Synthesis and SAR studies of fused oxadiazines as γ-secretase modulators for treatment of Alzheimer's disease

Article abstract of DOI:10.1021/ml300209g

Fused oxadiazines (3) were discovered as selective and orally bioavailable γ-secretase modulators (GSMs) based on the structural framework of oxadiazoline GSMs. Although structurally related, initial modifications showed that structure-activity relationsh

Full text of DOI:10.1021/ml300209g

Letter
pubs.acs.org/acsmedchemlett
Synthesis and SAR Studies of Fused Oxadiazines as γ‑Secretase
Modulators for Treatment of Alzheimer's Disease
Xianhai Huang,*,† Wei Zhou,† Xiaoxiang Liu,† Hongmei Li,† George Sun,† Mihirbaran Mandal,†
Monica Vicarel,† Xiaohong Zhu,† Chad Bennett,† Troy McCraken,† Dmitri Pissarnitski,† Zhiqiang Zhao,†
David Cole,† Gioconda Gallo,† Zhaoning Zhu,† Anandan Palani,† Robert Aslanian,† John Clader,†
Michael Czarniecki,† William Greenlee,† Duane Burnett,† Mary Cohen-Williams,‡ Lynn Hyde,‡
Lixin Song,§ Lili Zhang,§ Inhou Chu,∥ and Alexei Buevich⊥
‡
§
∥
†Department of Medicinal Chemistry, Department of In Vitro Biology, Department of In Vivo Biology, Drug Metabolism, and
⊥Structual Elucidation, Merck Research Laboratory, 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States
S
* Supporting Information
ABSTRACT: Fused oxadiazines (3) were discovered as
selective and orally bioavailable γ-secretase modulators
(GSMs) based on the structural framework of oxadiazoline
GSMs. Although structurally related, initial modifications
showed that structure−activity relationships (SARs) did not
translate from the oxadiazoline to the oxadiazine series.
Subsequent SAR studies on modifications at the C3 and C4
positions of the fused oxadiazine core helped to identify GSMs
such as compounds 8r and 8s that were highly efficacious in vitro and in vivo in a number of animal models with highly desirable
physical and pharmacological properties. Further improvements of in vitro activity and selectivity were achieved by the
preparation of fused morpholine oxadiazines. The shift in specificity of APP cleavage rather than a reduction in overall γ-secretase
activity and the lack of changes in substrate accumulation and Notch processing as observed in the animal studies of compound
8s confirm that the oxadiazine series of compounds are potent GSMs.
KEYWORDS: Alzheimer's disease, oxadiazine, amyloid precursor protein, γ-secreatase modulator, Notch processing, morpholine
lzheimer's disease (AD) is an age-related neurodegener-
Aative disorder that affects millions of elderly people in the
United States. It is estimated that more than 35 million people
suffer from AD worldwide, with an annual cost of over $600
billion, and the population may increase to more than 115
million by 2050.1 Because of clear unmet medical need, both
academic and industrial laboratories are working very
aggressively to develop therapies to halt or even reverse AD.
It is believed that the accumulation of amyloid-β (Aβ) peptide
and hyperphosphorylated protein τ contributes to AD
Figure 1. Sequential cleavage of APP by β- and γ-secretases.
progression.2−6 Aβ peptide is formed from a larger amyloid
precursor protein (APP) via sequential proteolytic cleavage by
β- and γ-secretases (Figure 1).7 γ-Secretase cleaves the APP C-
terminal fragment at multiple sites leading to Aβ peptides of
>10 for GSMs and <3 for GSIs. There are two major classes of
GSMs in clinical trials: one is the nonsteroidal anti-
37−42 amino acids of which Aβ42, the more hydrophobic form,
is the most amyloidogenic and neurotoxic. Although the
molecular mechanism of action remains largely unknown, γ-
secretase modulators (GSMs) are believed to act at an allosteric
site to shift the predominant site of γ-secretase cleavage toward
shorter, nonamyloidogenic peptides (e.g., Aβ38) by selectively
inhibiting Aβ42 formation without blocking overall γ-secretase
function. This potentially offers a better selectivity window over
γ-secretase inhibitors (GSIs)8−10 versus, for example, notch
processing.11 From an in vitro point of view, the Aβtotal/Aβ42
ratio can be used to distinguish GSMs from GSIs with a ratio of
inflammatory acids (NSAIDs),12,13 and the other is the non-
NSAIDs class such as lactam 1 from Eisai. In our effort in the
AD area, we have recently identified multiple structural
classes14−18 of GSMs with good in vitro potency and selectivity.
