The squalestatins: Decarboxy and 4-deoxy analogues as potent squalene synthase inhibitors

Article abstract of DOI:10.1021/jm9504969

Squalestatins without either the hydroxy group at C-4 or the carboxylic acid at C-3 or C-4 were prepared and evaluated for their ability to inhibit rat liver microsomal squalene synthase (SQS) in vitro. These modifications were well tolerated for compounds with the 4,6-dimethylectenoate ester at C- 6 (S1 series). However in analogues without the C-6 ester (H1 series), removal of the C-4 hydroxy group gave compounds with reduced potency, whereas decarboxylation at C-3 resulted in a dramatic loss of SQS inhibitory activity. In comparison with S1 1, C-4 deoxyS1 3 and C-3 decarboxyS1 10 have shorter in vivo durations of action on the inhibition of hepatic cholesterol biosynthesis in rats. C-4 deoxyS1 3 retains good serum cholesterol-lowering ability in marmosets, while C-3 decarboxyS1 10 showed only a marginal effect even at high dose.

Full text of DOI:10.1021/jm9504969

J . Med. Chem. 1996, 39, 207-216
207
Th e Squ a lesta tin s: Deca r boxy a n d 4-Deoxy An a logu es a s P oten t Squ a len e
Syn th a se In h ibitor s1
Chuen Chan,*,† Daniele Andreotti,† Brian Cox,† Brian W. Dymock,† J ulie L. Hutson,‡ Suzanne E. Keeling,†
Alun D. McCarthy,‡ Panayiotis A. Procopiou,† Barry C. Ross,† Meenu Sareen,‡ J an J . Scicinski,†
Peter J . Sharratt,† Michael A. Snowden,‡ and Nigel S. Watson†
GlaxoWellcome Research & Development, Medicines Research Centre, Gunnels Wood Road,
Stevenage, Hertfordshire SG1 2NY, U.K.
Received J uly 6, 1995X
Squalestatins without either the hydroxy group at C-4 or the carboxylic acid at C-3 or C-4
were prepared and evaluated for their ability to inhibit rat liver microsomal squalene synthase
(SQS) in vitro. These modifications were well tolerated for compounds with the 4,6-
dimethyloctenoate ester at C-6 (S1 series). However in analogues without the C-6 ester (H1
series), removal of the C-4 hydroxy group gave compounds with reduced potency, whereas
decarboxylation at C-3 resulted in a dramatic loss of SQS inhibitory activity. In comparison
with S1 1, C-4 deoxyS1 3 and C-3 decarboxyS1 10 have shorter in vivo durations of action on
the inhibition of hepatic cholesterol biosynthesis in rats. C-4 deoxyS1 3 retains good serum
cholesterol-lowering ability in marmosets, while C-3 decarboxyS1 10 showed only a marginal
effect even at high dose.
Sch em e 1
Atherosclerosis is a complex and progressive, multi-
factorial vascular disease2 that leads to narrowing and
occulsion of arterial vessels. Its clinical manifestations
include angina, hemorrhage, thrombosis, and cerebral
and myocardial infarction. Hypercholesterolemia and
hyperlipoproteinemia are major risk factors3,4 in the
progression of the disease, and a direct correlation
between low-density lipoprotein-cholesterol (LDL-C)
and the incidence of coronary heart disease has been
demonstrated.5 Over 70% of cholesterol in the body is
derived from de novo cholesterol biosynthesis, inhibition
of which is currently the most effective clinical means
of reducing plasma LDL-C levels.6 3-Hydroxy-3-meth-
ylglutaryl coenzyme A reductase (HMGR) inhibitors7
are currently the most effective therapeutic agents for
the control of hypercholesterolemia. Although no major
toxic effects are associated with this class of agents,
mevalonate, the product of HMGR catalysis, is a com-
mon precursor to all isoprenoids such as dolichol and
ubiquinone (Scheme 1). Squalene synthase (SQS) (far-
nesyl-diphosphate:farnesyl-diphosphate farnesyltrans-
ferase, EC 2.5.1.21), an enzyme that catalyzes the head
to head condensation of farnesyl diphosphate (FPP) to
presqualene diphosphate (PSPP) and its subsequent
rearrangement to squalene, is the first enzyme com-
mitted to sterol biosynthesis.8 Inhibitors of SQS are
therefore attractive in the regulation of steroidal genesis
as nonsterol pathways should be minimally affected.
an IC50 value of 26 nM against rat SQS and lowers
serum cholesterol level by 56% in marmosets when
adminstered iv for 7 days at 1 mg/kg/day.14 As part of
Recently we reported the isolation9 and structural
elucidation10 of the squalestatins, a novel class of highly
potent SQS inhibitors; squalestatin (S1, 1)11,12 possesses
an IC50 value of 12 nM against the rat enzyme and
lowers serum cholesterol levels by up to 75% when
administered 100 mg/kg/day for 7 days po to marmo-
sets13 and up to 86% at 1 mg/kg/day iv.14 We have also
reported on the biological activities of H1 (2), which has
our medicinal chemistry program to identify the requi-
site structural features for biological activity, we have
reported on modifications to both the C-1 side chain14,15
and the substituents at C-6 and C-716 and the role of
the 2,8-dioxabicyclo[3.2.1]octane moiety.17 The group
at Merck have also reported on modifications to C-1 and
C-6 side chains.18 In our recent communication19 de-
scribing a series of methyl esters of both S1 (1) and H1
(2), we reported that the C-5 carboxylic acid is essential
† Department of Medicinal Chemistry.
‡ Department of Molecular Science.
X Abstract published in Advance ACS Abstracts, November 15, 1995.
0022-2623/96/1839-0207$12.00/0 © 1996 American Chemical Society

208 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Chan et al.
Ch a r t 1
for SQS inhibitory activity and that, in the S1 series,
carboxylic acid groups are not required at C-3 or C-4
for potent SQS inhibitory activity to be retained. In
contrast C-3 and C-4 monomethyl esters of H1 show
reduced enzyme inhibitory activity compared with that
for the parent tricarboxylic acid H1 (2). Moreover C-3
hydroxymethyl analogues in the S1 series have been
shown to retain potent SQS inhibitory activities, whereas
the corresponding C-3 hydroxymethylH1 analogue pos-
sesses reduced enzyme inhibitory activity.20 Herein we
report on the synthesis of C-4 deoxy (3-7) and C-4
decarboxy (8 and 9) analogues together with their
biological evaluation and that of the corresponding C-3
decarboxy analogues21 (10 and 11). Differences in
structure-activity relationships (SAR) between the C-6
acyl and C-6 desacyl analogues derived for inhibition
of SQS and the in vivo activities of 4-deoxyS1 3 and
3-decarboxyS1 10 in comparison with S1 1 are high-
lighted.
determined as their C-7 hydroxy derivatives 17 and 18,
which were obtained by cleavage of the C-7 THP ether
with methanol in the presence of PPTS or Amberlyst-
15. Coupling constants from 1H-NMR spectroscopy
clearly established the identities of C-4 deoxyS1 (major)
and C-4 epideoxyS1 (minor) tri-tert-butyl esters 17 and
18, respectively. In the major isomer, the coupling
constant (11 Hz) between H-3 (δ 4.90) and H-4 (δ 2.95)
clearly indicated their diaxial (anti) relationship, thereby
establishing its structural identity as C-4 deoxyS1 tri-
tert-butyl ester 17. In the spectrum for the minor
isomer, the coupling constant of 4 Hz between H-3 (δ
4.67) and H-4 (δ 3.30) protons established their syn
relationship and confirmed the structure as C-4 epideox-
yS1 tri-tert-butyl ester 18.
Acid hydrolysis of 17 with formic acid gave C-4
deoxyS1 3. A minor byproduct was also isolated which
was identified as C-7 formyl C-4 deoxyS1 19. Removal
of the C-6 side chain in 3 with N-methylhydroxylamine16b
gave C-4 deoxyH1 4. The minor epimer 18 was simi-
larly treated to give C-4 epideoxyS1 5, C-7 formyl C-4
epideoxyS1 20, and C-4 epideoxyH1 6. Deacetylation
of C-4 deoxyS1 3 with concentrated HCl in methanol14
gave C-4 deoxyS2 7.
