Discovery of novel triazolobenzazepinones as γ-secretase modulators with central Aβ42 lowering in rodents and rhesus monkeys

Article abstract of DOI:10.1016/j.bmcl.2015.07.003

Abstract Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aβ42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aβ42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aβ42 levels.

Full text of DOI:10.1016/j.bmcl.2015.07.003

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3488–3494
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel triazolobenzazepinones as
c
-secretase modulators
with central Ab42 lowering in rodents and rhesus monkeys
a
a
a
a
a
Christian Fischer a, , Susan L. Zultanski , Hua Zhou , Joey L. Methot , Sanjiv Shah , Ikuo Hayashi ,
Bethany L. Hughes a, Christopher M. Moxham a, Nathan W. Bays a, Nadya Smotrov a, Armetta D. Hill a,
Bo-Sheng Pan a, Zhenhua Wu a, Lily Y. Moy a, Flobert Tanga a, Candia Kenific a, Jonathan C. Cruz a,
Deborah Walker a, Melanie Bouthillette a, George N. Nikov a, Sujal V. Deshmukh a,
Valentina V. Jeliazkova-Mecheva a, Damaris Diaz a, Maria S. Michener b, Jacquelynn J. Cook b,
Benito Munoz a, Mark S. Shearman a
⇑
a Merck Research Laboratories Boston, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
b Merck Research Laboratories West Point, 770 Sumneytown Pike, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 9 July 2015
Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are
presented in this communication. Starting from our azepinone leads, optimization studies toward
improving central lowering of Ab42 led to the discovery of novel benzo-fused azepinones. Several ben-
zazepinones were profiled in vivo and found to lower brain Ab42 levels in Sprague Dawley rats and trans-
genic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further
progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed
robust lowering of CSF Ab42 levels.
Keywords:
Alzheimer’s
c
-Secretase
GSM
Rhesus PK/PD
CSF Ab lowering
Ó 2015 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder which in 2015 is projected to affect more than 5 million
patients in the US alone.1 To date, only symptomatic treatments
are available, which provide short-term symptomatic benefit but
have no effect on overall disease progression. A popular hypothesis
for AD describes oligomers of amyloid b(Ab),2,3 and specifically the
longer fragment Ab42, as neurotoxic species that are formed by the
modulators (GSMs), and we have previously reported the discovery
and SAR of several exquisitely selective (Ab IC50 40/42 >100)
classes of GSMs such as piperidine carboxylic acids 2.11 In the area
of non-acid GSMs, following the early work from Neurogenetics,
Eisai disclosed aryl imidazoles, for example, 3 (E-2012).12 The past
decade has seen substantial activity in the pharmaceutical industry
on non-acid GSMs including in our labs.13 In our program aimed at
c
-secretase complex4 through proteolytic cleavage of C-99.5 Such
identifying novel non-carboxylic acids as modulators of c-secre-
oligomers have led to cell death and neurodegeneration in vitro
and in animal models of AD, causing symptoms such as cognitive
impairment.6
tase, we previously published our lead optimization strategy for
pyrimidine and purine derivatives (4) as GSMs.14,15 Additionally,
we detailed the discovery of novel non-acid GSMs such as quina-
zolinones16 and, more recently, aryl triazoles and triazolo lactams
(5).17,18
In parallel to the development of
c-secretase inhibitors (GSIs),
we and others have explored the possibility of modulating
c
-cleav-
age to favor production of shorter fragments, while not affecting
total Ab levels. This was postulated to be a safer approach to a dis-
ease-modifying therapy due to the sparing of Notch processing.7–9
(R)-Flurbiprofen (Tarenflurbil, Flurizan™) (1, Fig. 1)10 is an
We reported in our preceding letter18b SAR studies on azepanes
5 with Ab42 cellular potencies as low as single digit nM. We
continued our exploration of this series, with the goal to improve
their modest CNS activity, while maintaining their salient
features, for example, >1000-fold Notch selectivity. Several GSMs
of the general structure 5 were dosed orally in rodents where we
saw good plasma yet very low total brain exposures. We
envisioned a benzo-fused lactam to be an excellent starting point
for further optimization, based on early data suggesting that
example of the first generation of carboxylic acid
c-secretase
⇑
Corresponding author. Tel.: +1 617 922 2493; fax: +1 617 9922 2403.
