Novel 4-piperidinopyridine inhibitors of oxidosqualene cyclase-lanosterol synthase derived by consideration of inhibitor pKa

Article abstract of DOI:10.1016/S0960-894X(01)00423-1

Potent inhibition of rat microsomal oxidosqualene cyclase-lanosterol synthase (OSC) was maintained after structural modification of the 4-piperidinopyridine OSC inhibitor series. These novel analogues with a much lower pK_a range (5.8-6.7) gave

Full text of DOI:10.1016/S0960-894X(01)00423-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2213–2216
Novel 4-Piperidinopyridine Inhibitors of Oxidosqualene
Cyclase-Lanosterol Synthase Derived by Consideration of
Inhibitor pKa
George R. Brown,* Alan J. Foubister, Michael C. Johnson, Nicholas J. Newcombe,
David Waterson and Stuart L. Wells
AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Received 31 January 2001; revised 27 March 2001; accepted 12 June 2001
Abstract—Potent inhibition of rat microsomal oxidosqualene cyclase-lanosterol synthase (OSC) was maintained after structural
modification of the 4-piperidinopyridine OSC inhibitor series. These novel analogues with a much lower pKa range (5.8–6.7) gave
potent oral inhibition of rat cholesterol biosynthesis (8 ED80 0.7 mg/kg), and diminished effects on rat feeding after a 100 mg/kg
oral dose. # 2001 Elsevier Science Ltd. All rights reserved.
The lowering of raised plasma cholesterol levels with the
statin class of HMGCoA reductase inhibitor drugs has
been very successfully used in the treatment of cardio-
vascular disease.1,2 This widespread clinical use of the
statin drugs has led to a search for other inhibitors of
the cholesterol biosynthesis pathway, particularly at
later steps of the biosynthetic cascade. In this respect,
we3 (and others4) have identified 2,3-oxidosqualene
cyclase-lanosterol synthase (OSC, EC 5.4.99.7) as an
appropriate step to seek novel cholesterol biosynthesis
inhibitors. Recently we have described3 in detail a novel
series of N-pyridyl- and N-pyrimidinylpiperidines (e.g.,
1, 2) as inhibitors of microsomal rat and human OSC.
Due to this OSC inhibition, these compounds also
showed inhibition of rat cholesterol biosynthesis to a
similar extent to the statin drug simvastatin. When rats
(n=5) were given a higher oral dose of the pyridyl and
pyrimidinyl derivatives 1 and 2, however, there were
significantly different effects on food intake that were
typical of these series. A 50 mg/kg oral dose of pyridine
1 gave, after 24 h, a 64% reduction in rat food intake.
By contrast, 2 gave only a 17% reduction at 100 mg/kg
(rats dosed vehicle alone gave a 7% reduction). This
reduction in food intake found for the pyridine 1 would
prevent clinical development of compounds from the
pyridine series, because safety testing at higher doses
could not be completed. Herein we report the synthesis
of novel pyridine OSC inhibitors designed to have a
lower potential for feeding effects in rats (Fig. 1).
The mechanism of the effects of these compounds on
feeding is not known, and could not readily be deter-
mined. Thus, a basis was sought for the discovery of
pyridines with a limited effect on rat feeding, without
the need for the synthesis of large numbers of random
compounds supported by rat feeding tests. Examination
of the pKa values of the terminal pyridine and pyr-
imidine rings of 1 and 2 showed that as well as having
different feeding effects, they had very different pKa
*Corresponding author. Tel.: +44-1625-515918; fax: +44-1625-
516667; e-mail: george.r.brown@astrazeneca.com
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00423-1

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G. R. Brown et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2213–2216
values (Table 1). The pyridine 1 and a previously
described5 quinuclidine OSC inhibitor 3 had a much
higher pKa (>9) than the pyrimidine 2. In addition, a
known standard OSC inhibitor compound 4 with a
weak feeding effect in rats (4 15% reduction at 100 mg/
kg) also showed a lower pKa value. Thus two com-
pounds from different OSC inhibitory series have mar-
ginal feeding effects and much lower pKa values than the
pyridine inhibitors. It was postulated that lowering of
the pyridine ring pKa by appropriate ring substitution
might afford OSC inhibitory pyridines with a reduced
potential for feeding effects. This hypothesis was
approached in two ways. First the lead series (Fig. 2A)
was halogenated to give the 3-halogen-substituted pyr-
idines (e.g., B). This electron withdrawing halogen sub-
stituent should counteract the electron donating effect
of the 4-piperidino ring nitrogen atom present in A
(Table 3, 5–11). A pyrimidine was also similarly sub-
stituted (e.g., C). In the second approach (e.g., D), the
electron donating nitrogen atom in the piperidine ring
was replaced by a carbon atom to give analogues 12–15
(Table 4).
Scheme 1.
Table 1. Comparison of physical properties in OSC inhibitory series
Compd
pKa
IC50 nM inhib. rat OSC
1
2
3
4
9.2
6.1
9.1
4.7
228ꢁ49
116ꢁ21
58ꢁ19
90ꢁ14
Scheme 2.
Figure 2.
Biological Results and Discussion
Chemistry
The chloro- and bromo-substituted compounds 5–9 and
11 were prepared by halogenation (Scheme 1) of the
unsubstituted pyridine/pyrimidine ring with the appro-
priate N-halosuccinimide in MeCN at room tempera-
ture. Compound 10 was synthesised from the known6 3-
fluoro-4-iodopyridine and the piperidine3 16 by the
reported3 general method. The starting materials for 9,
and 11 are known.3 For 5–8 the starting materials were
synthesised by reaction of 17a in DMF at 100 ꢀC
(Scheme 2) with the 4-nitrophenylcarbamate derivative
of the known3 phenylsulphonylpiperazines of general
structure 18. Compounds 12–14 were prepared from 17b
in a similar manner. Selective reduction of 4,40-dipyridyl
19 by hydrogenation at 50 ꢀC in H2O over 5% Rh/C (5
atm, 7 h) gave the piperidine intermediate 17b. Com-
pound 15 was prepared by reaction of 17a and 20
(Scheme 2).
Inhibitor pKa was determined for examples of the two
approaches to pKa lowering, using a Beckman DU-7
spectrophotometer. Much lower pKa values were con-
firmed for the new compounds (Table 2), compared
with the pyridine 1. An unexpectedly high pKa value of
6.7 was found for the fluoro-substituted 10, compared
to the chloro derivative 5 (pKa 5.8). This higher pKa was
attributed to the absence of a twisting effect on the
Table 2. pKa measurements of typical pyridines
Compd
pKa
CLOGP
1
5
10
12
9.2
5.8
6.7
5.8
3.4
3.7
3.7
3.2