For the synthesis of C-4 decarboxysqualestatins 8 and
9 (Scheme 3), C-7 TBDMS-S1 tri-tert-butyl ester 21 was
employed which was readily prepared by silylation of
S1 tri-tert-butyl ester 1214 in quantitative yield. Selec-
tive acid hydrolysis19a gave the corresponding C-4 acid
22 (51%). Oxidative decarboxylation of 22 using N-ethyl-
2-chlorobenzoxazolium tetrafluoroborate and triethyl-
amine24 followed by reduction with sodium borohydride
provided access to the C-4 decarboxyS1 di-tert-butyl
ester 23 and its C-4 epi derivative 24 in overall yields
of 26% and 13%, respectively.25 The stereochemistry
Ch em istr y
The syntheses of C-3 decarboxysqualestatins 10 and
11 have been reported previously.21 In the preparation
of C-4 deoxysqualestatins 3-7 (Scheme 2), the readily
available S1 tri-tert-butyl ester 1214 was protected as
its C-7 THP ether 13 (85%). Deprotonation with NaH
followed by acylation with methyl oxalyl chloride gave
the C-4 methyl oxalate 14 in excellent yield (95%). Free
radical reduction22 with tri-n-butylstannane in the
presence of AIBN gave an epimeric mixture of C-4
deoxyS1 tri-tert-butyl esters 15 and 16 in the ratio of
4:123 in a total yield of 40% which were separable by
flash column chromatography. To avoid complications
from the diastereoisomeric pairs derived from the THP
protecting group, the identities of these epimers were

Decarboxy- and Deoxysqualestatins as SQS Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 209
Sch em e 2. Synthesis of C-4 Deoxysqualestatinsa
a
Conditions: (a) DHP, PPTS, CH2Cl2; (b) NaH, MeOCOCOCl, THF, room temperature; (c) n-Bu3SnH, AIBN, xylene, 90 °C; (d) PPTS,
MeOH, 60 °C; (e) Amberlyst-15, MeOH, room temperature; (f) HCO2H, room temperature; (g) Et3N, MeNHOH‚HCl, DMF, room temperature;
(h) concentrated HCl, MeOH.
of these epimeric alcohols was established by NMR
spectroscopy. NOE experiments on the isomer in which
H-6 resonates at δ 5.65 showed signal enhancement to
H-3 (δ 4.36) on irradiation of H-6 indicating their close
spatial proximity. The coupling constant of 10 Hz
between H-3 and H-4 (δ 3.88) confirmed their trans
diaxial relationship, thereby establishing its structure
as the C-4 epidecarboxyS1 di-tert-butyl ester 24.
A
similar NOE experiment on the other isomer showed
signal enhancements to H-3 (δ 4.72) and H-4 (δ 4.17)
on irradiation of H-6 (δ 5.45). These data together with
their coupling constant of 2 Hz confirmed the axial-
equatorial relationship of H-3 and H-4, thereby estab-
lishing its identity as the C-4 decarboxy isomer 23.
Fluoride-induced desilylation and hydrolysis with formic
acid thereby gave C-4 decarboxyS1 8 and its epimer 9.
Biologica l Resu lts a n d Discu ssion
The abilities of C-3 decarboxy-, C-4 deoxy-, and C-4
decarboxysqualestatins to inhibit the conversion of
[2-14C]farnesyl diphosphate (FPP) to [14C]squalene by
rat liver microsomal SQS were determined using our
published assay procedures (Table 1).13,26 As reported
previously,9,13,14 both S1 (1) and H1 (2) are highly potent
inhibitors of SQS. In the S1 series, compounds without
the carboxylic acid moieties at C-3 or C-4 (8-10) retain

210 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Sch em e 3. Synthesis of C-4 Decarboxysqualestatinsa
Chan et al.
a
Conditions: (a) TBDMSCl, imidazole, DMF, 65 °C; (b) HCl-dioxane; (c) 2-chloro-3-ethylbenzoxazolium tetrafluoroborate, Et3N, CH2Cl2;
(d) NaBH4, EtOH; (e) aqueous HF-MeCN; (f) HCO2H, room temperature; (g) TBAF, THF.
Ta ble 1. In Vitro Rat SQS Inhibitory Activities of
However, in the H1 series, C-3 decarboxyH1 11 is
without significant SQS inhibitory activity, supporting
Squalestatinsa
compd
no.
relative
our view that an acidic group at C-3 is essential for
maximal enzyme inhibitory activity in this series.19a
Removal of the C-4 hydroxyl in H1, to provide C-4
deoxyH1 4 and C-4 epideoxyH1 6, also resulted in a
significant loss of enzyme inhibitory activity, consistent
with our previous observations14,19a,20 that modifications
to the C-1 side chain and tricarboxylic acid moiety in
H1 substantially reduce biological activity. These dif-
ferences in SAR between S1 and H1 series show that
the C-6 dimethyloctenoate side chain critically affects
the in vitro SQS inhibitory activity for modifications
made in other parts of the squalestatin molecule. We
have suggested previously19a that analogues of S1 mimic
the biosynthetic intermediate PSPP while the related
H1 derivatives are FPP mimetics; in both series the
highly functionalized 2,8-dioxabicyclo[3.2.1]octane ring
system acts as a diphosphate mimetic.
trivial name
formulab
potencyc
S1 Series
1
3
5
7
8
S1
C
C
C
C
C
C
C
35H46O14
35H46O13‚H2O
1
1
C-4 deoxyS1
C-4 epideoxyS1
C-4 deoxyS2
C-4 decarboxyS1
C-4 epidecarboxyS1
C-3 decarboxyS1
35H46O13‚2H2O
33H44O12‚1.5H2O
34H46O12‚H2O
0.1
2
0.2
0.2
0.5
9
10
34H46O12‚1.3H2O
d
34H46O12
H1 Series
2
4
6
H1
C
C
C
C
25H30O13
0.5
C-4 deoxyH1
C-4 epideoxyH1
C-3 decarboxyH1
25H30O12‚2.5H2O
0.04
0.02
0.0005
25H30O12‚2.5H2O
d
11
24H30O11
a
Squalestatins were tested for their ability to inhibit the
conversion of [2-14C]farnesyl pyrophosphate (FPP) to [14C]squalene
by rat liver microsomal SQS using our published procedure.13,26
b
Unless otherwise stated, all compounds gave satisfactory mi-
croanalysis. c The concentration of test compounds required to
inhibit the FPP-squalene conversion by 50% was expressed as
an IC50 value which was determined on at least two different
occasions in duplicate with a minimum of five and a maximum of
eight dose levels. The relative potency of the test compound is
expressed as the quotient of the IC50 determined for S1 (typical
IC50 ) 12 ( 5 nM) divided by the IC50 for the test compound.
Having demonstrated their potent SQS inhibitory
activities, the potassium salts of C-4 deoxyS1 3 and C-3
decarboxyS1 10 were evaluated in vivo in rats and
marmosets. Their abilities to inhibit hepatic [14C]-
cholesterol biosynthesis from [14C]acetate in rats at 1 h
iv postdosing were studied. We reported previously13
that 1 possesses an ED50 of 0.1 mg/kg for inhibition of
cholesterol biosynthesis when administered iv in this
model. The relative potencies of C-4 deoxyS1 3 and C-3
decarboxyS1 10 compared with 1 are shown in Figures
1 and 2. The potency of C-4 deoxyS1 3 was found to be
the same as that for 1, whereas C-3 decarboxyS1 10 was
one-half as potent as 1. The duration of action of C-4
deoxyS1 3 relative to 1 was examined at an equipotent
dose of 1 mg/kg iv (Figure 3). Both compounds showed
maximal inhibition at 1 h postdose. Significant inhibi-
tion was observed for at least 7 h with S1 (1) and up to
d
Syntheses of these compounds have been reported.21
good inhibitory activities, consistent with our previous
findings19a for the C-3 and C-4 monomethyl esters of
S1. In compounds retaining the tricarboxylic acid
moiety, removal of the C-4 hydroxy group to give 3 did
not diminish SQS inhibitory activity, while the corre-
sponding epimer 5 showed 10-fold lower potency. The
corresponding C-1 modified allylic alcohol 7 retains
potent enzyme inhibitory activity; a similar finding has
been reported with the C-3 hydroxymethyl series.20

Decarboxy- and Deoxysqualestatins as SQS Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 211
F igu r e 1. Effect of S1 (1) and C-3 decarboxyS1 10 on
cholesterol biosynthesis in rats (n ) 8) at 1 h after iv
administration. *RP ) relative potency compared to 1. Figures
in brackets are fiducial ranges at p ) 0.95.
F igu r e 2. Effect of S1 (1) and C-4 deoxyS1 3 on cholesterol
biosynthesis in rats (n ) 8) at 1 h after iv administration. *RP
) relative potency compared to 1. Figures in brackets are
fiducial ranges at p ) 0.95.