E-mail address: Christian_fischer@merck.com (C. Fischer).
http://dx.doi.org/10.1016/j.bmcl.2015.07.003
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

C. Fischer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3488–3494
3489
CO2H
Me
O
Me
F
MeO
N
CO2H
N
N
Ar
F
N
R1 R2
Me
FlurizanTM ( )
E-2012 ( )
1
2
3
R1
N
R1
N
R2
O
N
N
R4
N
N
MeO
N
N
H
R2
N
N
R3
N
Me
Me
4
5
Me
Figure 1. Select gamma secretase modulators.
MeO
R1
N
R1
N
O
O
N
N
N
N
N
N
MeO
N
MeO
N
R2
N
N
5
6
Me
Me
Figure 2. Optimization of azepinones 5 to benzazepinones.
MeO
N
CHO
K2CO3, MeOH
MeO
N
MeO
F
CHO
Methylimidazole
N
N
K2CO3, DMF, Δ
Bestmann reagent
7
8
9
Me
Me
O
PCl5, 100oC
Benzene
NH
Cl
12
O
O
O
O
1) PCl5, cat. I2
2) Bromine
1) NH2OH.HCl, THF
2) PPA, 100oC
Br
NH
N3
NH
NaN3
DMF or DMSO
NH
CHCl3
10
11
13
15
O
1) TMEDA, TMSI
2) Iodine
I
NH
CHCl3
14
O
H
N
N
N
O
MeO
N
CuSO4
MeO
N
N3
NH
N
N
Na-ascorbate
EtOH/H2O
N
9
15
16
Me
Me
R1
N
O
O
H
N
N
N
N
N
N
N
R1OTf, CsCO3, THF, reflux
OR: R1Br, KHMDS, THF
MeO
N
MeO
N
N
N
16
17
Me
Me
Scheme 1. Synthesis of benzazepinone GSMs.

3490
C. Fischer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3488–3494
Table 1
Initial optimization of benzazepinones
N
N
N
MeO
N
hERG (MK-499)
IC50 (lM)
HEK
Ab42 IC50
SY5Y
(
lM)
Ab40 IC50
SY5Y
(lM)
Notch IC50
HELA
(lM)
Compound
N
Me
O
NH
18
19
0.39
0.43
4.72
3.08
33.0
>50
17.6
O
O
Me
F
N
N
N
4.06
3.22
20
0.44
3.14
>50
F
F
O
21
22
0.15
0.07
0.70
0.32
>50
>50
8.96
3.56
F3C
N
O
O
O
O
N
N
N
23
24
25
0.37
0.24
1.08
3.98
2.01
5.40
25.6
>50
>50
1.58
1.01
7.57
rac
N
rac
O
O
N
N
N
26
27
1.19
0.88
6.75
4.69
>50
>50
6.22
4.45
rac
N
rac
rac
O
O
N
Me
28
1.25
7.48
>50
18.1
compounds 6 (Fig. 2) showed improved total brain exposure and
modest Ab42 lowering.19 While it is understood that brain to
plasma ratios are a poor predictor for CNS programs, we empiri-
cally analyzed the ratio of total brain to total plasma exposure
and found that for this chemical series compounds with higher
brain to plasma ratios generally resulted in superior Ab42 lowering
in rodents.
Our initial exploration focused on simple benzazepinones and
related ring systems, which were prepared according to the general
chemistry described in Scheme 1.
