The discovery of pyridone and pyridazone heterocycles as γ-secretase modulators

Article abstract of DOI:10.1021/ml1000799

A series of novel pyridazone and pyridone compounds as γ-secretase modulators were discovered. Starting from the initial lead, structure?activity relationship studies were carried out in which an internal hydrogen bond was introduced to conformationally fix the side chain, and compounds with improved in vitro Aβ42 inhibition activity and good Aβtotal/Aβ42 selectivity were quickly discovered. Compound 35 displayed very good in vitro activity and excellent selectivity with good in vivo efficacy in both CRND8 mouse and nontransgenic rat models. This compound displayed a good overall profile in terms of rat pharmacokinetics and ancillary profile. No abnormal behavior and side effects were observed in all of the studies.

Full text of DOI:10.1021/ml1000799

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The Discovery of Pyridone and Pyridazone Heterocycles
as γ-Secretase Modulators
Xianhai Huang,*,† Robert Aslanian,*,† Wei Zhou,† Xiaohong Zhu,† Jun Qin,†
William Greenlee,† Zhaoning Zhu,† Lili Zhang,‡ Lynn Hyde,‡ Inhou Chu,§
Mary Cohen-Williams,‡ and Anandan Palani†
Departments of †Medicinal Chemistry, ‡Neurobiology, and §Pharmacology, Merck Research Laboratory,
2015 Galloping Hill Road, Kenilworth, New Jersey 07033
ABSTRACT A series of novel pyridazone and pyridone compounds as γ-secretase
modulators were discovered. Starting from the initial lead, structure-activity
relationship studies were carried out in which an internal hydrogen bond was
introduced to conformationally fix the side chain, and compounds with improved
in vitro Aβ42 inhibition activity and good Aβtotal/Aβ42 selectivity were quickly
discovered. Compound 35 displayed very good in vitro activity and excellent
selectivity with good in vivo efficacy in both CRND8 mouse and nontransgenic rat
models. This compound displayed a good overall profile in terms of rat pharma-
cokinetics and ancillary profile. No abnormal behavior and side effects were
observed in all of the studies.
KEYWORDS Pyridazone, pyridone, γ-secretase, modulator, Alzheimer's disease
lzheimer's disease (AD) is an age-related neurodegen-
erative disorder (cognitive impairment, loss of mem-
ory, and language ability) that affects millions of older
γ-secretase modulator compound (3) that was discovered
based on the idea of mimicking the double bond in compound
2 with intramolecular hydrogen bonding between the hydroxyl
and the pyridazone carbonyl groups. Herein, we report our
structure-activity relationship (SAR) effort based on this lead to
quickly identify compounds with good in vitro and in vivo
activity with good Aβtotal/Aβ42 selectivity.
A
people in the United States.1,2 To date, there is still an unmet
medical need for treatments of this disease, and research
laboratories across academia and pharmaceutical companies
are working aggressively to identify new therapeutic agents to
cure or, at a minimum, retard progression of it. Although the
cause of the AD is still debated,3-5 short Aβ peptides generated
from the cleavage of Aβ peptides in the brain are believed to be
one of the main contributors to the disease.6 γ-Secretase is a
biochemically complex aspartyl protease enzyme, which, when
coupled with β-secretase, can process amyloid precursor pro-
tein (APP) to produce Aβ peptides. Soluble oligomeric forms of
Aβ peptides have been proposed as the neurotoxic agents.
Inhibition of these enzymes would reduce production of Aβ,
which should slow or halt the progression of cell death and
cognitive decline. γ-Secretase cleavage of CTFβ leads to Aβ
peptides of 37-42 amino acids of which Aβ42, the more
hydrophobic form, is most toxic.7 γ-Secretase modulators shift
the γ-secretase cleavage toward short peptides by selectively
inhibiting Aβ42 without blocking overall γ-secretase function
(Aβtotal). Modulation as opposed to outright inhibition should
offer a potentially improved side effect profile;for example,
versus notch processing.8-10 Because of this advantage, re-
search in this field has heated up in recent years in the pharma-
ceutical industry. There are two major classes of γ-secretase
modulators in clinical trials. One is structure-related to nonster-
oidal anti-inflammatory acids (NSAIDs),11 , 1 2 such as 1 from
Myriad. The other is the non-NSAIDs class, such as 2 from Eisai.