4 h with C-4 deoxyS1 3. Studies with C-3 decarboxyS1
10 relative to 1 (1 mg/kg) at 6 h iv postdosing showed
no significant effect on cholesterol biosynthesis in rats
at doses up to 10 mg/kg (data not shown). The related
C-3 hydroxymethylS1 25 has been shown similarly to
possess a short in vivo duration of action compared to
S1 1 due to rapid excretion through the biliary duct.27
We have reported previously14 that 1 lowers serum
cholesterol levels in marmosets by up to 60% during 7
days after a single iv dose of 1 mg/kg. In the present
studies the serum cholesterol-lowering effects of C-4
deoxyS1 3 (10 mg/kg) and C-3 decarboxyS1 10 (50 mg/
kg) in marmosets were similarly compared with 1 (1 mg/
kg) (Figure 4). These results again demonstrated the
profound and extended cholesterol-lowering effect of 1
in marmosets. C-4 deoxyS1 3 retains good lipid lower-
ing ability, while C-3 decarboxyS1 10 has only a
marginal effect on cholesterol levels even when dosed
at 50 mg/kg.
F igu r e 3. Duration of effect of S1 (1) and C-4 deoxyS1 3 on
cholesterol biosynthesis in rats (n ) 8) after iv administration.
An asterisk indicates significantly (p < 0.05) below control.
deoxyS1 3 and C-3 decarboxyS1 10 have shorter in vivo
durations of action in the inhibition of cholesterol
biosynthesis in rats compared with 1. In comparison
with 1, which shows an excellent and extended reduc-
tion of serum cholesterol levels in marmosets following
administration of a single iv dose, C-4 deoxyS1 3
retained good lipid-lowering activity, whereas removal
of C-3 carboxylic acid in S1, 10, resulted in a dramatic
loss in its ability to lower serum cholesterol levels.
Con clu sion
C-4 deoxy- and C-3 and C-4 decarboxysqualestatins
were prepared and evaluated for their abilities to inhibit
rat microsomal SQS in vitro. These modifications were
well tolerated in the S1 series, and potent SQS inhibi-
tory activities were retained. However in the H1 series
removal of the hydroxy group at C-4 significantly
reduced their enzyme inhibitory activities, while the
removal of carboxylic acid at C-3 resulted in a dramatic
loss in its enzyme inhibitory activities. Both C-4
Exp er im en ta l Section
Organic solutions were dried over MgSO4, and column
chromatography was performed on silica gel 60 (Merck, Art

212 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Chan et al.
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â(2R*S*)]]-1-
[4-(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-4,6-
dih ydr oxy-7-[(2-tetr ah ydr opyr an yl)oxy]-2,8-dioxabicyclo-
[3.2.1]octa n e-3,4,5-tr ica r boxylic Acid , 6-(4,6-Dim eth yl-2-
octen oa te), 3,4,5-Tr is(1,1-d im eth yleth yl) Ester (13).
A
solution of 1214 (6.60 g, 7.70 mmol) in CH2Cl2 (150 mL) was
treated with 3,4-dihydro-2H-pyran (15 mL, 161 mmol) and
pyridinium p-toluenesulfonate (1 g, 4 mmol). After 20 h of
stirring at room temperature, the mixture was diluted with
CH2Cl2 (220 mL) and washed with a saturated aqueous
solution of NaHCO3 (100 mL) and a saturated aqueous solution
of NH4Cl (100 mL). The organic phase was dried and
concentrated to give a brown oil. This was purified by flash
column chromatography on silica gel eluting with EtOAc:
cyclohexane (2:8) to give 13 (6.10 g, 85%): NMR (CDCl3) δ 1.02
(d, 3H, J ) 7 Hz, CHdCHCHCH3), 1.40, 1.42, 1.45, 1.65, and
1.70 (5s, 27H, 3 t-BuO2C), 2.1 (s, 3H, CH3CO2), 2.75 (2dd, 1H,
J ) 5, 13 Hz for both, one proton of PhCH2), 3.45 and 3.80
(2m, 2H, CH2O of THP), 4.05 and 4.08 (s, 1H, OH), 4.08 and
4.21 (d, 1H, J ) 2 Hz, H-7), 4.70 and 4.90 (2bt, 1H, OCHO of
THP), 4.98 (bs, 2H, CdCH2), 5.07 and 5.10 (s, 1H, H-3), 5.15
(d, 1H, J ) 5 Hz, CHOAc), 5.75 (d, 1H, J ) 15.5 Hz,
CHdCHCO), 6.45 and 6.60 (d, 1H, J ) 2 Hz, H-6), 6.90 (dd,
1H, J ) 8, 15.5 Hz, CHdCHCO), 7.1-7.3 (m, 5H, C6H5); MS
(DCI, NH3, +ve) m/ z 960 (M + NH4)+, 943 (M + H)+. Anal.
(C52H78O15) C, H.
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â(2R*S*)]]-1-
[4-(Acetyloxy)-5-m eth yl-3-m eth ylen e-6-p h en ylh exyl]-4,6-
dih ydr oxy-7-[(2-tetr ah ydr opyr an yl)oxy]-2,8-dioxabicyclo-
[3.2.1]octa n e-3,4,5-tr ica r boxylic Acid , 6-(4,6-Dim eth yl-2-
oct en oa t e), 4-(Met h oxy-2-oxoa cet a t e), 3,4,5-Tr is(1,1-d i-
m et h ylet h yl) E st er (14), [1S-[1r(4R*,5S*),3r,4â,5r,6r-
(2E,4R*,6R*),7â(2R*S*)]]-1-[4-(Acetyloxy)-5-m eth yl-3-m e-
t h ylen e-6-p h en ylh exyl]-6-h yd r oxy-7-[(2-t et r a h yd r op y-
r a n yl)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 et h yl-2-oct en oa t e), 3,4,5-Tr is-
(1,1-d im et h ylet h yl) E st er (15), a n d [1S-[1r(4R*,5S*),-
3r,4r,5r,6r(2E,4R*,6R*),7â(2R*S*)]]-1-[4-(Acet yloxy)-5-
m eth yl-3-m eth ylen e-6-p h en ylh exyl]-6-h yd r oxy-7-[(2-tet-
r a h yd r op yr a n yl)oxy]-2,8-d ioxa bicyclo[3.2.1]octa n e-3,4,5-
t r ica r b oxylic Acid , 6-(4,6-Dim et h yl-2-oct en oa t e), 3,4,5-
Tr is(1,1-d im eth yleth yl) Ester (16). A solution of 13 (5.20
g, 5.6 mmol) in dry THF (80 mL) was treated with NaH (60%
oil dispersion, 1.7 equiv, 0.38 g, 9.5 mmol) at room tempera-
ture. After 10 min, methyl oxalyl chloride (1.3 mL, 14 mmol)
was added. After 10 min, saturated aqueous NH4Cl solution
(100 mL) and Et2O (200 mL) were added. The organic phase
was dried and evaporated to give 14 (5.2 g, 95%): NMR
(CDCl3) δ 1.02 (d, 3H, J ) 7 Hz, CHdCHCHCH3), 1.42, 1.45,
1.55, and 1.60 (4s, 27H, 3 t-BuO2C), 2.1 (s, 3H, CH3CO2), 3.8
(s, 3H, CH3OCOCOO), 4.70 and 4.82 (bt, 1H, OCHO of THP),
4.95 (bs, 2H, CdCH2), 5.21 (d, 1H, J ) 5 Hz, CHOAc), 5.80 (d,
1H, J ) 15.5 Hz, CHdCHCO), 6.40 and 6.52 (d, 1H, J ) 2 Hz,
H-6), 6.90 (dd, 1H, J ) 8, 15.5 Hz, CHdCHCO), 7.10-7.30
(m, 5H, C6H5); MS (DCI, NH3, +ve) m/ z 1028 (M + NH4)+ for
F igu r e 4. Effect of S1 (1), C-4 deoxyS1 3, and C-3 decar-
boxyS1 10 on serum cholesterol levels of marmosets (n ) 6)
after a single iv dose. An asterisk indicates significantly (p <
0.05) below control.
no. 9385). Analytical HPLC was performed on a Spherisorb
ODS-2 column (20 cm × 0.4 cm) using CH3CN/H2O containing
0.15 mL/L concentrated H2SO4 or 0.1% TFA as eluent, at a
flow rate of 1.5 mL/min and detecting at 210 nm. Preparative
HPLC was conducted on either a Spherisorb ODS-2 column
(25 cm × 2.5 cm i.d.) at a flow rate of 10 mL/min (column A)
or Spherisorb ODS-2 column (25 cm × 5 cm i.d.) at a flow rate
of 40 mL/min (column B) using CH3CN/H2O containing 0.15
mL/L concentrated H2SO4 or 0.1% TFA as eluent and detecting
at 210 nm. The appropriate fractions from each run were
combined, the CH3CN was removed in vacuo (bath tempera-
ture < 40 °C), and the remainder was extracted with EtOAc.
