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inhibit the cellular steroidgenesis in Hep G2cells
(human hepatoma cell line) cultured with 5% lipopro-
tein deficient serum containing medium for 48 h in vitro.
All biological tests were also conducted under the same
experimental conditions with pravastatin as reference
for direct comparison. The activities were determined by
decreased incorporation of sodium [2-14C] acetate into
non-saponifiable lipids. Selected compounds were fur-
ther evaluated for their ability to inhibit hepatic choles-
terol synthesis in a male Sprague–Dawley rat after oral
administration, as determined by decreased incorpora-
tion of intraperitoneally injected sodium [2-14C] acetate
into serum non-saponifiable lipids.
these five-member ring-fused nitrogen-containing
pyridine and pyrimidine compounds can be used
as a surrogate for the structurally complex hexa-
hydronaphthalene ring present in the naturally
occurring HMG-CoA reductase inhibitors.
2. However, the variations at other positions of the
nucleus affected their substantial activities in a
different manner in each series. In particular,
many compounds with a variety of substituents for
R2 and R3 showed significant activities in the pyr-
azolopyridine series. This work shows that further
modulation and improvement in potency at inhi-
biting HMG-CoA reductase may be obtained with
a variety of additional substituents in this series.
On the other hand, the optimum potency was
retained in the simplest compound with no R2 and
R3 substituents in the thienopyridine series. This
fact may allow the design of a compound with
high potency without any introduction of meta-
bolically unfavorable substituents.
3. While no clear correlation exists between the sub-
stantial activity in cell free system, inhibitory
activity in cell culture, and the in vivo potency of
these sets of compounds, the pharmacokinetic
properties of the compounds such as bioavail-
ability, membrane permeability, and metabolic
degradation do have an effect on these parameters.
In a limited number of compounds prepared, potent
inhibitors which exceeded pravastatin’s activity in vitro
and showed marked steroidgenesis inhibitory activities
in vivo were found largely in pyrazolopyridine series 2a.
In general, replacement of the isopropyl substituent for
R1 by cyclopropyl resulted in an appreciable increase in
potency (2a–1 vs 2a–2, 2a–3 vs 2a–4, 2a–5 vs 2a–6, 2c–5
vs 2c–6, 2c–8 vs 2c–9, 2d–1 vs 2d–2). Summary of results
obtained in each series: (i) Pyrazolopyridine (2a–1 to
2a–18); many compounds in this series were exceedingly
potent inhibitors in cell free test; their IC50 values were
in the order of nM. Some compounds were more active
than pravastatin by factors of over 100 in the Hep G2
test (2a–2 and 2a–10). (ii) Isoxazolopyridine series (2b–
1, 2b–2); although the compounds in this series were
almost equipotent or more potent than pravastatin in
cell free system, their inhibitory activities diminished in
Hep G2system and in vivo. (iii) Thienopyridine series
(2c–1 to 2c–11); whereas significant loss of activity was
observed by incorporation of phenyl into the scaffold
(2c–4, 2c–7), many compounds in this series were nearly
half as active as pravastatin in cell free test and were
more active by a factor of over 20 in the Hep G2 test.
Alkyl or phenyl substituents for R2,R3 were not essen-
tial to increase the in vitro potency. The most potent
compound in this series was R2,R3 unsubstituted ana-
logue 2c–1, which showed marked inhibitory activity in
vivo. (iv) Pyrazolopyrimidine series (2d–1 to 2d–9);
introduction of the bulky alkyl group or aryl group for
the R2 substituent led to a slight reduction in potency in
cell free test (cf. 2d–1 and 2d–2 with 2d–3, 2d–4, 2d–5,
and 2d–6). Generally, the compounds in this series
showed diminished activities in Hep G2tests.
References and Notes
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Conclusion
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1. In agreement with literature findings, prominent
activity resides in compounds possessing the 4-(4-
fluorophenyl) and isopropyl (cyclopropyl) sub-
stituent in these scaffolds. Most compounds in
these series were equipotent or more potent than
pravastatin in vitro and some of them showed
marked inhibitory activities in vivo. Therefore,