1310 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Brown et al.
3:1) to give an oil 10a (1.6 g, 52%): 1H NMR (CDCl3) δ 1.44
(m, 1H), 1.59 (m, 1H), 1.76 (m, 1H), 1.91 (m, 1H), 2.12 (m,
1H), 2.81 (m, 4H), 2.96 (m, 1H), 3.48 (m, 2H), 6.02 (m, 1H),
7.26 (m, 2H), 7.56 (d, 1H); EI-MS m/z 298 (M + H). The oil
DMSO as follows: molecular weight/1000 × 7.875 ) mg/mL
required to give a 10-4 M stock solution. Dilutions were made
from the stock to give 10-5, 10-6 M, etc. concentrations of test
compound. Phosphate-buffered tween (281 µL) was placed in
5 mL disposable vials, and compound solution (4 µL) was added
and mixed well. Microsomes (14.6 µL) were added and the vials
preincubated for 10 min at 37 °C. Oxidosqualene mix (15 µL)
was added and the mixture incubated for another 1 h. The
reaction was terminated by the addition of 16% KOH in 20%
aqueous ethanol (315 µL). The samples were heated at 80 °C
for 2 h, hexane (2 × 5 mL) was added, and the samples were
“whirl-mixed” for 10 s. The hexane phases were separated and
blown down with N2, and the residue was dissolved in CH3CN/
iPrOH (300 µL, 4:1). The samples were chromatographed using
a Hichrom 3ODS1 column with an isocratic elution using
CH3CN/iPrOH (95:5, flow rate 1 mL/min). The output from the
UV detector was connected to a radiochemical detector to
visualize radiolabeled sterol intermediates. The reaction rate
was measured as the conversion of oxidosqualene to lanosterol,
and the effects of compounds were expressed as an inhibition
of this process. IC50 values were obtained using the “Origin”
curve-fitting program supplied by MicroCal Software Inc., with
a tight binding background.
i
(300 mg) was dissolved in PrOH (2 mL), and 11 M HCl (0.25
mL) and Et2O were added to give a colorless hydrochloride
(210 mg): mp 214-215 °C. Anal. (C13H16BrNS‚HCl) C, H, N.
Typ ica l Syn th esis of a 3-Oxy Lin k ed Qu in u clid in e
(Mu lt ip le-P a r a llel Syn t h esis). 3-[(4-Ch lor ob en zoyl)-4-
p h en oxy]qu in u clid in e (13a ). Compound 11a , (0.64 g, 5
mmol) in THF (4.5 mL) and DMF (2.5 mL) was added to a
stirred solution of 4-chloro-4′-hydroxybenzophenone (1.29 g,
5.5 mmol), PPh3 (1.70 g, 6.5 mmol), and DEAD (0.94 mL, 6.0
mmol) in THF (4 mL) at 5-10 °C, and the mixture was stirred
for 18 h. The solvent was evaporated and the residue dissolved
in MTBE (8 mL); the solution was extracted with 2 M HCl,
and the extracts were washed with MTBE. The extract was
made basic with 4 M NaOH to pH ) 11, and the mixture was
stirred for 1 h and filtered to give a solid, which was triturated
in EtOH to give, on crystallization from, EtOH 13a (0.37 g,
1
22%): mp 147-149 °C; H NMR (CDCl3) δ 1.4 (m, 1H), 1.67
(m, 2H), 2.0 (m, 1H), 2.20 (m, 1H), 2.95 (m, 5H), 3.32 (m, 1H),
4.47 (m, 1H), 6.90 (d, 2H), 7.34 (d, 2H), 7.75 (q, 4H); EI-MS
m/z 342 (M + H). Anal. (C20H20ClNO2) C, H, N.