The combined extracts were washed with brine and evapo-
rated; the residue was dissolved in H2O/dioxane and freeze-
dried. IR spectra were recorded on a Nicolet 5SXC FTIR
spectrometer. NMR spectra were recorded on a Bruker AM
500 or AM 250 or Varian VXR 400 spectrometer using
standard pulse sequences. Chemical shifts are expressed as
parts per million downfield from internal tetramethylsilane.
Desorption chemical ionization mass spectrometry using NH3
(DCI, NH3, +ve) and negative ion FAB mass spectrometry (-ve
FAB) were performed on Finnigan 4600 and 8400 spectrom-
eters, respectively. Positive ion thermospray mass spectrom-
etry (thermospray, +ve) was carried out with an HP 5989A
engine, and high-resolution LSI mass spectrometry [HRMS
(LSI, +ve)] was performed on a VG Autospec S spectrometer.
Elemental analyses were determined with a Perkin-Elmer
240C or Carlo-Erba 1106 elemental analyzer.
C
55H80O18
.
A solution of 14 (5.1 g, 5.5 mmol) in dry xylene (80 mL) at
90 °C was treated with tributyltin hydride (2.2 mL, 8 mmol)
and AIBN (50 mg). Further portions of AIBN (50 mg) and
tributyltin hydride (0.7 mL, 2.5 mmol) were added after 3 h
with continued heating at 90 °C. After a total of 16 h, heating
was stopped, and the reaction mixture was allowed to cool and
then purified by flash column chromatography on silica gel
eluting with EtOAc:cyclohexane (5:95,1:9,1:4) to give 15 (1.56
g, 32%): NMR (CDCl3) δ 1.02 (2d, 3H, J ) 6.5 Hz for both,
CHdCHCHCH3), 2.1 (2s, 3H, CH3CO2), 2.95 and 2.96 (2d, 1H,
J ) 12 Hz for both, H-4), 3.45 and 3.80 (2m, 2H, CH2O of THP),
4.02 and 4.20 (2d, 1H, J ) 2 Hz for both, H-7), 4.75 and 4.90
(2t, 1H, OCHO of THP), 4.92 and 4.95 (bs, 2H, CdCH2), 4.94
and 4.98 (2d, 1H, J ) 12 Hz for both, H-3), 5.10 and 5.15 (2d,
1H, J ) 5 Hz for both, CHOAc), 5.75 and 5.80 (2d, 1H, J ) 15
Hz for both, CHdCHCO), 6.23 and 6.38 (2d, 1H, J ) 2 Hz for
both, H-6), 6.90 (2dd, 1H, J ) 7.5, 15 Hz for both, CHdCHCO),
Biologica l Tests. Squalestatins were tested for their
ability to inhibit the conversion of [2-14C]farnesyl pyrophos-
phate (FPP) to [14C]squalene by rat liver microsomal SQS
using our published procedure.13,26 Effects of iv administration
of 1, C-4 deoxyS1 3, and C-3 decarboxyS1 10 on cholesterol
biosynthesis in rats and on serum cholesterol levels in
marmosets were carried out according to our published
methods.13,14

Decarboxy- and Deoxysqualestatins as SQS Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 213
7.1-7.3 (m, 5H, C6H5); MS (DCI, NH3, +ve) m/ z 944 (M +
NH4)+. Anal. (C52H78O14) C, H.
CHdCHCO), 5.94 (s, 1H, H-6), 6.95 (dd, 1H, J ) 15, 8 Hz,
CHdCHCO), 7.20-7.38 (m, 5H, C6H5).
16 (0.4 g, 8%): NMR (CDCl3) δ 1.02 (d, 3H, J ) 7 Hz,
CHdCHCHCH3), 2.1 (s, 3H, CH3CO2), 2.7 (bdd, 1H, one proton
of PhCH2), 3.28 and 3.32 (d, 1H, J ) 3.5 Hz, H-4), 3.95 and
4.11 (d, 1H, J ) 1.5 Hz, H-7), 4.62 and 4.66 (d, 1H, J ) 3.5
Hz, H-3), 4.95 and 4.98 (bs, 2H, CdCH2), 5.51 and 5.71 (d,
1H, J ) 1.5 Hz, H-6), 5.80 and 5.81 (d, 1H, J ) 15 Hz,
CHdCHCO), 6.91 (dd, 1H, J ) 7, 15 Hz, CHdCHCO), 7.1-
7.3 (m, 5H, C6H5).
[1S-[1r(4R*,5S*),3r,4â,5r,6r,7â]]-1-[4-(Acetyloxy)-5-m eth -
y l-3-m e t h y le n e -6-p h e n y lh e x y l]-6,7-d i h y d r o x y -2,8-
d ioxa bicyclo[3.2.1]octa n e-3,4,5-tr ica r boxylic Acid (4). A
solution of 3 (70 mg, 0.105 mmol) in dry DMF (2 mL) was
treated with Et3N (63 µL, 0.47 mmol) and N-methylhydroxy-
lamine hydrochloride (26 mg, 0.311 mmol) at room tempera-
ture. After stirring for 16 h, the solvent was evaporated under
reduced pressure. The residue was purified by reverse-phase
HPLC (column A) eluting with 43% MeCN-H2O and acidified
with 0.15 mL/L concentrated H2SO4 to give 4 (30 mg, 55%):
NMR (CD3OD) δ 0.85 (d, 3H, J ) 7 Hz, CH3CH), 2.10 (s, 3H,
CH3CO2), 2.7 (dd, 1H, J ) 6, 14 Hz, one proton of PhCH2),
2.95 (d, 1H, J ) 12 Hz, H-4), 4.0 (d, 1H, J ) 1 Hz, H-7), 7.1-
7.3 (m, 5H, C6H5); MS (-ve FAB) m/ z 521 (M - H)-. Anal.
(C25H30O12‚2.5H2O) C, H.
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â]]-1-(4-Hy-
d r oxy-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl)-6,7-d ih y-
d 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) (7). A solution of 3 (25
mg, 0.037 mmol) in acetone:H2O (3:1, 1 mL) at room temper-
ature was treated with concentrated H2SO4 (0.05 mL, 0.92
mmol). After stirring for 14 days, the organic solvent was
evaporated and the residue was purified by reverse-phase
HPLC (column A) eluting with 60% MeCN-H2O acidified with
0.15 mL/L concentrated H2SO4 to give 7 (17 mg, 70%): NMR
(CD3OD) δ 1.0 (d, 3H, J ) 7 Hz, CHdCHCHCH3), 2.75 (dd,
1H, J ) 6, 12.5 Hz, one proton of PhCH2), 3.05 (d, 1H, J ) 11
Hz, H-4), 3.90 (d, 1H, J ) 5 Hz, CH(OH)CdCH2), 4.03 (d, 1H,
J ) 1.5 Hz, H-6), 5.80 (d, 1H, J ) 15.5 Hz, CHdCHCO), 6.85
(dd, 1H, J ) 8.5, 15.5 Hz, CHdCHCO), 7.1-7.3 (m, 5H, C6H5).
Anal. (C33H44O12‚1.5H2O) C, H.
[1S-[1r(4R*,5S*),3r,4r,5r,6r(2E,4R*,6R*),7â]]-1-[4-(Acety-
loxy)-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl]-6-h 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), 3,4,5-Tr is(1,1-d im eth yleth -
yl) Ester (18). A mixture of 16 (450 mg, 0.486 mmol) and
Amberlyst-15 (1 g) in MeOH (20 mL) was stirred at room
temperature for 3 days. The methanolic solution was evapo-
rated to give a residue which was purified by flash column
chromatography to afford 18 (200 mg, 49%): NMR (CDCl3) δ
1.02 (d, 3H, J ) 6.5 Hz, CHdCHCHCH3), 1.42-1.55 (3s, 27H,
3 t-BuO2C), 2.1 (s, 3H, CH3CO2), 2.7 (dd, 1H, J ) 13, 5 Hz,
one proton of PhCH2), 2.93 (d, 1H, J ) 2.5 Hz, CHOH), 3.3 (d,
1H, J ) 4 Hz, H-4), 3.95 (t, 1H, J ) 2.5 Hz, H-7), 4.67 (d, 1H,
J ) 4 Hz, H-3), 4.95 (s, 2H, CdCH2), 5.02 (d, 1H, J ) 2.5 Hz,
H-6), 5.1 (d, 1H, J ) 5 Hz, CHOAc), 5.8 (d, 1H, J ) 15 Hz,
CHdCHCO), 6.9 (dd, 1H, J ) 15, 8 Hz, CHdCHCO), 7.1-7.3
(m, 5H, C6H5); MS (DCI, NH3, +ve) m/ z 860 (M + NH4)+ for
C47H70O13; analytical HPLC (90% MeCN-H2O) showed 85%
purity (tR ) 5.9 min).
