Discovery of fused 5,6-bicyclic heterocycles as γ-secretase modulators

Article abstract of DOI:10.1016/j.bmcl.2010.12.012

We herein report the discovery of four series of fused 5,6-bicyclic heterocycles as γ-secretase modulators. Synthesis and SAR of these series are discussed. These compounds represent a new class of γ-secretase modulators that demonstrate moderate to good

Full text of DOI:10.1016/j.bmcl.2010.12.012

Bioorganic & Medicinal Chemistry Letters 21 (2011) 664–669
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of fused 5,6-bicyclic heterocycles as c-secretase modulators
Jun Qin a, , Pawan Dhondi a, Xianhai Huang a, Mihirbaran Mandal a, Zhiqiang Zhao a, Dmitri Pissarnitski a,
Wei Zhou a, Robert Aslanian a, Zhaoning Zhu a, William Greenlee a, John Clader a, Lili Zhang b,
Mary Cohen-Williams b, Nicholas Jones b, Lynn Hyde b, Anandan Palani a
⇑
a Department of Medicinal Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Department of Neurodegenerative Diseases, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein report the discovery of four series of fused 5,6-bicyclic heterocycles as
Synthesis and SAR of these series are discussed. These compounds represent a new class of
modulators that demonstrate moderate to good in vitro potency in inhibiting Ab42 production.
c
-secretase modulators.
-secretase
Received 20 October 2010
Revised 1 December 2010
Accepted 2 December 2010
Available online 13 December 2010
c
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
c
-Secretase modulator
Amyloid-b peptide
Notch receptor
Despite Alzheimer’s disease being identified more than
100 years ago, it still remains an urgent medical challenge.1–3
Alzheimer’s disease is a neurodegenerative disorder with clinical
symptoms of cognitive impairment and progressive loss of bodily
functions and language ability. The disease is characterized patho-
logically by neuronal loss, neurofibrillary tangle (NFT) formation,
and extracellular deposition of amyloid-b (Ab) peptide plaques.
Although there are several hypotheses about the cause of the dis-
ease, a predominant one is the amyloid hypothesis.4,5 According
to this hypothesis, reduced clearance and/or increased aggregation
of the Ab peptides, especially Ab40 and Ab42, play a key role in the
pathogenesis of Alzheimer’s disease.
side effects such as thymus atrophy and intestinal goblet cell
hyperplasia.8,9
On the other hand,
inhibit the production of Ab42 without blocking the overall func-
tion of -secretase on CTF and other substrates such as Notch.
c-secretase modulators (GSMs) selectively
c
Therefore this class of molecules should not cause Notch-related
side effects and could offer a better safety profile than GSIs.10 It
is believed that GSMs interact with c-secretase at an allosteric site,
inducing a conformational change in the protease and causing a
shift of cleavage specificity to preferentially produce shorter, more
soluble Ab-peptides such as Ab38
of this allosteric site on -secretase remains unknown. Non-steroi-
.
11–16 However, the exact location
c
Ab peptides are generated by initial cleavage of the amyloid
precursor protein (APP) by b-secretase to form a C-terminal frag-
dal anti-inflammatory drugs (NSAIDs) such as ibuprofen, indo-
methacin, sulindac sulfide and tarenflurbil were the first class of
ment (CTF), which is subsequently proteolyzed by
c
-secretase to
GSMs reported to selectively reduce the production of Ab42 in favor
17–19
produce Ab peptides ranging from 37 to 42 amino acids in length.
Despite accounting for only 10% of the overall Ab peptide produc-
tion, Ab42 is the major component of amyloid plaques. Several
of shorter peptides such as Ab38
.
In addition, they did not in-
hibit the cleavage of other transmembrane protein substrates such
as Notch by
c
-secretase.20 However, they demonstrated limited
classes of
c
-secretase inhibitors (GSIs) have been reported to
potency (Ab42 IC50 = 25–200 lM) and did not improve cognition
demonstrate efficacy in reducing overall production of Ab peptides
in clinical trials in AD patients.21,22
in plasma, cerebrospinal fluid (CSF) and brain.6,7 However,
Eisai reported a GSM 1 which contains a cyclic cinnamamide
motif (Scheme 1).23–25 It demonstrated good in vitro potency and
selectivity as a GSM in our hands (Cell Ab42 IC50 = 64 nM, Abtotal
IC50/Ab42 IC50 ratio = 226).26 This compound also showed robust
in vivo efficacy to reduce CSF and brain Ab42 (À55% CSF Ab42 and
À44% brain Ab42, 3 h after a single oral dose of 30 mg/kg).27
Inspired by this novel structure, we decided to modify the core
of 1 to identify new chemical entities with improved potency
and ancillary profile, while removing the cinnamamide double
c-secretase inhibitors can cause mechanism-based toxicity due
to disruption of the processing of other -secretase substrates such
as Notch and ERB4. Cleavage of Notch is important for cellular gene
transcription, and inhibition of Notch cleavage by GSIs results in
c
⇑
Corresponding author. Tel.: +1 908 740 3904; fax: +1 908 740 7664.
E-mail addresses: jun.qin@merck.com, jun2005qin@yahoo.com (J. Qin).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.012