In Vivo Assa y for In h ibition of Ch olester ol Biosyn -
th esis in Ra ts. Female rats (130 ( 20 g) were acclimatized
to reverse lighting and compounds orally dosed (n ) 1) in
DMSO/HPMC. One hour later, tritiated mevalonate (2.5 µCi)
was administered, and after another 1 h the rats were
sacrificed. A weighed piece of liver (ca. 0.5 g) was saponified
in KOH/EtOH (2 mL, 1:9 w/v) at 80 °C for 2 h, and the mixture
was diluted (2 mL) before extraction with isohexane (5 mL).
The solvent was evaporated under N2 at 40 °C, and the residue
(+) a n d (-) 3-[(4-Ch lor oben zoyl)-4-p h en oxy]qu in u cli-
d in e (13b) a n d (13c). These compounds were prepared in a
manner similar to that of 13a , but starting from (+) and
(-)quinuclidin-3-ols 11b and 11c, respectively, to give 13b and
13c as colorless solids. 13b (22%): mp 124-125 °C; [R2D5] )
+32.5° (c ) 0.49, EtOH). Anal. (C20H20ClNO2) C, H, N. 13c
(20%): mp 124-125 °C; [R2D5] ) -33.5° (c ) 0.49, EtOH).
Anal. (C20H20ClNO2) C, H, N. The 1H NMR data found for these
enantiomers were identical to that found for 13a .
i
was dissolved in PrOH/CH3CN (300 µL, 1:4). Aliquots of this
solution (100 µL) were examined by HPLC (Spherisorb column
S3ODS1-1590; 10 cm × 4.6 mm) and the column eluates
monitored by an on-line radiochemical detector. In this way
an HPLC lipid profile was obtained for each compound relating
radioactivity counts to elution times. Chromatographic peaks
obtained at different retention times were identified by
comparison to the retention time of standard intermediates
in the cholesterol biosynthesis pathway (e.g., oxidosqualene
4.8 min; squalene 7.0 min; farnesyl pyrophosphate 2.0 min;
cholesterol 13.1 min), so that a selectivity profile could be
obtained in addition to the extent of the inhibition of the
biosynthesis of cholesterol from peak area.
Typ ica l Syn th esis of a 3-Eth yn yl Lin k ed Qu in u clid in e.
3-[4-(4-Br om oph en ylsu lfon yl)ph en yleth yn yl]qu in u clidin -
3-ol (14e). Bis(4-bromophenyl)sulfone (1.5 g, 4 mmol) and
dichlorobis(triphenylphosphine) palladium(II) (60 mg, 0.085
mmol) were added to a solution of 3-ethynyl-3-trimethylsilyl-
oxyquinuclidine (0.444 g, 1.995 mmol) in NEt3 (6 mL) and DMF
(2 mL), and the mixture was heated under reflux at 70 °C for
4 h. The cooled reaction mixture was added to H2O (20 mL),
and the mixture was extracted with EtOAc/Et2O (1:1). The
extract was washed with H2O, dried, and evaporated. The
residue was dissolved in EtOAc and filtered through alumina,
eluting with EtOAc/MeOH (1:1) to give, after crystallization
from THF-n-hexane, 14e as a colorless solid (254 mg, 25%):
mp 218-219 °C; 1H NMR (CDCl3) δ 1.42 (m, 1H), 1.63 (m, 2H),
1.91 (m, 2H), 2.03 (m, 1H), 2.84 (m, 3H), 3.05 (d, 1H), 3.29 (d,
1H), 7.53 (d, 2H), 7.55 (d, 2H), 7.78 (d, 2H), 7.88 (d, 2H); EI-
MS m/z 445 (M+). Anal. (C21H20BrNO3S) C, H, N.
Refer en ces
(1) These results were reported, in part, orally at the Royal Society
of Chemistry 8th Symposium on Medicinal Chemistry in Eastern
England, Hatfield, U.K., April 17, 1997.
(2) Shepherd, J .; Cobbe, S. M.; Ford, I.; Isles, C. G.; Lorimer, A. R.;
Macfarlane, P. W.; McKillop, J . H.; Packard, C. J . Prevention of
Coronary Heart Disease with Pravastatin in Men with Hyper-
cholesterolemia. New Engl. J . Med. 1995, 333, 1301-1307.