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â]]-1-[4-(Acety-
loxy)-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl]-6-h 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), 3,4,5-Tr is(1,1-d im eth yleth -
yl) Ester (17). A solution of 15 (1.34 g, 1.44 mmol) in MeOH
(100 mL) at 60 °C was treated with pyridinium p-toluene-
sulfonate (0.1 g, 0.4 mmol). After 16 h of stirring at 60 °C,
the solvent was evaporated and EtOAc (150 mL) was added.
The organic phase was washed with a saturated aqueous
solution of NaHCO3 (30 mL) and a saturated aqueous solution
of NH4Cl (30 mL). The organic phase was dried and evapo-
rated to give 17 (1.15 g, 94%): NMR (CDCl3) δ 1.02 (d, 3H, J
) 7 Hz, CHdCHCHCH3), 1.44, 1.45, and 1.47 (s, 27H, 3
t-BuO2C), 2.10 (s, 3H, CH3CO2), 2.70 (dd, 1H, J ) 5, 13 Hz,
one proton of PhCH2), 2.95 (d, 1H, J ) 11 Hz, H-4), 3.07 (bd,
1H, OH), 3.98 (bs, 1H, H-7), 4.90 (d, 1H, J ) 11 Hz, H-3), 4.93
and 4.95 (bs, 2H, CdCH2), 5.10 (d, 1H, J ) 5 Hz, CHOAc),
5.72 (d, 1H, J ) 2 Hz, H-6), 5.75 (d, 1H, J ) 15 Hz,
CHdCHCO), 6.9 (dd, 1H, J ) 7, 15 Hz, CHdCHCO), 7.10-
7.30 (m, 5H, C6H5); MS (DCI, NH3, +ve) m/ z 860 (M + NH4)+.
Anal. (C47H70O13‚1.5H2O) C, H.
[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-ph en ylh exyl]-6,7-dih ydr 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 et h yl-2-oct en oa t e) (3) a n d [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]-6,7-dih ydr oxy-2,8-dioxabicyclo-
[3.2.1]octa n e-3,4,5-tr ica r boxylic Acid , 6-(4,6-Dim eth yl-2-
octen oa te), 7-Meth a n oa te (19). A solution of 17 (1.1 g, 1.32
mmol) in formic acid (10 mL) was stirred at room temperature.
After 16 h, the formic acid was evaporated; the residue was
purified by reverse-phase HPLC (column B) eluting with 68%
MeCN-H2O and acidified with 0.15 mL/L concentrated H2-
SO4 to give 3 (0.385 g, 43%): NMR (CD3OD) δ 0.83-0.91 (m,
9H, 3 CH3), 1.03 (d, 3H, J ) 6.5 Hz, CHdCHCHCH3), 2.09 (s,
3H, CH3CO2), 2.67 (dd, 1H, J ) 13.5, 6.5 Hz, one proton of
PhCH2), 3.08 (d, 1H, J ) 10.5 Hz, H-4), 4.01 (d, 1H, J ) 2 Hz,
H-7), 4.98 and 5.01 (2s, 2H, CdCH2), 5.06 (d, 1H, J ) 5 Hz,
CHOAc), 5.82 (d, 1H, J ) 16 Hz, CHdCHCO), 5.86 (d, 1H, J
) 2 Hz, H-6), 6.87 (dd, 1H, J ) 16, 8 Hz, CHdCHCO), 7.09-
7.30 (m, 5H, C6H5); MS (-ve FAB) m/ z 673 (M - H)-. Anal.
(C35H46O13‚H2O) C, H.
19 (0.121 g, 13%): NMR (CD3OD) δ 2.1 (s, 3H, CH3CO2),
2.66 (dd, 1H, J ) 6, 14 Hz, one proton of PhCH2), 3.02 (d, 1H,
J ) 11 Hz, H-4), 4.93 (d, 1H, J ) 11 Hz, H-3), 5.05 (d, 1H, J
) 5 Hz, CHOAc), 5.27 (d, 1H, J ) 2 Hz, H-7), 5.80 (d, 1H, J )
15 Hz, CHdCHCO), 6.20 (d, 1H, J ) 2 Hz, H-6), 6.85 (dd, 1H,
J ) 9, 15 Hz, CHdCHCO), 7.10-7.30 (m, 5H, C6H5), 8.25 (s,
1H, OCHO); MS (-ve FAB) m/ z 701 (M - H)-. Anal.
(C36H46O14‚1.3H2O) C, H.
[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-ph en ylh exyl]-6,7-dih ydr 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), Tr ip ota ssiu m Sa lt (Tr ip o-
ta ssiu m Sa lt of 3). KHCO3 (3 equiv, 170 mg, 1.7 mmol) in
H2O (5 mL) was added slowly to a stirring solution of 3 (380
mg, 0.56 mmol) in a mixture of dioxane (20 mL) and H2O (2
mL). Stirring was continued until the cloudy solution became
clear. The resultant solution was freeze-dried to give the
potassium salt of 3 as a white solid (quantitative): NMR (D2O)
δ 0.80 (t, 3H, J ) 9 Hz, CH2CH3), 0.81 (d, 3H, J ) 7 Hz,
CHCH3), 0.91 (d, 3H, J ) 7 Hz, CHCH3), 1.01 (d, 3H, J ) 6.5
Hz, CHdCHCHCH3), 2.18 (s, 3H, CH3CO2), 2.60 (d, 2H, J ) 7
Hz, PhCH2CH), 2.69 (d, 1H, J ) 11 Hz, H-4), 3.90 (s, 1H, H-7),
4.72 (d, 1H, J ) 11 Hz, H-3), 4.89 (d, 1H, J ) 5 Hz, CHOAc),
4.97 and 5.03 (2s, 2H, CdCH2), 5.93 (d, 1H, J ) 15 Hz,
[1S-[1r(4R*,5S*),3r,4r,5r,6r(2E,4R*,6R*),7â]]-1-[4-(Acety-
loxy)-5-m eth yl-3-m eth ylen e-6-ph en ylh exyl]-6,7-dih ydr 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 et h yl-2-oct en oa t e) (5) a n d [1S-[1r(4R*,5S*),-
3r,4r,5r,6r(2E,4R*,6R*),7â]]-1-[4-(Acetyloxy)-5-m eth yl-3-
m eth ylen e-6-ph en ylh exyl]-6,7-dih ydr oxy-2,8-dioxabicyclo-
[3.2.1]octa n e-3,4,5-tr ica r boxylic Acid , 6-(4,6-Dim eth yl-2-
octen oa te), 7-Meth a n oa te (20). 18 (200 mg, 0.237 mmol)
was dissolved in formic acid (5 mL) and stirred at room
temperature overnight. Removal of solvent gave a foam which
was purified by preparative HPLC (column A) eluting with
60% MeCN-H2O and acidified with 0.15 mL/L concentrated
H2SO4 (tR ) 10.69 min) to give 5 (108 mg, 67%): NMR (CD3-
OD) δ 0.82-0.92 (m, 9H, 3 CH3), 1.06 (d, 3H, J ) 7.2 Hz,
CHdCHCHCH3), 2.09 (s, 3H, CH3CO2), 2.67 (dd, 1H, J ) 13.5,
6.5 Hz, one proton of PhCH2), 3.61 (d, 1H, J ) 3.5 Hz, H-4),
3.99 (d, 1H, J ) 2.5 Hz, H-7), 4.94 and 5.01 (2s, 2H, CdCH2),
5.06 (d, 1H, J ) 5 Hz, CHOAc), 5.43 (d, 1H, J ) 2.5 Hz, H-6),
5.85 (d, 1H, J ) 15.5 Hz, CHdCHCO), 6.90 (dd, 1H, J ) 15.5,
8 Hz, CHdCHCO), 7.09-7.30 (m, 5H, C6H5); MS (-ve FAB)
m/ z 673 (M - H)-, 629 (M - CO2H)-. Anal. (C35H46O13‚2H2O)
C, H.