In our effort in the AD area, we have recently identified an initial
Initial SARs focusing on the pyridazone core and modifi-
cation of the left-hand side imidazole suggested that the
methyl group was important for in vitro activity. When there
was no methyl substitution on the imidazole, the Aβ42
inhibition decreased by at least 2-fold (4, Table 1). We then
turned our attention to the central linker modification. When
the hydroxyl methyl group was converted to a carbonyl group,
the activity increased by about 3-fold (5). Hypothesizing that
an NH group as the linker might hydrogen bond with the
pyridazone carbonyl group and lock the side chain in the
same conformation as the double bond in the lead structure
2 (Figure 1, 3 to 30), we prepared compound 6, which
showed improved activity. Although the desired H-bond
Received Date: April 16, 2010
Accepted Date: May 18, 2010
Published on Web Date: May 24, 2010
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Table 1. SAR Studies of the Pyridazone Series
Table 2. Initial SAR Studies of the Pyridone Series
a Each IC50 value is an average of at least two determinations.
position. As summarized in Table 1, introduction of alkyl ethers
at C6 was tolerated and gave reasonably good in vitro activity
(11-13). Bulky substituents did not affect the activity too
much and gave comparable Aβ42 inhibition and Aβtotal/42
selectivity (14). This suggests that there is space available for
further modification at this position. C4 substitution with an
amine linker proved to be important for in vitro activity. When
the biaryl aniline was moved from the C4 to the C5 position,
we saw more than a 10-fold decrease of activity (15). When an
oxygen linker was introduced (16), the activity was 4-fold less
potent as compared to its amino analogue (11).
a Each IC50 value is an average of at least two determinations. b n.d.=
not determined.
Figure 1. Employing intramolecular hydrogen bonding to lock
the confirmation.
To further improve the in vitro activity, we decided to turn
our attention to core modifications and began by adjusting the
electron density of the core. We quickly identified that the
pyridone core was a suitable substitution of the pyridazone
core. We first introduced the amide linker (17 and 18, Table 2)
in this series. Compound 17 showed better activity than 18,
which had a reversed amide linker, but both were not as good
as the pyridazone analogues. On the other hand, when the
amino group was used as the linker, compound 19 displayed
obviously improved in vitro activity and selectivity. We then
decided to focus on modification of the C5 position with the
amino group as the linker. Ester (20) and aldehyde (21) groups
were tolerable at this position and gave good Aβ42 inhibition
and Aβtotal/42 selectivity. When a series of amide groups
were introduced (22-25), only moderate in vitro activity was
effect was not observed, we thought that the C5 methoxy
group might diminish the effect because of steric repulsion.
Further improvement of the activity was indeed achieved by
removing the C5 methoxy group, and the IC50 value had a
dramatic improvement to 345 nM (7), a 10-fold increase. This
result suggests that the bulkyl group at C5 is not amenable to
improving the in vitro activity, and intramolecular hydrogen
bonding may indeed have a beneficial effect. This observation
was further confirmed with the preparation of 9 and 10. A
6-fold loss of activity was observed when the bromine atom
was introduced at C5. When the methyl group at the right-
hand benzylic position (N2) was installed, the in vitro activity
did notchange significantly (8). To further improve the activity,
we next turned our attention to modification of the C6
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Table 3. SAR Studies of the Pyridone Series Focusing on the C5
Modification
Scheme 1. Synthesis of Pyridone Analogue 35
SAR effort was focused on the compounds with the S confi-
guration. A carboxylic acid group at the C5 position (32) was
not a good choice in terms of improving the Aβ42 inhibition.
Smaller electron-withdrawing groups seemed to improve
activity. With the introduction of a bromine atom, the com-
pound showed much improved Aβ42 IC50 value (33, 60 nM)
and excellent selectivity over Aβtotal (251 fold). A hydroxy
methyl group was tolerated and further improved the in vitro
activity (34, 48 nM). When a difluoromethyl group was intro-
duced (35), further improvement in selectively was ob-
served. A stronger electron-withdrawing group such as the
trifluoromethyl group seemed not very helpful at improving
the activity (36). At this point, we wanted to find out whether
the C5 and N1 substitutions had a synergistic effect on the in
vitro potency. We therefore introduced polar groups on the
methyl side chain at the N1 position. Even with a bulky
TBDMS group at the right-hand side, racemic compound 37
showed 3-fold better activity than compound 29. This sug-
gests that a large cavity may be available at the N1 position
for further SAR modification. Interestingly, the more polar,
and smaller, hydroxy methyl benzyl group at the N1 position
was tolerated, and compound 38 had an Aβ42 IC50 value at
114 nM as a racemic mixture.