Aldehyde 8 was readily prepared from commercially available
4-fluoro-3-methoxybenzaldehyde (7), 4-methyl-1H-imidazole,
and potassium carbonate as the inorganic base by heating the reac-
tion mixture overnight in DMF. Separation of the undesired

C. Fischer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3488–3494
3491
Table 2
Modulation of Ab42/40 processing by azepinones 29–39
N
N
N
MeO
N
hERG (MK-499)
IC50 (lM)
HEK
Ab42 IC50
SY5Y
(lM)
Ab40 IC50
SY5Y
(lM)
Notch IC50
HELA
(lM)
Compound
N
Me
F3C
O
N
29
0.04
0.17
36.6
6.68
Me
O
F3C
N
30
31
0.06
0.58
0.24
6.38
28.4
>50
4.75
6.00
S
F3C
O
N
Me
F3C
O
N
32
0.03
0.07
>50
0.90
O
O
CF3
N
N
33
34
0.04
0.03
0.19
0.13
34.1
39.8
2.68
2.61
F
CF3
F
O
O
O
CF3
N
35
0.04
0.15
>50
1.83
F
F
CF3
N
36
37
0.01
0.15
0.05
0.94
>50
0.53
>50
Br
F
CF3
N
41.8
N
O
O
CF3
N
N
38
39
0.18
0.07
1.34
0.47
>50
>50
8.74
9.00
N
Ph

3492
C. Fischer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3488–3494
regioisomer was achieved through a wash procedure and subse-
quent purification on silica gel.20
albeit with an improvement in hERG binding. We continued to pro-
file compounds 29–39 in rat PK as well as rodent PK/PD studies to
differentiate them in vivo. Compound 34 was particularly effica-
cious when we profiled it in APP-YAC29 transgenic mice as well
as Sprague-Dawley (SD) rats where an ED50 of 15 mpk was deter-
mined for both models with oral dosing.30 At the highest dose
(100 mpk) of triazole 34 in the SD rat dose–response experiment
Alkynylation of aldehyde 8 with the Bestmann–Ohira reagent
gave the desired alkyne 9 in quantitative yield. A click reaction21
of alkyne
copper(II)sulfate and sodium ascorbate as the reductant, furnished
triazoles 16 in good to excellent yields. The required -azido lac-
tams 15 were generally prepared from the corresponding -halo
lactams 12–14 and sodium azide in polar aprotic solvents, such
as DMF or DMSO, at room temperature. -Halogenation of lactams
9 with azidolactams 15 in ethanol/water, with
a
a
a total brain level of 35 lM 6 h after dosing was observed with a
reduction in brain Ab42 levels of 92% relative to vehicle control.
While other compounds in Table 2 were also efficacious in our
rodent models we found that compound 34 performed most con-
sistently in both rodent species and additionally showed excellent
exposures at higher doses. Chart 1 details some of the in vitro and
in vivo data for compound 34.
Further studies with compound 34 first addressed the concern-
ing hERG binding; in patchXpress the half maximal inhibition of IKr
was 12.9 lM.
a
11 was accomplished using the standard methods described in
Scheme 1; lactams 11 were either commercially available or pre-
pared using conventional two-step ring-expansion chemistry from
commercially available tetralones 10. We found the Beckmann
reaction to provide lactams in reproducibly high yields and regioi-
someric purity. Installation of a substituent on nitrogen was gener-
ally accomplished through alkylation of 16 to give 17 through
standard chemistry.
We first decided to explore the influence of the lactam N-sub-
stituent on cellular potency, Notch selectivity and hERG binding.
In our earlier amide and lactam series we had found a strong influ-
ence on potency and hERG binding and thus explored both broadly
and with our earlier SAR in mind. Cell biochemical data for the
inhibition of Ab42/4022 production and Notch processing23 as well
as binding affinity for the hERG channel24 for our initial set of ben-
zazepinones 18–28 are reported in Table 1.
While stability in hepatocytes was only modest we were able to
establish reasonable PK in SD rats at low doses and excellent expo-
sure at 100 mpk in Wistar-Han rats where a normalized AUC of
8.8
l
MÁhÁkg/mg was calculated. Free fractions were reasonable
across species and compound 34 was also not a substrate for human
Aβ42/40 IC50 (nM):
31/131
39780
2609
Notch IC50 (nM):
hERG (MK_499) IC50 (nM):
Compounds 18–23 were resolved and data for the more active
enantiomer is shown, whereas for compounds 24–28 data for the
racemates is reported. Overall the trends we observed were similar
to our earlier azepinone series, where hydrophobic groups proved
superior in terms of potency but remained challenging when trying
to attenuate off-target activities such as hERG binding. Installation
of polarity on the amide nitrogen side chain was met with consid-
erable loss in potency and only marginal improvements in ion
channel binding. Additionally, the added polarity tended to
increase susceptibility for PgP-mediated efflux (data not shown).
Triazole 22 showed the most promising on-target potency, with
a >500-fold window over Notch processing. Therefore we decided
to continue our exploration, keeping the N-trifluoroethyl group
constant while altering both the azepane and benzo-fused ring.
We continued to utilize the facile ring-expansion using the
Beckmann reaction, which gave exclusively the desired regio-iso-
mer for all benzo-fused tetralone derivatives. Selected compounds
are shown in Table 2 with their respective Ab42/40, Notch and
hERG potencies. All compounds in this table were chirally resolved
and only data for the most active isomer is shown. We saw a pref-
erence for one enantiomer, with the active enantiomer being 5- to
15-fold more potent than its less potent congener.25 Compound 22
had modest metabolic stability (35 min t½ in rat liver microsomes)
and we began to explore the azepane ring first with blocking
attempts on positions that we suspected as metabolic soft spots.