20 (20 mg, 8%): NMR (CD3OD) δ 0.81-0.92 (m, 9H, 3 CH3),
1.05 (d, 3H, J ) 7 Hz, CHdCHCHCH3), 2.09 (s, 3H, CH3CO2),

214 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Chan et al.
2.68 (dd, 1H, J ) 14, 6 Hz, one proton of PhCH2), 3.69 (d, 1H,
J ) 3.5 Hz, H-4), 5.05 (d, 1H, J ) 5 Hz, CHOAc), 5.19 (d, 1H,
J ) 2 Hz, H-7), 5.64 (d, 1H, J ) 2 Hz, H-6), 5.86 (d, 1H, J )
15 Hz, CHdCHCO), 6.90 (dd, 1H, J ) 15, 9 Hz, CHdCHCO),
7.12-7.30 (m, 5H, C6H5), 8.19 (s, 1H, OCHO); MS (-ve FAB)
[1S-[1r(4R*,5S*),3r,4r,5r,6r(2E,4R*,6R*),7â]]-1-[4-(Acety-
loxy)-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl]-4,6,7-t r ih y-
dr oxy-2,8-dioxabicyclo[3.2.1]octan e-3,5-dicar boxylic Acid,
6-(4,6-Dim et h yl-2-oct en oa t e) (8) a n d [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-d ih yd r oxy-7-[[(1,1-d im eth -
yle t h yl)d im e t h ylsila n yl]oxy]-2,8-d ioxa b icyclo[3.2.1]-
octan e-3,5-dicar boxylic Acid, 6-(4,6-Dim eth yl-2-octen oate),
3,5-Bis(1,1-d im eth yleth yl) Ester (24). To a stirring solid
mixture of 22 (1.046 g, 1.14 mmol) and 2-chloro-3-ethylben-
zoxazolium tetrafluoroborate (1.5 equiv, 0.476 g, 1.71 mmol)
under nitrogen at room temperature was added a solution of
Et3N (3 equiv, 0.48 mL, 3.43 mmol) in CH2Cl2 (2 mL).24 After
1 h, the mixture was cooled to 0 °C and sodium borohydride
(130 mg, 3.42 mmol) in EtOH (10 mL) was added. After a
further 2 h, the reaction was quenched with 10% citric acid (9
mL) and the mixture diluted with H2O (50 mL) and extracted
with Et2O (3 × 50 mL). Combined ethereal extracts were
washed with brine, dried, and concentrated to a light brown
oil (1.316 g) which was purified by flash column chromatog-
raphy eluting with EtOAc:cyclohexane (1:9-1:4) and then
MeOH:CHCl3 (1:4) to give initially an isomeric mixture
containing 24 (0.139 g): Rf ) 0.34 (SiO2/15% EtOAc:cyclohex-
ane). This material was further purified by preparative HPLC
(column A) eluting with 90% MeCN-H2O/0.1% TFA (tR ) 35.9
min) to give 24 as a colorless gum (65 mg, 12% based on the
recovery of 22): NMR (CDCl3) δ 1.02 (d, 3H, J ) 7 Hz,
CHdCHCHCH3), 1.41 and 1.45 (2s, 18H, 2 t-BuO2C), 2.05 (s,
3H, CH3CO2), 2.62 (dd, 1H, J ) 14, 6 Hz, one proton of PhCH2),
3.88 (d, 1H, J ) 10 Hz, H-4), 4.07 (d, 1H, J ) 2 Hz, H-7), 4.36
(d, 1H, J ) 10 Hz, H-3), 5.01 (d, 1H, J ) 5 Hz, CHOAc), 5.65
(d, 1H, J ) 2 Hz, H-6), 5.83 (d, 1H, J ) 16 Hz, CHdCHCO),
6.89 (dd, 1H, J ) 16, 9 Hz, CHdCHCO), 7.01-7.27 (m, 5H,
C6H5); HRMS (C48H76O12Si + Na)+ calcd 895.5059, found
895.5063.
m/ z 701 (M - H)- for C36H46O14
.
[1S-[1r(4R *,5S*),3r,4r,5r,6r,7â]]-1-[4-(Ace t yloxy)-5-
m et h yl-3-m et h ylen e-6-p h en ylh exyl]-6,7-d ih yd r oxy-2,8-
d ioxa bicyclo[3.2.1]oct a n e-3,4,5-t r ica r b oxylic Acid (6).
Et3N (6 equiv, 63 µL, 0.66 mmol) followed by N-methylhy-
droxylamine (3 equiv, 24 mg, 0.33 mmol) was added to a
solution of 5 (75 mg, 0.111 mmol) in dry DMF (2 mL). The
mixture was stirred at room temperature overnight. Removal
of volatile components under high vacuum gave a residue
which was purified by preparative HPLC (column A) eluting
with 40% MeCN-H2O and acidified with 0.15 mL/L concen-
trated H2SO4 (tR ) 4.41 min) to give 6 (42 mg, 72%): NMR
(CD3OD) δ 0.85 (d, 3H, J ) 7 Hz, CH3), 2.1 (s, 3H, CH3CO2),
2.7 (dd, 1H, J ) 13, 5 Hz, one proton of PhCH2), 3.51 (d, 1H,
J ) 3.5 Hz, H-4), 3.98 and 4.17 (2s, 2H, J ) 2 Hz, H-6, H-7),
4.65 (d, 1H, J ) 3.5 Hz, H-3), 4.95 and 5.0 (2s, 2H, CdCH2),
5.1 (d, 1H, J ) 5 Hz, CHOAc), 7.1-7.3 (m, 5H, C6H5); MS
(-ve FAB) m/ z 521 (M
(C25H30O12‚2.5H2O) C, H.
-
H)- for C25H30O12
.
Anal.
[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-ph en ylh exyl]-4,6-dih ydr oxy-
7-[[(1,1-d im et h ylet h yl)d im et h ylsila n yl]oxy]-2,8-d ioxa -
bicyclo[3.2.1]octa n e-3,4,5-tr ica r boxylic Acid , 6-(4,6-Dim -
et h yl-2-oct en oa t e), 3,4,5-Tr is(1,1-d im et h ylet h yl) E st er
(21). To a solution of 1214 (9.84 g, 11.45 mmol) in dry DMF
(30 mL) under nitrogen at 50 °C was added imidazole (18
equiv, 14 g, 206 mmol) and tert-butyldimethylchlorosilane (9
equiv, 15.46 g, 102.6 mmol). After heating at 80 °C for 5 h,
the mixture was cooled to room temperature and stirred for a
further 17 h. The mixture was diluted with Et2O (500 mL),
washed with H2O (100 mL), aqueous HCl (1 M, 100 mL), and
saturated aqueous NH4Cl solution (2 × 100 mL), dried, and
concentrated to an orange gummy oil (19.26 g) which was
purified by flash column chromatography eluting with EtOAc:
cyclohexane (1:9-3:7) to give 21 as a pale orange gum (11.5
g, quantitative): Rf ) 0.47 (SiO2, 25% EtOAc:cyclohexane); IR
(KBr) 1729 cm-1; NMR (CDCl3) δ 0.05 and 0.10 (2s, 6H, (CH3)2-
Si), 0.75-0.97 (m, 18H, t-BuSi, 3 CH3), 1.02 (d, 3H, J ) 6 Hz,
CHdCHCHCH3), 1.41, 1.44, and 1.56 (3s, 27H, 3 t-BuO2C),
2.09 (s, 3H, CH3CO2), 2.72 (dd, 1H, J ) 13, 5.5 Hz, one proton
of PhCH2), 4.06 (s, 1H, 4-OH), 4.07 (d, 1H, J ) 1.5 Hz, H-7),
4.96 and 4.98 (2s, 2H, CdCH2), 5.09 (s, 1H, H-3), 5.13 (d, 1H,
J ) 5 Hz, CHOAc), 5.79 (d, 1H, J ) 15.5 Hz, CHdCHCO),
6.34 (d, 1H, J ) 1.5 Hz, H-6), 6.92 (dd, 1H, J ) 15.5, 8 Hz,
CHdCHCO), 7.1-7.3 (m, 5H, C6H5); MS (thermospray, +ve)
m/z 990 (M + NH4)+. Anal. (C53H84O14Si‚2H2O) C, H.
[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-ph en ylh exyl]-4,6-dih ydr oxy-
7-[[(1,1-d im et h ylet h yl)d im et h ylsila n yl]oxy]-2,8-d ioxa -
bicyclo[3.2.1]octa n e-3,4,5-tr ica r boxylic Acid , 6-(4,6-Dim -
eth yl-2-octen oa te), 3,5-Bis(1,1-d im eth yleth yl) Ester (22).