J. Qin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 664–669
665
Scheme 1. Design of fused 5,6-bicyclic c-secretase modulators.
bond, which could be a potential Michael acceptor. We explored
core modifications by forming an additional five-membered ring
between the cinnamamide double bond and the carbonyl group
in the central core. This modification led to four series of com-
pounds containing a fused 5,6-bicyclic structure as the central core
as depicted in Scheme 1. We conducted SAR on the R region of each
of these series. Herein we discuss the synthesis and SAR data for
each of these series.
We first looked at the tetrahydrotriazolopyrimidine series, and
the synthesis is outlined in Scheme 2. The acid 2 was coupled with
hydrazinopyrimidine to afford 3. With pyridine as solvent at
elevated temperature, 3 underwent intramolecular cyclization to
form triazolopyrimidine core 4, which was then alkylated with
1-(1-bromoethyl)-4-fluorobenzene to afford 5. Analogues 6–24
with different N-substitution were prepared by a similar route to
5, and SAR data of these compounds is summarized in Table 1.
Compound 5 with a 1-(4-fluorophenyl)ethyl group had moderate
activity to inhibit Ab42. Longer alkyl chains attached to the benzylic
carbon improved the potency and selectivity (6 Ab42 IC50 = 156 nM,
7 Ab42 IC50 = 145 nM). Replacement of the simple alkyl chain at the
benzylic carbon with a cyclopropyl group (8, 9) was not tolerated.
Compound 10 with hydrogen substitution at the benzylic carbon
showed potency similar to that exhibited by 5. Substitutions on
the phenyl ring also affected the activity. Halogen substitutions
generally were better than non-halogen substitutions (13–14 vs
15–16). Compound 12 with a 1-(3,4,5-trifluorophenyl)ethyl group
and 14 with a 3,4-dichlorophenyl ring were the most potent ana-
logues in this series (12 Ab42 IC50 = 116 nM, 14 Ab42 IC50 = 126 nM).
Longer N-alkyl substitutions as in the case of 17 and 18 did not
help improve the potency. Amide derivatives 19–22 were generally
not favored, although 21 had moderate activity. Urea compound 23
was not active up to 20 lM. N-Arylsulfonamide analogue 24
showed weak potency. Changing the R from the 1-(4-fluoro-
phenyl)ethyl of 5 to a hydrogen atom (4) completely eliminated
the activity. Overall, N-benzyl derivatives were favored over urea,
amide and N-arylsulfonamide compounds. In order to determine
the ability of compounds 12 and 14 to reduce the amount of
Ab42 in CSF, each compound was dosed orally in rats at 30
mg/kg. Unfortunately, neither compound showed statistical effi-
cacy in this in vivo model, despite good in vitro potency. Examina-
tion of the pharmacokinetics of 12 and 14 revealed that each had
limited brain exposure (12 Pl. C3h = 9.00
lM, brain C3h = 0.49 lM;
14 Pl. C3h = 22.8 M, brain C3h = 0.52
l
l
M).28
Given the disappointing in vivo data from the tetrahydrotriazol-
opyrimidine series, we decided to work on tetrahydroisoxazol-
o[3,4-b]pyridine and tetrahydroisoxazolo[5,4-b]pyridine series,
which could offer better brain penetration due to lower basicities
and higher c log P values. The preparation of the tetrahydroisoxaz-
olo[3,4-b]pyridine series is depicted in Scheme 3. A Sonogashira
coupling of 25 with pent-4-yn-1-ol, followed by protection of the
alcohol afforded 26. Cycloaddition of 26 with nitrile oxide, gener-
ated in situ from dibromoformaldoxime, gave 27. Deprotection of
O
O
N
N
N
N
H
N
O
N
O
N
N
O
N
O
N
a
OH
N
H
N
N
b
N
NH
c
HN
F
N
N
N
N
2
3
4
5
Scheme 2. Reagents and conditions: (a) 2-hydrazino-1,4,5,6-tetrahydropyrimidine, HATU, i-Pr2NEt, DMF, 88%; (b) pyridine, 120 °C, 54%; (c) NaH, 1-(1-bromoethyl)-4-
fluorobenzene, DMF/THF, 4%.