Substitution in the 5-position (e.g., 29)26 was generally tolerated
and the 5-carbon could also be substituted with heteroatoms
(e.g., 30).27 Substitution to form the C3 quaternary carbon (com-
pound 31), however, was met with a significant loss in potency.28
While compound 29 had an improved in vitro metabolic stability
(72 min t½ in rat liver microsomes) its in vivo PK was not
improved over compound 22.
12900
hERG patch Xpress IC50 (nM):
CYP-3A4 (inh):
42% (@ 10 μM)
μ
CYP-2C9 (inh):
68% (@ 10 M)
28% (@ 10 μM)
> 30000
CYP-2D6 (inh):
PXR EC50 (nM):
CYP3A4TDI (%lost @ 50 μM)
Sol pH 7/2 (μM) / logD / PSA:
18%
5.6(180) / 3.3 / 76
Free fraction(%) rat:3.7 dog:5.8 rhesus:5.7 human:3.6
Hepatocyte Stability Half-Lives
rat (36 min), dog (67 min), rhesus (49 min), human (61 min)
PGP and passive permeability
Human mdr1: 1.2
Rat mdr1a: 1.3
P
app: 25x10-6 cm/s
PK Species
SD Rat
Dog
Rhesus
Dose (iv; po) (mg/kg)
Clp (mL/min/kg)
Vdss (L/Kg)
1; 2 0.25; 0.5 0.25; 0.5
38
4.8
0.8
190
1.6
82
14
6.2
<5
17
2.0
1.2
<5
t½ (iv) (h)
%F
0.1
ND
AUCN 0–24 po (μM h kg/mg)
Plasma concentrations (μM) of compound 34 after a 100 mg/kg
p.o. dose in Wistar Han rats
100
10
1
Rat 1
Rat 2
Rat 3
Mean
Next, we turned to exploring the benzo-fused ring and found
that substitution in the 6- and 7-positions as well as 6,8- and
6,7-disubstitution were favored whereas 8- and 9-substitution
led to flat SAR or slight potency loss (data not shown). While 6,7-
disubstitution (e.g., 36) or fused rings such as 32 gave rise to com-
pounds with promising cell potencies, their off target profile as
0.1
0
5
10
15
20
25
30
Time (Hour)
exemplified by sub-lM hERG binding was less than ideal.
Heteroaromatic analogs 37 and 38 showed a loss in cellular activity
Chart 1. Detailed profiling of compound 34.

C. Fischer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3488–3494
3493
Figure 3. Compound 34 pilot PK/PD study in CMP rhesus monkey (n = 3): 6 + 44 mpk (bolus + 4 h iv infusion). AUC 183
Geomean baseline adjusted.
lMÁh (Plasma) 2.2
lMÁh (CSF). Data analysis:
or rodent PgP (no directional transport was observed in an
mdr1/mdr1a expressing vs. control cell line with efflux ratios of
transporter (IC50: 32 nM).32 We hypothesized the privileged ben-
zazepinone scaffold might be responsible for this off-target activity
and proceeded to profile compounds which lack the benzo-fused
ring (e.g., compound 39) or heteroaromatic analogs (such as 37).
Gratifyingly pyridyl compound 37 and cyclohexane-fused com-
pound 39 maintained significant intrinsic cellular activity as well
as selectivity over Ab40 and Notch while at the same time no effect
on the serotonin transporter was observed.
1.2–1.3)
and
displayed
good
LLC-PK1
permeability
(25 Â 10À6 cm sÀ1).
While rodent PK for compound 34 was acceptable and good oral
bioavailability was observed even at low doses, dog PK was signif-
icantly inferior which was also true for other compounds in
Table 2; a liability which we subsequently addressed.33 We did
not in greater depth interrogate the low oral bioavailability of com-
pound 34 in dogs, however, a contribution of first pass metabolism
may be a contributing factor given the high plasma clearance of
almost twice the liver blood flow.
