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M. Sawada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 633–637
Scheme 1. (a) BrCH2CO2Et, KI, K2CO3, acetone, reflux, quant.; (b) NaBH4, EtOH, 0 ꢀC, 63%; (c) TBDMSCl, imidazole, DMF, 0 ꢀC, quant.; (d)
MeMgBr, THF, 0 ꢀC, quant.; (e) (1) NaH, DMF, 0 ꢀC to rt; (2) 3-bromomethylthiophene, 0 ꢀC to rt, 67%; (f) TBAF, THF, 0 ꢀC, 82%; (g) MsCl,
Et3N, THF, quant.; (h) (E)-N-ethyl-6,6-dimethyl-2-hepten-4-yn-1-amine, K2CO3, DMF, rt, 54%; (i) (1) HCl/AcOEt, rt; (2) recryst. from Hex/
AcOEt, 63%.
[14C]acetate was added. After 2 h of incubation, the cells
were lysed and saponified with ethanolic KOH. Digito-
2. Synthesis
The synthesis of our new SE inhibitor, FR194738, is
outlined as shown in Scheme 1. Alkylation of m-hydro-
xybenzaldehyde with ethyl bromoacetate, followed by
aldehyde reduction with sodium borohydride afforded
the primary alcohol 3 in 63% yield over two steps. Pro-
tection of the alcohol as TBDMS, followed by reaction
with excess methylmagnesium bromide afforded the
tertiary alcohol 5 in quantitative yield. Alkylation of
the alcohol moiety of 5 with 3-bromomethylthiophene
in the presence of NaH–DMF, followed by deprotection
of the silyl protecting group with TBAF in tetra-
hydrofuran afforded the alcohol 7 in good yield.
Finally, activation of the alcohol as the mesylate (MsCl–
Et3N), followed by coupling with (E)-N-ethyl-6,6-dime-
thyl-2-hepten-4-yn-1-amine in the presence of potassium
carbonate and conversion of the resulting free amine
into the hydrochloride salt, afforded FR194738 as a
white crystalline solid. The derivatives shown in Table 1
and 2 were prepared in a similar manner.
nin was added to the non-saponifiable lipids. Radio-
activity of the digitonin coprecipitable sterols was
counted.
3.3. Induction of HMG-CoAreductase activity in HepG2
cells
HepG2 cells were incubated for 18 h in a medium con-
taining 10% human lipoprotein-deficient serum with or
without drug. Thereafter, the cells were washed, scalped
and suspended in buffer containing potassium phos-
phate, EDTA, KCl and Brij 97. After incubation for 15
min at37 ꢀC, the suspension was centrifuged at 12,000g
for 15 min and the supernatant was used for measuring
HMG-CoA reductase activity. HMG-CoA reductase
Table 1. Squalene epoxidase inhibitory activity of compounds with
heteroatom-based linkers
3. Biological methods
3.1. In vitro SE assay
Compd
A
R
Squalene epoxidase
inhibitory activity
IC50 (nM)
ClogP
Dog or rat liver microsomes and the homogenate of
HepG2 cells, which are considered to be a suitable
model for investigating lipid metabolism in human
liver,13 were solubilized with 2% Triton X-100. The
assay mixture containing microsomes or cell homo-
genate, AMO-1680, FAD, NADPH, EDTA, [3H]squa-
lene and drug was incubated for 90 min at 37 ꢀC. The
reaction was terminated by the addition of ethanolic
KOH. Non-saponifiable materials were extracted and
separated on a silica gel TLC plate, and the radio-
activity of 2,3-oxidosqualene was counted.
Human
Dog
9
Et23
Et5.4
Me
370
34
7.48
10a
11
12
7.30
11
31
7.2
77
51
43
6.78
7.83
8.19
Pr
3.2. Cholesterol synthesis in HepG2 cells
HepG2 cells were incubated overnight in a medium
containing 10% human lipoprotein-deficient serum. The
cells were preincubated for 1 h with drug and then
NB-598
a Free form of FR194738.