As summarized in Table 3, compound 35 showed one of the
best in vitro profiles in terms of enzyme activity and Aβ42
selectivity over Aβtotal. Therefore, it was further profiled in in
vivo studies. This compound showed very good in vivo efficacy
in a CRND8 mouse model, giving over 85% reduction of Aβ42
in plasma at 30 mpk with little effect on the Aβtotal. In the
nontransgenic rat in vivo model,14 this compound displayed a
40% reduction of Aβ42 in the CSF at 100 mpk and a 26%
reduction of Aβ42 in brain, while the Aβtotal only had a 7%
reduction in the CSF. Compound 35 had good rat PK with an
AUC1-6h of 7.5 μM.h at 10 mpk and favorable brain concen-
tration (347.3 ng/g) at the 6 h time point. No abnormal
behavior or side effects were observed in those studies.
To demonstrate the synthesis of the pyridone analogues,
the synthetic route to compound 35 is illustrated in Scheme
1. Starting from commercially available compound 39, a
Mitsunobu reaction with (R)-1-hydroxy-1-(4-fluorophenyl)-
ethane gave enantiomerically pure 40. The ester group was
converted to alcohol 41 in three steps15 since direct reduc-
tion with LiAlH4 resulted in a complex mixture. Compound
41 was converted to difluoro compound 42 in two steps via
Dess-Martin oxidation and fluorination. A final coupling
a Each IC50 value is an average of at least two determinations.
observed; an aromatic ring (22) and alkyl group (23) did not
change the IC50 value significantly. In comparison, when a
smaller group such as a cyano groupwas introduced, compound
26 showed improved activity and selectivity even though it had
similar potency and selectivity as the unsubstituted compound
19. A methyl group at the right-hand side benzylic position (N1)
again did not affect the activity too much, and compound 27
showed similar in vitro activity to 26.However,themethylgroup
did have some impact on the pharmacokinetic (PK) profile of
these compounds. The rat oral PK13 at 10 mpk (0-6 h) showed
compound 27 had a better brain concentration (917.3 ng/g) at
the 6 h time point than compound 26 (391.3 ng/g), which is
desirable in the current project.
Because of the improved PK properties provided by the
methyl group, we next focused our SAR efforts on modifica-
tion of the C5 position with the R-methylbenzyl group
installed at the right side N1 position in the hope to identify
compounds with a good overall profile (Table 3). When there
was no substitution at C5, compound 28 retained good in
vitro activity and selectivity. The configuration of the sub-
stituent on N1 had a clear impact on activity. Racemic
compound 29 had an IC50 value of 163 nM. Its enantiomers,
however, displayed different activity. The R isomer (30) was
3-fold less active than the S isomer (31). Thus, our further
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Scheme 2. Synthesis of Pyridazone Analogues
(3)
(4)
(5)
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reaction using Pd(OAc)2 gave the desired product in moder-
ate yield. Other pyridone compounds were prepared in a
similar fashion. The synthesis of pyridazone compounds
was straightforward and is shown in Scheme 2. Bromides
44 and 45 were obtained from compound 43 by treatment
with NH2NHBn. Methylation of compound 45 furnished 46,
which was coupled with aniline 47 using a catalytic amount
of Pd(OAc)2 to give the final product 11.
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In summary, we have indentified a series of novel pyrida-
zone and pyridone compounds as γ-secretase modulators.
Starting from the initial lead, we have carried out SAR studies
employing a strategy that utilized an internal hydrogen bond
to lock the conformation of the side chain present in the lead
structure. The new analogues displayed an improved in vitro
Aβ42 activity and good Aβtotal/Aβ42 selectivity. Compound
35 displayed very good in vitro activity and excellent selec-
tivity with good in vivo efficacy in both the CRND8 mouse
and the nontransgenic rat models. This compound had a
good overall profile in terms of rat PK and ancillary profile
such as clean hERG, clean hPXR, acceptable P450 inhibition
profile, and good human hepatocyte clearance data (2.9 μL/
m/M cell). Further profiling is in progress, and the result will
be reported in due course.
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Nomeir, A. A.; Josien, H.; Bara, T.; Clader, J. W.; Zhang, L.;
Parker, E. M. Studies to investigate the in vivo therapeutic
window of the γ-secretase inhibitor N2-[(2S)-2-(3,5-difluoro-
phenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-di-
hydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide (LY411,575)
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(14) See the Supporting Information for the detailed assay proce-
dure.
(15) Kokotos, G.; Noula, C. Selective one-pot conversion of
carboxylic acids into alcohols. J. Org. Chem. 1996, 61, 6994.
SUPPORTING INFORMATION AVAILABLE Experimental
procedures and spectral data for compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. (X.H.) Tel: 908-740-3487. Fax: 908-740-7664. E-mail:
xianhai.huang@spcorp.com. (R.A.) Tel: 908-740-3514. Fax: 908-
740-7152. E-mail: robert.aslanian@spcorp.com.
ACKNOWLEDGMENT We thank Drs. Ismail Kola and Malcolm
MacCoss for their strong support of the program.
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