In summary, we have reported the discovery and SAR of novel
benzazepinones as modulators of c-secretase. Starting from previ-
ously reported azepinones we were able to optimize potency as
well as overall properties. A novel series of potent and selective
benzazepinone GSMs was subsequently discovered. Through SAR
studies within this series, we identified lactam 34, which we pro-
filed extensively in vitro and in vivo. Compound 34 was found to
be highly efficacious in a rhesus PK/PD study and presents an
example of a non-acid GSMs to show central lowering of Ab42 in
the CSF of higher species. While we were able to develop highly
efficacious GSMs, their overall PK profiles in preclinical species,
particularly in dog, were less than ideal and we continued our
studies in this series with changes to the arylimidazole portion of
the scaffold which we present in an accompanying
communication.33
After identifying GSM 34 as a key compound with good brain
exposure and central Ab42 lowering in transgenic mice as well as
SD rats, we proceeded to initiate a pilot study to assess whether
GSM 34 could also lower Ab levels in the CSF of non-human pri-
mates. Rhesus PK for compound 34 showed moderate plasma
clearance, but unfortunately displayed no oral bioavailability.
GSM 34 was instead dosed in an acute PK/PD study through iv infu-
sion (bolus of 6 mpk followed by a 4 h infusion of 44 mpk for a
total dose of 50 mpk) in our cisterna-magna-ported rhesus model31
which we previously described as a model allowing repeat CSF
sampling (Fig. 3).
We were pleased to observe significant lowering of Ab42 levels
in rhesus CSF while Ab40 levels were significantly less impacted,
which is consistent with the proposed mechanism of action.
Following 20 h after the infusion of compound 34, Ab42 did not
yet reach a pre-dose baseline level which suggests an extended
pharmacodynamic effect.
Having established robust pharmacodynamic effects in rodents
and rhesus monkeys we continued to further profile compound 34
and found it to be a potent inhibitor of the serotonin re-uptake
References and notes
1. Alzheimer’s Association: 2014 Alzheimer’s disease facts and figures http://www.
alz.org/downloads/Facts_Figures_2014.pdf.
2. Thinakara, G.; Koo, E. H. J. Biol. Chem. 2008, 283, 29615.
3. (a) Li, H.; Wolfe, M. S.; Selkoe, D. J. Cell Struct. 2009, 17, 326; (b) Osenkowski, P.;
Li, H.; Ye, W.; Li, D.; Aeschbach, L.; Fraering, P. C.; Wolfe, M. S.; Selkoe, D. J.; Li,
H. J. Mol. Biol. 2009, 385, 642; Recently, a new 4.3 Å cryo-EM structure was
published: (c) Sun, S.; Zhao, L.; Yang, G.; Yan, C.; Zhou, R.; Zhou, X.; Xie, T.;
Zhao, Y.; Wu, S.; Li, X.; Shi, Y. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 6003.

3494
C. Fischer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3488–3494
4. For example: (a) Selkoe, D. J.; Wolfe, M. S. Cell 2007, 131, 215; (b) Wolfe, M. S.
Curr. Top. Med. Chem. 2008, 8, 2; (c) Steiner, H.; Fluhrer, R.; Haass, C. J. Biol.
Chem. 2008, 283, 29627; (d) Wolfe, M. S. Semin. Cell Dev. Biol. 2009, 20, 219.
5. C-99 is the C-terminal fragment of APP, which is formed from APP by BACE1
mediated proteolysis.
Bays, N. W.; Smotrov, N.; Hill, A. D.; Pan, B.-S.; Wu, Z.; Moy, L. Y.; Tanga, F.;
Kenific, C.; Cruz, J. C.; Walker, D.; Nikov, G. N.; Deshmukh, S. V.; Jeliazkova-
Mecheva, V. V.; Diaz, D.; Cook, J. J,; Munoz, B.; Goulet, M. T.; Shearman, M. S.
Benzoazepinones as Modulators of c-Secretase; Presented at EFMC/ISMC Brussels
2010.
6. For example: (a) Hung, L. W.; Ciccotosto, G. D.; Giannakis, E.; Tew, D. J.; Perez,
K.; Masters, C. L.; Cappai, R.; Wade, J. D.; Barnham, K. J. J. Neurosci. 2008, 28,
11950; (b) Moreno, H.; Yu, E.; Pigino, G.; Hernandez, A. I.; Kim, N.; Moreira, J. E.;
Sugimori, M.; Llinas, R. R. PNAS 2009, 106, 5901; (c) Abramowski, D.; Rabe, S.;
Upadhaya, A. R.; Reichwald, J.; Danner, S.; Staab, D.; Capetillo-Zarate, E.;
Yamaguchi, H.; Saido, T. C.; Wiederhold, K.-H.; Thal, D. R.; Staufenbiel, M. J.