G. R. Brown et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2213–2216
2215
piperidine ring from the fluoro atom of 10. Thereby a
greater electron donation into the pyridine ring would
occur from the piperidine ring nitrogen atom.
Table 4. Oral inhibition of rat cholesterol biosynthesis and rat OSC
in vitro after carbon substitution
The strategy employed for evaluation of compounds as
OSC inhibitors has been fully described.3,5 Compounds
were assessed first in oral cholesterol biosynthesis tests
in rats before confirmation in vitro that the in vivo
activity found was derived from inhibition of the target
OSC enzyme. Compounds were assessed for inhibition
of cholesterol biosynthesis in rats dosed with tritiated
mevalonate. At 1 h later an oral dose of test compound
was administered. First, the cholesterol biosynthetic
precursor pattern was found from an HPLC analysis
(radiochemical detector) of extracts of saponified liver
samples. This lipid profile was compared with the same
profile from control animals. These HPLC chromato-
grams indicated which steps of cholesterol biosynthesis
had been inhibited, i.e., whether the enzyme inhibition
was selective for the OSC step. Second, the level of
cholesterol present was also measured from the same
HPLC chromatograms, and initially expressed as an
oral ED50 or ED80 for the inhibition (n=5). Confirma-
tion of OSC inhibition was carried out in a rat micro-
somal OSC assay3 at compound concentrations of 100
nanomolar. Food intake was determined 24 h after an
oral dose of test compound to chow pellet fed rats
(n=5). The percentage reductions in food eaten were
found by comparison with the food intake in the same
animals dosed the previous day with vehicle alone.
Compd
R2
X
ED50
(mg/kg)
ED80
(mg/kg)
OSC% inhibition
(100 nM)
12
13
14
15
4-Cl
4-Br
4-F
N
N
N
C
1.7
0.4
0.5
0.5
3.6
1.8
—
91
94
97
100
4-Br
4.7
approach (Table 4), gave similar results for rat OSC and
cholesterol biosynthesis inhibition. Overall the inhibi-
tory potency of 12–15 for cholesterol biosynthesis was
lower than with the halogen-substituted pyridines in
Table 3. Here 13 was the best oral inhibitor of rat cho-
lesterol biosynthesis with an ED80 of 1.8 mg/kg. Com-
pounds 5 and 13 were tested in the rat feeding test as
examples of pyridines from the two pKa lowering
approaches followed. Both compounds gave an
improved feeding effect at oral doses of 100 mg/kg
(5=14%; 13=23%; vehicle dosed control=7%). Thus,
structural modification in the pyridine series of OSC
inhibitors has led to a significant widening of the ratio
of oral inhibition of cholesterol biosynthesis and feeding
inhibition.
The effective doses for the inhibition of rat cholesterol
biosynthesis shown in Table 3 indicate that a consider-
able lowering of pyridine pKa had not diminished the very
potent oral inhibition of rat cholesterol biosynthesis.3
In summary the pyridine OSC inhibitors have been
modified to give analogues with a lower pKa. These
novel pyridines have similar inhibitory potency to the
pyrimidine 2 in terms of the oral inhibition of rat cho-
lesterol biosynthesis and rat microsomal OSC in vitro.
In addition the effect on rat feeding seen with the origi-
nal pyridine series was considerably alleviated. The new
pyridine analogues with a lower pKa were candidates for
development as novel cholesterol lowering agents.
Table 3. Oral inhibition of rat cholesterol biosynthesis and rat OSC
in vitro after halogen substitution
Acknowledgements
Compd R1
R2
X
ED50 ED80
(mg/kg) (mg/kg)
OSC% inhibition
(100 nM)
The authors thank our colleagues Donald J. Mirrlees,
Fergus McTaggart and John W. Myatt for the cholesterol
biosynthesis and OSC inhibition measurements.
5a
6a
7a
8a
9a
10a
11a
1
Cl
Cl
Cl
Cl
Br
F
Cl
Br
F
CF3
Br
CF3
Br
N
N
N
N
C
C
C
0.6
1.1
1.1
0.2
0.5
0.4
>2.0
2.0
0.4
1.4
2.3
2.3
0.7
—
1.0
—
>2.0
1.4
87
100
100
100
89
100
47
100
100
Br
References and Notes
2
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inhibitory in rats at the 2 mg/kg test dose. The second

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G. R. Brown et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2213–2216
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