Chan, C.; Inglis, G. G. A.; Lester,
M. G.; Srikantha, A. R. P.; Sidebottom, P. J .; Watson, N. S. The
Squalestatins: Cleavage of the Bicyclic Core via the Novel 6,8-
dioxabicyclo[3.2.1]octane Ring System. J . Chem. Soc., Perkin
Trans. 1 1995, 1341-1347.
(19) Cox, B.; Hutson, J . L.; Keeling, S. E.; Kirk, B. E.; Srikantha, A.
R. P.; Watson, N. S. The Squalestatins: Potent Inhibitors of
Squalene Synthase, 3-hydroxymethyl Derivatives. BioMed. Chem.
Lett. 1994, 4, 1931-1936.
(20) Bamford, M. J .; Chan, C.; Craven, A. P.; Dymock, B. W.; Green,
D.; Henson, R. A.; Kirk, B. E.; Lester M. G.; Procopiou, P. A.;
Snowden, M. A.; Spooner, S. J .; Srikantha, A. R. P.; Watson, N.
S.; Widdowson, J . A. The Squalestatins: Synthesis and Biologi-
cal Activity of Some C3-Modified Analogues; Replacement of a
Carboxylic Acid or Methyl Ester with an Isoelectronic Hetero-
cyclic Functionality. J . Med. Chem. 1995, 38, 3502-3513.
(21) Lester, M. G.; Evans, G. L.; Henson, R. A.; Procopiou, P. A.;
Sareen, M.; Snowden, M. A.; Spooner, S. J .; Srikantha, A. R. P.;
Watson, N. S. The Squalestatins: Effects of Changes at the
Allylic Centre in the C1 sidechain. BioMed. Chem. Lett. 1994,
4, 2683-2688.
Ack n ow led gm en t. We are indebted to the Struc-
tural Chemistry Department, Glaxo Research and De-
velopment (Greenford), for spectral and analytical data,
to Mrs. Surriya Faulkes and Mrs. J oselyn F. Roberts of
Molecular Science Department for expert technical
assistance in conducting the rat enzyme inhibition
assays, and to Dr. Mark J . Bamford for providing us
with a sample of compound 21.
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J M950893J