Neurosci. 2012, 32, 1273.
20. Careful work-up and purification was necessary to remove the unwanted
regioisomer. See Ref. 17 for detailed synthetic procedures.
21. For a review on click chemistry, see for example: Kolb, H. C.; Finn, M. G.;
Sharpless, K. B. Angew. Chem. Int. Ed. 2004, 2001, 40.
22. IC50 measurements for Ab40 and Ab42 were determined using
electrochemiluminescent detection of peptides secreted by SH-SY5Y cells
stably overexpressing the b-APP C-terminal fragment SPA4CT. All compounds
were tested at least twice in independent experiments.; Consistent with the
7. For
a
review on secretase biology and modes of
c-secretase
inhibition/modulation, see: De Strooper, B.; Vassar, R.; Golde, T. Nat. Rev.
Neurol. 2010, 6, 99.
profile of
selected GSMs were confirmed in
c
-secretase modulators, total Ab peptide levels were constant;
SELDI experiment confirming the
a
8. For selected reviews on GSMs, see: (a) Peretto, I.; La Porta, E. Curr. Top. Med.
Chem. 2008, 8, 38; (b) Breher, D. Curr. Top. Med. Chem. 2008, 8, 34; (c) Imbimbo,
B. P. Curr. Top. Med. Chem. 2008, 8, 54; (d) Tomita, T. Expert Rev. Neurother. 2009,
9, 661; (e) Hall, A.; Patel, T. R. Prog. Med. Chem. 2014, 53, 101; (f) Crump, C. J.;
Johnson, D. S.; Li, Y.-M. Biochemistry 2013, 52, 3197.
9. For a review of the GSM chemical space until 2010, see for example: Oehlrich,
D.; Berthelot, D. J.-C.; Gijsen, H. J. M. J. Med. Chem. 2011, 54, 669.
10. Flurizan is the COX-inactive (COX1 and 2) enantiomer of (S)-Flurbiprofen.
11. (a) For patents in this area, see: Hannam, J. C.; Hartmann, S.; M., Andrew; R.,
Mark P. WO 2007110667; (b) Madin, A.; Ridgill, M. P.; Kulagowski, J. J. WO
2007116228; (c) Garcia, Y.; Hannam, J. C.; Harrison, T.; Hamblett, C. L.; Hubbs,
J. L.; Kulagowski, J. J.; Madin, A.; Ridgill, M. P.; Seward, E. WO 2007125364; (d)
Stanton, M. G.; Munoz, B.; Sloman, D. L.; Hubbs, J. L.; Hamblett, C. L. WO
2008030391; (e) Munoz, B.; Hubbs, J. L.; Hamblett, C. L.; Zhou, H.; Martinez, M.
WO 2008085301; For a recent comparison with GSIs, see also: (f) Mitani, Y.;
Yarimizu, J.; Saita, K.; Uchino, H.; Akashiba, H.; Shitaka, Y.; Ni, K.; Matsuoka, N.
J. Neurosci. 2012, 32, 2037.
appearance of shorter fragments while Ab42 formation was suppressed. (a)
Best, J. D.; Jay, M. T.; Otu, F.; Ma, J.; Nadin, A.; Ellis, S.; Lewis, H. D.; Pattison, C.;
Reilly, M.; Harrison, T.; Shearman, M. S.; Williamson, T. L.; Atack, J. R. J.
Pharmacol. Exp. Ther. 2005, 313, 902; (b) Clarke, E. E.; Shearman, M. S. J.
Neurosci. Methods 2000, 102, 61; (c) Dyrks, T.; Dyrks, E.; Monning, U.;
Urmoneit, B.; Turner, J.; Beyreuther, K. FEBS Lett. 1993, 335, 89.
23. All compounds were also profiled at least twice in independent experiments to
assess their effect on Notch processing: HeLa cells were made to co-express
nonfunctional halves of luciferase, one fused to Notch
-Secretase mediated cleavage of Notch E results in release of Notch
intracellular domain (NICD)/N-terminal luciferase, which translocates to the
nucleus and binds the RBP-J /C-terminal luciferase to form functional
DE and the other to RBP-
Jj.
c
D
j
a
luciferase enzyme. This split-luciferase complementation system is used to
detect NICD levels by measuring total luminescence upon addition of luciferin
to lysed cells. Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2005, 77, 1295.