To an ice-cold solution of 21 (2.37 g, 2.44 mmol) in dioxane
(10 mL) was added dropwise a solution of HCl (6.4 M in
dioxane, 3.8 mL). After 2 h, the mixture was warmed to 10
°C and a further aliquot of HCl (6.4 M in dioxane, 3.8 mL)
was added. After another 1.5 h, the mixture was warmed to
room temperature and concentrated in vacuo to give a pale
yellow gummy foam (1.9 g) which was purified by gravity
column chromatography eluting with CHCl3:MeOH (9:1) to
give 22 as an off-white foam (1.15 g, 51%): Rf ) 0.54 (SiO2/
CHCl3:MeOH, 5:1); IR (KBr) 3566, 1724 cm-1; NMR (CD3OD)
δ 0.0 and 0.1 (2s, 6H, (CH3)2Si), 0.70-0.95 (m, 18H, t-BuSi, 3
CH3), 1.00 (d, 3H, J ) 7 Hz, CHdCHCHCH3), 1.40 and 1.43
(2s, 18H, 2 t-BuO2C), 2.07 (s, 3H, CH3CO2), 2.65 (dd, 1H, J )
13, 5 Hz, one proton of PhCH2), 4.02 (d, 1H, J ) 1 Hz, H-7),
4.93 and 4.97 (2s, 2H, CdCH2), 5.05 (d, 1H, J ) 5 Hz, CHOAc),
5.22 (s, 1H, H-3), 5.8 (d, 1H, J ) 15.5 Hz, CHdCHCO), 6.67
(d, 1H, J ) 1 Hz, H-6), 6.88 (dd, 1H, J ) 15.5, 8 Hz,
CHdCHCO), 7.05-7.25 (m, 5H, C6H5); MS (thermospray, +ve)
m/z 934 (M + NH4)+. Anal. (C49H76O14Si‚2H2O) C, H.
A second component from the flash column chromatography
was 23 (0.159 g, 30% based on the recovery of 22): Rf ) 0.20
(SiO2/EtOAc:cyclohexane, 3:17); NMR (CDCl3) δ -0.03 and 0.0
(2H, 6H, (CH3)2Si), 0.76-0.91 (m, 18H, 3 CH3, t-BuSi), 1.02
(d, 3H, J ) 7 Hz, CHdCHCHCH3), 1.41 and 1.49 (2s, 18H, 2
t-BuO2C), 2.08 (s, 3H, CH3CO2), 2.70 (dd, 1H, J ) 13, 5 Hz,
one proton of PhCH2), 2.73-2.85 (bm, 1H, OH), 4.06 (d, 1H, J
) 2.5 Hz, H-7), 4.1-4.2 (m, 1H, H-4), 4.72 (d, 1H, J ) 2.5 Hz,
H-3), 4.94 and 4.98 (2s, 2H, CdCH2), 5.1 (d, 1H, J ) 5 Hz,
CHOAc), 5.45 (d, 1H, J ) 2.5 Hz, H-6), 5.8 (d, 1H, J ) 15.5
Hz, CHdCHCO), 6.95 (dd, 1H, J ) 15.5, 8 Hz, CHdCHCO),
7.1-7.3 (m, 5H, C6H5).
23 (101 mg, 0.116 mmol) was dissolved in formic acid (10
mL) and allowed to stand at room temperature for 17 h.
Removal of solvent gave a light brown oil which was dissolved
in MeCN (5 mL). To this solution was added dropwise aqueous
hydrofluoric acid (40%, 2.12 mL). After 41 h, the reaction was
quenched with saturated aqueous NaHCO3 and the mixture
extracted with EtOAc, dried, and evaporated to give a gummy
residue (63 mg). Preparative HPLC (column A) eluting with
40% MeCN-H2O/0.1% TFA rising to 95% MeCN-H2O/0.1%
TFA over 25 min (tR ) 17.5 min) gave 8 as an off-white solid
(15 mg, 20%): NMR (CD3OD) δ 0.81-0.91 (m, 9H, 3 CH3), 1.04
(d, 3H, J ) 7 Hz, CHdCHCHCH3), 2.10 (s, 3H, CH3CO2), 2.69
(dd, 1H, J ) 13, 6 Hz, one proton of PhCH2), 4.01 (d, 1H, J )
2 Hz, H-7), 4.29 (d, 1H, J ) 3 Hz, H-4), 4.96 and 5.08 (2s, 2H,
CdCH2), 5.08 (d, 1H, J ) 5 Hz, CHOAc), 5.27 (d, 1H, J ) 2
Hz, H-6), 5.83 (d, 1H, J ) 15.5 Hz, CHdCHCO), 6.88 (dd, 1H,
J ) 15.5, 8.5 Hz, CHdCHCO), 7.07-7.29 (m, 5H, C6H5); MS
(thermospray, +ve) m/z 664 (M + NH4)+. Anal. (C34
H46O12‚H2O) C, H.
-
The starting material 22 (0.49 g, 49%) was also recovered
from the reaction mixture.
[1S-[1r(4R*,5S*),3r,4â,5r,6r(2E,4R*,6R*),7â]]-1-[4-(Acety-
loxy)-5-m et h yl-3-m et h ylen e-6-p h en ylh exyl]-4,6,7-t r ih y-
dr oxy-2,8-dioxabicyclo[3.2.1]octan e-3,5-dicar boxylic Acid,
6-(4,6-Dim eth yl-2-octen oa te) (9). To a solution of 24 (47
mg, 0.054 mmol) in THF (1 mL) was added tetra-n-butylam-
monium fluoride (1 M solution in THF, 60 mL, 60 mmol). After
stirring at room temperature for 0.5 h, the mixture was diluted
with EtOAc (10 mL), washed with 2 M HCl (5 mL) and brine

Decarboxy- and Deoxysqualestatins as SQS Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 215
G.; Lane, S. J .; Noble, D.; Piercey, J . E.; Sidebottom, P. J .; Webb,
G. The Squalestatins: Novel Inhibitors of Squalene Synthase
Produced by a Species of Phoma. V. Minor Metabolites. J .
Antibiot. 1994, 47, 741-755.
(2 × 5 mL), dried, and concentrated to a colorless gum (40 mg).
Flash column chromatography (SiO2/EtOAc:cyclohexane, 1:3)
gave the desilylated product as a colorless gum (33 mg, 81%):
NMR (CDCl3) δ 0.75-0.90 (m, 9H, 3 CH3), 1.05 (d, 3H, J ) 7
Hz, CHdCHCHCH3), 1.51 and 1.55 (2s, 18H, 2 t-BuO2C), 2.09
(s, 3H, CH3CO2), 2.70 (1H, dd, J ) 14, 5 Hz, one proton of
PhCH2), 3.15 (s, 1H, OH), 3.71 (s, 1H, OH), 4.01 (d, 1H, J )
2.5 Hz, H-7), 4.07 (d, 1H, J ) 9.5 Hz, H-4), 4.34 (d, 1H, J )
9.5 Hz, H-3), 4.93 and 4.96 (2s, 2H, CdCH2), 5.09 (d, 1H, J )
5 Hz, CHOAc), 5.24 (d, 1H, J ) 2.5 Hz, H-6), 5.77 (d, 1H, J )
16 Hz, CHdCHCO), 6.92 (dd, 1H, J ) 16, 9 Hz, CHdCHCO),
7.1-7.3 (bm, 5H, C6H5); MS (thermospray, +ve) m/z 776 (M
+ NH4)+. Anal. (C42H62O12) H; C: calcd, 66.47; found, C 67.17.
This desilylated compound (12 mg, 0.016 mmol) was dis-
solved in formic acid (1 mL). After stirring at room temper-
ature for 2 h, the solvent was evaporated. The residue was
purified by preparative HPLC (column A) eluting with 60%
MeCN-H2O/0.1% TFA (tR ) 23 min) to give 9 as a colorless
gum (6 mg, 59%): NMR (CD3OD) δ 1.04 (d, 3H, J ) 7 Hz,
CHdCHCHCH3), 2.09 (s, 3H, CH3CO2), 2.66 (dd, 1H, J ) 14,
6 Hz, one proton of PhCH2), 3.96 (d, 1H, J ) 10 Hz, H-4), 4.05
(d, 1H, J ) 2 Hz, H-7), 4.45 (d, 1H, J ) 10 Hz, H-3), 4.93 and
4.97 (2s, 2H, CdCH2), 5.04 (d, 1H, J ) 5 Hz, CHOAc), 5.59 (d,
1H, J ) 2 Hz, H-6), 5.83 (d, 1H, J ) 16 Hz, CHdCHCO), 6.87
(dd, 1H, J ) 16, 9 Hz, CHdCHCO), 7.1-7.3 (m, 5H, C6H5);
HRMS (C34H45O12)+ calcd 645.2911, found 645.2889; analytical
HPLC (60% MeCN-H2O/0.1% TFA) showed 99.6% purity (tR
) 9.58 min). Anal. (C34H46O12‚1.3H2O) C, H.