(3) Pedersen, T. R.; Kjekshus, J .; Berg, K.; Haghfelt, T.; Fargeman,
O.; Thorgeirsson, G.; Pyorala, K.; Mertinen, T.; Olsson, A. G.;
Wedel, H.; Wilhelmsmen, L. Randomised trial of cholesterol
lowering in 4444 patients with coronary heart disease: the
Scandinavian Simvastatin Survival Study (4S). Lancet 1994,
344, 1383-1389.
(4) Adams, J . L.; Metcalf, B. Therapeutic Consequences of the
Inhibition of Sterol Metabolism. Comprehensive Medicinal Chem-
istry; Sammes, P. G., Taylor, J . B., Eds.; Pergamon Press:
Oxford, 1990; Vol. 2, pp 333-364.
(5) Biller, S. A.; Neuenschwander, K.; Ponpipom, M. M.; Poulter,
C. D. Squalene Synthase Inhibitors. Curr. Pharm. Des. 1996, 2,
1-40.
(6) Alberts, A. W.; Berger, G. D.; Meade, B.; Bergstrom, J . D.
Squalene Synthase Inhibitors. US Appl. 5135935. Chem. Abstr.
1992, 117, 245613.
Meyer -Sch u st er R ea r r a n gem en t R ea ct ion . (Z)-3-[4-
(4-Br om oben zoyl)p h en a cylid en e]qu in u clid in e (16). 3-[2-
(4-Bromobenzophenone)ethynyl]quinuclidin-3-ol (0.5 g, 1.2
mmol) was added to 98% H2SO4 (5 mL) with stirring. The
reaction mixture was stirred at ambient temperature for 18
h, and H2O (10 mL) was added cautiously. The diluted solution
was poured onto ice/10 M NaOH to precipitate a solid, and
the solid was extracted into CH2Cl2 (2 × 50 mL). The extracts
were combined, washed with brine, dried, and evaporated. The
residue was purified by flash chromatography on silica gel,
eluting with EtOH/EtOAc/Et3N (80:20:3) to give 16 as a
colorless solid (0.25 g, 50%): mp 182-183 °C; 1H NMR (DMSO-
d6) δ 1.6-1.7 (m, 2H), 1.8-1.95 (m, 2H), 2.7 (m, 1H), 2.7-
2.95 (m, 4H), 3.95 (s, 2H), 7.1 (m, 1H), 7.6-7.9 (m, 6H), 8.1
(m, 2H); EI-MS m/z 410 (M + H). Anal. (C22 H20BrNO2‚
0.25H2O) C, H, N.
B. Biologica l Assa ys. In Vitr o Assa y for In h ibition of
R a t Micr osom a l OSC. Rat microsomes (1 mL) containing
protein (15-20 mg/mL) were diluted with 50 mM phosphate
buffer (722 µL, pH 7.4), and tween (0.1 g) was added to 50
mM phosphate buffer (100 mL). A solution of “cold” oxido-
squalene in ethanol (1 mL, 0.65 mg/mL) was added to an
ethanolic solution (1 mL) of radiolabeled oxidosqualene (stock
solution 18 µL, 1 mCi/mL), and compounds were dissolved in
(7) Amin, D.; Morris, R. L.; Neuenschwander, K.; Scotese, A. C.
Preparation of Azabicyclooctyloxymethylbenzenes and Related
Compounds as Squalene Synthase Inhibitors. PCT Int. Appl. WO
9215579 AI 920917. Chem. Abstr. 1993, 118, 124402.
(8) Brown, G. R.; Clarke, D. S.; Foubister, A. J .; Freeman, S.;
Harrison, P. J .; J ohnson, M. C.; Mallion, K. B.; McCormick, J .;
McTaggart, F.; Reid, A. C.; Smith, G. J .; Taylor, M. J . Synthesis
and Activity of a Novel Series of 3-Biarylquinuclidine Squalene
Synthase Inhibitors. J . Med. Chem. 1996, 39, 2971-2979.