666
J. Qin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 664–669
Table 1
Cell Ab42 IC50 and
4–24
Table 1 (continued)
c-secretase modulator selectivity of tetrahydrotriazolopyrimidines
a
Compound
R
Ab42 IC50 (nM)
Abtotal IC50/Ab42
IC50 ratio
O
N
N
O
N
R
19
20
19,316
1919
1
N
N
F
N
F
F
O
O
10
a
Compound
R
Ab42 IC50 (nM)
Abtotal IC50/Ab42
IC50 ratio
21
22
23
485
2069
41
10
1
4
H
20,000
1
O
O
5b
509
156
39
( )3
F
F
F
20,000
N
O
6
128
138
O
O
S
24
2215
9
7
8
145
F
a
b
c
IC50 was reported as an average of multiple determinations (n P 2).
c log P = 4.5.
c log P = 4.7.
c log P = 5.4.
d
16,374
1
F
F
TBS ether 27, followed by oxidation of the resulting alcohol to an
aldehyde and the reductive amination of this aldehyde with (S)-
1-(4-fluorophenyl)ethanamine provided 28. Final compound 29
was delivered after ring closure at elevated temperature and
displacement of aromatic bromide with 4-methyl imidazole.
Compounds 30–32 were synthesized by a similar method to 29.
The SAR data in Table 2 showed that this tetrahydroisoxazol-
o[3,4-b]pyridine series showed moderate but reduced activity rel-
ative to 1. Varying the R substitution of 29 to that of 30–32 had
little effect on potency. Compound 31 was dosed orally at 30 mg/
kg in rats, but did not show statistical efficacy. Meanwhile, the
tetrahydroisoxazolo[5,4-b]pyridine series was synthesized as
shown in Scheme 4. N-Alkylation of 33 with 1-(1-bromoethyl)-
4-fluorobenzene afforded 34, which was converted to 35 by Aldol
condensation with 3-methoxy-4-(4-methyl-1H-imidazol-1-yl)
benzaldehyde and Swern oxidation of the resulting alcohol inter-
mediate. Finally, oxime ether formation and dehydrative ring
closure delivered the product 36, which was resolved by chiral
HPLC to provide its two enantiomers 37 and 38.29 The latter was
3-fold more potent than the former. The SAR data for the rest of
the analogues 39–4129 is presented in Table 2. Changing the R sub-
stitution from the 1-(4-fluorophenyl)ethyl of 38 to a 1-(4-fluoro-
9
20,000
649
1
31
58
10
11
F
F
342
F
F
F
12c
116
165
F
F
13
552
126
36
F
Cl
14d
159
phenyl)propyl (39) or to
a 1-(3,5-difluorophenyl)ethyl (40)
Cl
CN
O
decreased potency 2-fold, whereas the 1-(3,4,5-trifluorophe-
nyl)ethyl derivative 41 maintained similar activity to that of 38.
Overall, the tetrahydroisoxazolo[3,4-b]pyridine and tetrahydrois-
oxazolo[5,4-b]pyridine series displayed moderate in vitro activity
and did not offer a better profile than the tetrahydrotriazolopyrim-
idine series. Although these two series of compounds have
relatively lower basicities (higher c log P value) than tetrahydro-
triazolopyrimidine series in general, which suggest a possibility
of better brain penetration, compound 31 did not show improved
in vivo potency. The lack of in vivo activity could be partially
15
16
2531
1409
3
14
F
17
18
619
32
14
( )3
1406