(11) Subsequent to our publications, Merck also reported on the
isolation of zaragozic acids in which zaragozic acid A has the
same structure as S1 1. (a) 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. (b) Hensens, O. D.;
Dufresne, C.; Liesch, J . M.; Zink, D. L.; Reamer, R. A.; Van-
Middlesworth, F. The Zaragozic Acids: Structure Elucidation
of a New Class of Squalene Synthase Inhibitors. Tetrahedron
Lett. 1993, 34, 399-402.
(12) The group at Tokyo Noko University-Mitsubishi has also isolated
S1 from a different microorganism; see: Hasumi, K.; Tachikawa,
K.; Sakai, K.; Marakawa, S.; Yoshikawa, N.; Kumazawa, S.;
Endo, A. Competitive Inhibition of Squalene Synthase by Squal-
estatin 1. J . Antibiot. 1993, 46, 689-691.
(13) 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.
(14) 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.; Sapra, M.; Scicinski, J . J .; Sharratt, P. J .;
Snowden, M. A.; Watson, N. S.; Williams, R. J . The Squalest-
atins: Novel Inhibitors of Squalene Synthase. Enzyme Inhibitory
Activities and in vivo Evaluation of C1-Modified Analogues. J .
Med. Chem. 1994, 37, 3274-3281.
(15) Procopiou, P. A.; Bailey, E. J .; Hutson, J . L.; Sharratt, P. J .;
Spooner, S. J .; Watson, N. S. The Squalestatins: Novel Inhibi-
tors of Squalene Synthase. The Optimal C1 Chain-length
Requirements. BioMed. Chem. Lett. 1993, 3, 2527-2532.
(16) (a) 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. (b)
Lester, M. G.; Giblin, G. M. P.; Inglis, G. G. A.; Procopiou, P. A.;
Ross, B. C.; Watson, N. S. Structurally simplified Squalest-
atins: A Convenient Route to a 6,7-Unsubstituted Derivative.
Tetrahedron Lett. 1993, 34, 4357-4360.
(17) (a) Sharratt, P. J .; Hutson, J . L.; Inglis, G. G. A.; Lester, M. G.;
Procopiou, P. A.; Watson, N. S. Structurally Simplified
Squalestatins: Monocyclic 1,3-Dioxane Analogues. BioMed.
Chem. Lett. 1994, 4, 661-666. (b) Andreotti, D.; Procopiou, P.
A.; Watson, N. S. The Squalestatins: Cleavage of the Bicyclic
Core via the Novel 2,8,9-Trioxabicyclo[3.3.1]nonane Ring Sys-
tem. Tetrahedron Lett. 1994, 35, 1789-1792.
(18) Ponpipom, M. M.; Girotra, N. N.; Bugianesi, R. L.; Roberts, C.
D.; Berger, G. D.; Burk, R. M.; Marquis, R. W.; Parsons, W. H.;
Bartizal, K. F.; Bergstom, J . D.; Kurtz, M. M.; Onishi, J . C.; Rew,
D. J . Structure-Activity Relationships of C1 and C6 Side Chains
of Zaragozic Acid A Derivatives. J . Med. Chem. 1994, 37, 4031-
4051.
Ack n ow led gm en t. We are indebted to Miss Belinda
J . Fitzgerald and Mrs. Surriya Faulkes for expert
technical assistance in conducting the rat enzyme
inhibition assays, Mr. Sean Lynn and Mr. Andy Roberts
for NMR studies, and Dr. P. J . Sidebottom for expert
assistance in NMR interpretations.
Refer en ces
(1) Chan, C.; Andreotti, D.; Cox, B.; Dymock, B. W.; Inglis, G. G.
A.; Keeling, S. E.; McCarthy, A. D.; Procopiou, P. A.; Ross, B.
C.; Sareen, M.; Scicinski, J . J .; Sharratt, P. J .; Srikantha, A. R.
P.; Watson, N. S. The Squalestatins: Decarboxy and 4-Deoxy
Analogues as Potent Squalene Synthase Inhibitors. 207th ACS
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(5) Grundy, S. M. Cholesterol and Coronary Heart Disease - Future
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(7) (a) Alberts, A. W. Effects of HMG CoA Reductase Inhibitors on
Cholesterol Synthesis. Drug Invest. 1990, 2 (Suppl. 2), 9-17.
(b) Maher, V. M. G.; Thompson, G. R. HMG CoA Reductase
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Lovastatin and an Appraisal of Simvastatin and Pravastatin.
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(8) (a) Poulter, C. D.; Rilling, H. C. In Biosynthesis of Isoprenoid
Compounds; Porter, J . W., Spurgeon, S. L., Eds.; Wiley: New
York, 1981; Vol. 1, Chapter 1. (b) Poulter, C. D.; Rilling, H. C.
Ibid. Chapter 8.
(9) Dawson, M. J .; Farthing, J . E.; Marshall, P. S.; Middleton, R.
F.; O’Neill, M. J .; Shuttleworth, A.; Stylli, C.; Tait, R. M.; Taylor,
P. M.; Wildman, G.; Buss, A. D.; Langley, D.; Hayes, M. V. The
Squalestatins, Novel Inhibitors of Squalene Synthase Produced
by a Species of Phoma I. Taxonomy, Fermentation, Isolation,
Physico-Chemical Properties and Biological Activity. J . Antibiot.
1992, 45, 639-647.
(19) (a) 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. (b) The group at Merck has
also reported their findings on esters of carboxylic acid of S1;
see: Biftu, T.; Acton, J . J .; Berger, G. D.; Bergstom, J . D.;
Dufresne, C.; Kurtz, M. M.; Marquis, R. W.; Parsons, W. H.; Rew,
D. R.; Wilson, K. E. Selective Protection and Relative Importance
of Carboxylic Acid Groups of Zaragozic Acid A for Squalene
Synthase Inhibition. J . Med. Chem. 1994, 37, 421-424.
(20) 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.
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(21) 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
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(10) (a) 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. (b) Blows, W. M.; Foster,
(23) The influence of different esters of S1 and the source of hydrogen
radical on the diastereoselectivities in this deoxygenation reac-
tion will form the basis of a future communication.

216 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Chan et al.
(24) Mukaiyama, T.; Echigo, Y. A New Method for the Preparation
of Ketones by Decarboxylation of R-Hydroxycarboxylic Acids with
2-Chlorobenzoxazolium Salt. Chem. Lett. 1978, 49-50.
a
mixture of two compounds (≈1:1) which were tentatively
assigned as the diastereoisomers of the lactol C (6%).
(25) Oxidative decarboxylation of S1 3,5-di-tert-butyl ester without
the C-7 TBDMS protecting group followed by NaBH4 reduction
gave a mixture containing the 3,7-lactone A (2%) and the 3,4-
diepi C-4 decarboxysqualestatin
B (11%). The structure of
lactone A was determined from the 1D and 2D NMR experi-
ments. A key correlation was that of the CdO (δ 167.3) with
H-7 (δ 4.70, s) in a long range heteronuclear correlation experi-
ment which established the lactone linkage between C-3 and
C-7. The stereochemistry at C-4 was established from NOE
difference experiments. Thus irradiation at H-4 (δ 4.36, d, J )
7 Hz) gave signal enhancements at H-3 (δ 4.76, d, J ) 7 Hz)
and OH at 4 (δ 3.64, bs), and irradiation at H-6 (δ 5.68, s) gave
signal enhancements to the OH at 4 and H-7 (δ 4.70, s). The
structure of B was determined from the MS [m/ z 647 (C34H61O11
+ H)+] and 1D and 2D NMR experiments. Thus irradiation at
H-3 (δ 4.68, d, J ) 7 Hz) gave signal enhancement at H-4 (δ
4.55, d, J ) 7 Hz); irradiation of the HO at 4 (δ 3.32) gave signal
enhancements at H-4 and H-6 (δ 5.92, d, J ) 2 Hz). A third
component was also isolated whose 1H-NMR spectrum showed
(26) Tait, R. M. Development of a Radiometric Spot-Wash Assay for
Squalene Synthase. Anal. Biochem. 1992, 203, 310-316.
(27) Unpublished results from these laboratories.
J M9504969