Herein, we report our continued structure−activity relationship
Special Issue: Alzheimer's Disease
Received: July 22, 2012
Accepted: September 4, 2012
Published: September 4, 2012
© 2012 American Chemical Society
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Figure 2. Design rationale of oxadiazine GSMs.
Figure 3. Comparison between oxadiazoline/oxadiazine of initial SAR studies. Each IC50 value is an average of at least two determinations.
a
Table 1. SAR Studies of C3-Monosubstituted Oxadiazines
a
Each IC50 value is an average of at least two determinations. Racemic at C3 except 7i, which was the S-isomer as prepared from chiral starting
material.
(SAR) effort focusing on novel fused oxadiazine core structures,
which resulted in the identification of compounds with potent
in vitro and in vivo activity and highly desirable physicological
and physical properties.
Focusing on the non-NSAID GSM class represented by
compound 1 to further optimize its overall pharmacological and
pharmacokinetic (PK) profile and address its lenticular toxicity
issue,7 we have recently identified cyclic hydroxyamidines such
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a
Table 2. SAR Studies of C4-Monosubstituted Oxadiazines
a
Each IC50 value is an average of at least two determinations. The stereochemistry of the more active enantiomer is as shown in the table. The
absolute stereochemistry was confirmed by synthesis from chiral starting material.
as oxadiazolines (2, Figure 2) as highly efficacious GSMs in
both in vitro studies and in vivo animal models. These
oxadiazolines were designed as novel isosteric replacements of
amides with a consideration of hydrogen-bonding character-
istics and lack of strong basicity. They were found to not only
be chemically stable but also possess highly desirable PK and
toxicological profiles.17 With the validation of five-membered
cyclic hydroxyamidines as lactam carbonyl isosteric replace-
ments and to further improve properties of this series of
compounds, we became interested in fused oxadiazines (3),
which are modestly basic and could offer opportunities to
improve activity and enable salt formation for ease of
formulation for rapid absorption.
Equipped with SAR information from the oxadiazoline series,
we believed that it would be possible to identify a lead
compound from the oxadiazine series considering the similarity
of the two core structures. Because the left-hand imidazolyl-
phenyl unit is necessary for potency,17 we chose to focus our
SAR studies on the modification of the right-hand side. Because
of better activity of C3-disubstituted compound to mono-
substituted oxadiazolines,17 we first prepared C4-disubstituted
oxadiazine compounds 4−6 (racemic, Figure 3). To our
surprise, it turned out that the SAR clearly did not translate
from the oxadiazoline to the oxadiazine series. C4 methyl 4-
fluorophenyl-substituted compound 4 lost in vitro potency by
4-fold vs 4′, and the spiro compound 6 was about 7-fold less
active as compared to 6′. More dramatically, hydroxylmethyl
oxadiazine 5 was much less active than oxadiazoline compound
5′. This SAR trend prompted us to consider that the two series
might have different conformational interactions with the
enzyme such that substitution at C3 of the oxadiazine core was
preferred. Therefore, we decided to turn our effort to C3
modifications and carry out focused SAR studies (third degree
SAR studies).19
Indeed, more tractable SAR was realized with the
modifications at C3 (Table 1). We first introduced a
hydroxymethyl group for ease of functionalization. Although
this compound (7a) was not very potent, its TBDPS protected
form (7b) caught our attention due to its much improved Aβ42
inhibition even though the Aβtotal/Aβ42 selectivity was not
desirable. We then explored the benzyl (7c) and phenyl (7d)
ethers, which displayed moderated activity, with 7d having
improved selectivity as compared to compound 7b. This
suggested that the aromatic ring might be playing a role in the
activity. By moving the aryl group closer to the oxadiazine ring
(7e), we saw much improved binding activity and selectivity as
compared to the primary alcohol 7a. The hydroxyl group did
not appear to be crucial for activity. Compound 7f retained
similar potency and selectivity. When the methylene group of
7f was eliminated, a slightly decreased activity was observed
(7g). Aryl modifications such as naphthyl (7h) were tolerated
and retained good activity and selectivity. Further improvement
of potency was achieved by introduction of the trifluorophenyl
group, and compound 7i showed very good Aβ42 activity and
good selectivity. With this improved in vitro profile, 7i was
further studied in in vivo, PK, and ancillary profile evaluations.