J. Qin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 664–669
667
OTBS
( )3
O
N
O
N
O
O
O
I
Br
Br
a, b
c
d, e
f, g
Br
Br
OTBS
Br
N
H
Br
O
27
28
25
26
F
N
O
N
h, i
N
F
N
29
Scheme 3. Reagents and conditions: (a) pent-4-yn-1-ol, PdCl2(PPh3)2, CuI, DIEA, THF; (b) TBSCl, imidazole, CH2Cl2, 99% for two steps; (c) dibromoformaldoxime, NaHCO3,
CH2Cl2, 50 °C, 30%; (d) HF, THF, 99%; (e) (COCl)2, DMSO, CH2Cl2, À78 °C; (f) (S)-1-(4-fluorophenyl)ethanamine, MgSO4, CH2Cl2; (g) NaBH4, MeOH, 80% for three steps; (h)
BEMP, CH3CN, 180 °C, 15%; (i) 4-methyl imidazole, Cu2O, Cs2CO3, 4,7-dimethoxy-1,10-phenanthroline, polyethylene glycol, butyronitrile, 30%.
attributed to its limited in vitro potency (31, Ab42 IC50 = 356 nM).
Therefore, we need to identify compound with not only low basi-
city but also good in vitro potency for displaying in vivo activity.
Having explored the previous three series, we turned our atten-
tion to the tetrahydropyrazolopyridine series. Synthesis of this
tetrahydropyrazolopyridine series is outlined in Scheme 4. From
intermediate 35, analogue 42 was obtained through hydrazone for-
mation and cyclization in one pot.30 Compound 42 showed good
in vitro activity (Ab42 IC50 = 164 nM) (Table 3). Changing the R sub-
stitution from the 1-(4-fluorophenyl)ethyl of 42 to a 1-(3,4,5-triflu-
orophenyl)propyl (43) maintained similar activity. Enantiomer
4431 was examined in our rat in vivo model, where it demonstrated
moderate, but statistically significant efficacy in reducing rat CSF
Ab42 (À26% Ab42, 3 h after 30 mg/kg po dosing). The improved
in vivo activity of 44 seems to correlate well with its good
in vitro potency and lower basicity (c log P = 6.6) than the previous
Table 2
Cell Ab42 IC50 and c-secretase modulator selectivity of tetrahydroisoxazolo[3,4-b]pyridines 29–32 and tetrahydroisoxazolo[5,4-b]pyridines 37–41
X
Y
O
N
R
N
N
a
Compound
X
O
Y
R
Ab42 IC50 (nM)
Abtotal IC50/Ab42 IC50 ratio
29b
N
465
38
F
30
O
O
N
N
537
356
37
48
31c
F
F
32
O
N
440
46
F
37
N
N
O
O
1392
448
14
29
F
F
38d
39
N
O
875
17
F
(continued on next page)

668
J. Qin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 664–669
Table 2 (continued)
a
Compound
X
Y
R
Ab42 IC50 (nM)
Abtotal IC50/Ab42 IC50 ratio
F
40
N
O
935
21
F
F
F
F
41
N
O
540
37
a
IC50 was reported as an average of multiple determinations (n P 2).
b
c
c log P = 5.8.
c log P = 5.5.
c log P = 5.8.
d
N
O
O
O
O
O
O
O
N
a
b, c
d, e
N
N
NH
N
F
N
N
F
F
N
36
33
34
35
f
42
Scheme 4. Reagents and conditions: (a) NaH, 1-(1-bromoethyl)-4-fluorobenzene, THF, 57%; (b) t-BuLi, 3-methoxy-4-(4-methyl-1H-imidazol-1-yl)benzaldehyde, À78 °C, 50%,
THF; (c) (COCl)2, DMSO, Et3N, CH2Cl2, 94%; (d) NH2OHÁHCl, KOAc, MeOH, 89%; (e) P2O5, EtOH, 80 °C, 29%; (f) NH2NH2, P2O5, EtOH, 75 °C, 21%.
three series. With this encouraging preliminary data in hand, we
plan to explore the potential of this series and the results will be
reported in the near future.
Table 3
Cell Ab42 IC50 and
42–45
c-secretase modulator selectivity of tetrahydropyrazolopyridins
In summary, we have explored four series of 5,6-bicyclic het-
erocycles as
c-secretase modulators. Compounds from all series
N
NH
demonstrated moderate to good in vitro potency. Among these ser-
ies, we have identified the tetrahydropyrazolopyridine core as a
promising new lead series. Specifically, compound 44 demon-
O
N
R
N
strated good in vitro and moderate in vivo efficacy as a
modulator.
c-secretase
N
Acknowledgments
a
Compound
R
Ab42 IC50 (nM) Abtotal IC50/Ab42 IC50 ratio
We thank Drs. Chad Bennett, Jared Cumming, Andrew Stamford
and Eric Parker for proofreading and comments on the preparation
of the letter.
42b
164
201
46
99
F
References and notes
F
F
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F
45
a
b
c
IC50 was reported as an average of multiple determinations (n P 2).
c log P = 5.9.
c log P = 6.6.

J. Qin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 664–669
669
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27. In vivo assay method: Male CD rats (ꢀ100 g; Charles River Laboratories) were
orally administered
a compound (formulated in 20% hydroxypropyl b
cyclodextin; 5 ml/kg) and 3 h later tissues were collected. Immediately
following euthanasia with excess CO2, cerebrospinal fluid (CSF) was collected
from the cisterna magna and frozen on dry ice; only clear samples were
analyzed. Blood (from the vena cava) and brains were collected from the
animals immediately following CSF collection. Blood was briefly kept on ice
until it was spun at 6000 rpm for about 10 min and plasma was removed.
Brains were immediately frozen on dry ice. All samples were stored at À80 °C
until analysis. Rat CSF Ab40 and Ab42 were analyzed using AlphaLISA Amyloid
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homogenized and extracted in 5 M guanidine–HCl/50 mM Tris–HCl, pH 8.
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extraction matrix in 96-well format, the HLB plate (Waters). The samples
eluted from the HLB plate were dried and resuspended in freshly prepared PBS/
0.5% Tween 20. Ab40 and Ab42 were measured using AlphaLISA amyloid assay
kits.
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a
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26. Cell based in vitro assay method: Human embryonic kidney (HEK) 293 cells
stably transfected with APPsw-lon in pcDNA3.1 vector (Invitrogen) were
treated with GSM compounds for 5 h. Ab in conditioned media was measured
using MesoScale Discovery (MSD) technology-based sandwich immunoassays.
28. Korfmacher, W. A.; Cox, K. A.; Ng, K. J.; Veals, J.; Hsieh, Y.; Wainhaus, S.; Broske,
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29. Absolute configuration was assigned based on chiral HPLC retention time when
correlated to closely related series of enantiomers of known absolute
configuration. HPLC condition: chiral OD column, hexane/i-PrOH = 4:1.
30. The exact isomeric structures of pyrazoles were not determined.
31. Absolute configuration was assigned based on chiral HPLC retention time when
correlated to closely related series of enantiomers of known absolute
configuration. HPLC condition: chiral AD column, hexane/i-PrOH/
Et2NH = 100:25:0.1.