Compound 7i displayed 45% inhibition of Aβ42 in the rat
cerebrospinal fluid (CSF) at the 3 h time point following acute
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oral administration at the 10 mg/kg dose. An oral rat PK study
at a dose of 10 mpk showed high exposure with a plasma AUC
of 24300 nM h, substantial brain concentration (1885 ng/g) at
6 h, and a brain/plasma ratio of 1.0. It did not inhibit CYP
P450s or induce PXR in in vitro studies; however, 83%
inhibition of hERG was observed in a function assay at a
concentration of 10 μM. In an attempt to improve the hERG
profile, we introduced polar groups such as heteroaryl
substitutions at the C3 position. Compounds 7j and 7k
showed much reduced Aβ42 activity. A carbonyl substitution at
C4 (7l) also resulted in decreased activity.
potent in vitro and in vivo activity and excellent rat PK profile
(bioavailability, 100%; T1/2, 3.5 h; Vdss, 0.4 L/kg; and CL, 1.4
mL min−1 kg−1), compound 8s was further profiled. This
compound was found to dose dependently reduce Aβ42
preferentially over Aβtotal and Aβ40 upon acute oral admin-
istration to rats,17 thus confirming modulator activity in vivo.
Compound 8s displayed pKa values of 7.4 and 2.7, indicating
that the oxadiazine ring was modestly basic, which conferred to
a highly desirable aqueous solubility of the HCl salt of >100
mM at pH 3.5. Compound 8s was highly permeable in a Caco-
2 membrane permeability assay and did not appear to be a
substrate for PGP efflux. Even though a 76% inhibition of
hERG activity was observed at 10 μM, no affect on QTc
intervals in dogs at sufficient exposure multiples over exposures
required for pharmacological effects were observed (data not
shown). Compound 8s did not affect Notch processing in
HEK293 cells expressing the human Notch1 protein at
concentrations up to 50 μM. There was no evidence of
Notch-related side effects or biomarker changes following
repeated administration of the highest tolerable doses of 8s to
rats.17
To further improve the overall profile of this series of
compounds, we also prepared fused morpholine oxadiazines
(9−12) by replacing the C8 carbon with an oxygen atom. This
modification could block potential metabolism at C8 and, by
modifying the overall electronic proterties of the molecule,
might also offer advantageous properties. On the basis of SAR
studies of the morpholine oxadiazoline series,21 the C7 methyl
group was installed to further improve activity. As shown in
Figure 4, difluorophenyl derivatives (9 and 10) showed
To further improve the in vivo activity and overall profile of
7i, we decided to revisit substitutions at the C4 position by
systematically focusing on monosubstitution.19 As summarized
in Table 2, the absence of substitution at C3 and C4 (8a)
resulted in loss of activity. The carbonyl group at C4 did not
improve activity (8b). However, when small alkyl groups such
as isopropyl (8c) were introduced, improved activity was
observed. Steric bulk was tolerated as both isobutyl and tert-
butyl substitutions gave compounds 8d and 8e with good
activity and reasonable selectivity. Tetrahydropyran-substituted
compound 8f showed decreased activity, which might be due to
the introduction of the polar oxygen atom. A similar SAR trend
was observed with C3 modification. The hydroxyl group (8g)
did not improve activity, but benzyl ether (8h), aryl-substituted
secondary alcohol (8i), and simple benzyl substitution (8j)
showed improved activity. To modify the physical properties of
these compounds, polar groups were introduced at C4 position
to decrease the clog D value. However, all of these changes
resulted in much decreased activity [thiozole 8k (6400 nM),
sulfone 8l (19000 nM), amide 8m (820 nM), morpholine 8n
(3700 nM), and reverse amide 8o (1500 nM)]. On the other
hand, when aryl substitutions were introduced, consistently
good activity and selectivity (8p−t) were achieved. In
particular, the introduction of halogens to the aryl groups
further improved the binding activity and Aβtotal to Aβ42
selectity (8q−t). To differentiate these compounds for further
profiling, they were characterized in PK and in vivo studies. As
summarized in Table 3, compounds 8r and 8s showed highly
Table 3. Rat PK Profile20 and in Vivo Activity of
Compounds 8p−t
brain
B/P
CSF Aβ42
brain
e
plasma
e
a
b
c
d
compd AUC0−6h
concn
ratio
reduction (%) level
level
8p
8q
8r
8s
8t
17620
86460
24580
87410
22680
539
2846
1340
2907
268
0.9
0.8
1.1
0.4
39
53
62
62
26
2.8
6.2
6.7
7.7
Figure 4. Identification of highly potent and selective fused
morpholine oxadiazine GSMs. Each IC50 value is an average of at
least two determinations.
f
f
f
ND
ND
ND
7.2
10.7
6.3
1.1
a
b
nM h with 10 mpk, po dosing. ng/g at 6 h from rapid rat PK studies.
c
d
comparable in vitro activity to the trifluorophenyl analogs (11
and 12). In both cases, the (4S,7S)-isomers had better Aβ42
inhibition and Aβtotal/Aβ42 selectivity than the (4S,7R)-
isomers.22 Compound 12 represented one of the most potent
and selective GSMs that we identified. This compound
displayed excellent in vivo activity, reducing Aβ42 in rat (60%
reduction of CSF Aβ42 at 3 h with 10 mpk acute oral dosing),
and provided opportunities for future SAR development.
In summary, in an effort to identify GSMs to treat AD, we
discovered a series of fused oxadiazines (3) as selective and
orally bioavailable GSMs based on the structural framework of
oxadiazolines GSMs. Although structurally related, initial
studies showed that SAR did not translate from the
At 6 h from rapid rat PK studies. Average of multiple testings at the
e
3 h time point with 10 mpk acute oral dosing in PD studies. μM,
average of multiple testings at the 3 h time point with 10 mpk acute
oral dosing in PD studies. Not determined.
f
potent in vivo activity with both compounds resulting in 62%
reduction of CSF Aβ42 following oral dosing of 10 mg/kg. On
the basis of the correlation between the decreased in vivo
activity of compounds 8p and 8t and their lower brain exposure
at the 6 h time point in rapid rat PK studies and at the 3 h time
point in the PK/PD studies (for 8p), sufficient brain exposure
seemed important for improved CSF activity. Because of its
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Letter
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ASSOCIATED CONTENT
* Supporting Information
■
S
Biological assay protocols, general experimental descriptions
and procedures, and characterization of final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +1 732 594 4734. E-mail: xianhai.huang@merck.com.
■
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M.; Jones, N. T.; Josien, H.; Hyde, L. A.; Lee, J.; McKittrick, B.; Song,
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Andrew Stamford for comments on the
preparation of the manuscript. We thank Dr. Eric Parker for
his strong support of the program. We thank Dr. Li-Kang
Zhang for technical support.
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