Typical transition-state analogues (e.g., L-685,458) do not show a window over
Ab 40/42 processing in this assay.
12. For a key patent, see: Kimura, T.; Kawano, K.; Doi, E.; Kitazawa, N.; Shin, K.;
Miyagawa, T.; Kaneko, T.; Ito, K.; Takaishi, M.; Sasaki, T.; Hagiwara, H. WO
2005115990.
13. For a recent review of the GSM patent space, see for example: Pettersson, M.;
Stepan, A. F.; Kauffman, G. W.; Johnson, D. S. Expert Opin. Ther. Pat. 2013, 23,
1349.
14. (a) Blurton, P.; Fletcher, S.; Teall, M.; Harrison, T.; Munoz, B.; Rivkin, A.;
Hamblett, C.; Siliphaivanh, P.; Otte, K. WO 2008099210; (b) Rivkin, A.; Ahearn,
S. P.; Chichetti, S. M.; Hamblett, C. L.; Garcia, Y.; Martinez, M.; Munoz, B. WO
2010019392.
15. (a) Rivkin, A.; Ahearn, S. P.; Chichetti, S. M.; Kim, Y. R.; Li, C.; Rosenau, A.;
Kattar, S. D.; Jung, J.; Shah, S.; Hughes, B. L.; Crispino, J. L.; Middleton, R. E.;
Szewczak, A. A.; Munoz, B.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2010, 20,
1269; (b) Rivkin, A.; Ahearn, S. P.; Chichetti, S. M.; Hamblett, C. L.; Garcia, Y.;
Martinez, M.; Hubbs, J. L.; Reutershan, M. H.; Daniels, M. H.; Siliphaivanh, P.;
Otte, K. M.; Li, C.; Rosenau, A.; Surdi, L. M.; Jung, J.; Hughes, B. L.; Crispino, J. L.;
Nikov, G. N.; Middleton, R. E.; Moxham, C. M.; Szewczak, A. A.; Shah, S.; Moy, L.
Y.; Kenific, C. M.; Tanga, F.; Cruz, J. C.; Andrade, P.; Angagaw, M. H.; Shomer, N.
H.; Miller, T.; Munoz, B.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2010, 20,
2279.
24. For a description of the MK-499/hERG radioligand displacement assay, see:
Butcher, J. W.; Claremon, D. A.; Connolly, T. M.; Dean, D. C.; Karczewski, J.;
Koblan, K. S.; Kostura, M. J.; Liverton, N. J.; Melillo, D. G. WO 200205860.
25. We were unable to obtain a single crystal X-ray for compounds in Table 2;
however, for a structurally related compound which will is the subject of an
accompanying publication,33 the absolute stereochemistry was established as
3S.
26. Larger substituents were also tolerated, for example, phenyl (data not shown)
or disubstitution such as 5,5-dimethyl.
27. Other heteroatoms such as O or NR were also tolerated in this position (data
not shown).
28. C4 substitution was not explored.
29. Hsiao, K.; Chapman, P.; Milsen, S.; Eckman, C.; Harigaya, Y.; Younkin, S.; Yang,
F.; Cole, G. Science 1996, 274, 99.
30. Compound 34 was dosed po as a single dose at 100, 30, 10, 1, 0.1 mpk. After 6 h
all animals (5 per group) were sacrificed and compound levels in plasma and
brain as well as brain Ab40 and Ab42 levels determined.
31. (a) Cook, J. J.; Wildsmith, K. R.; Gilberto, D. B.; Holahan, M. A.; Kinney, G. G.;
Mathers, P. D.; Michener, M. S.; Price, E. A.; Shearman, M. S.; Simon, A. J.; Wang,
J. X.; Wu, G.; Yarasheski, K. E.; Bateman, R. J. J. Neurosci. 2010, 30, 6743; (b)
Michener M. S.; Holahan M.; Gilberto D. B.; Sitko G.; Shearman M. S.; Harrison
T.; Lewis H.; Sankaranarayanan S.; Wu G.; Pietrak B.; Crouthamel M.; Simon A.
J., and Cook J. J. Nonhuman primate model for prediction of human Ab effects.
16. Fischer, C.; Shah, S.; Hughes, B. L.; Nikov, G. N.; Crispino, J. L.; Middleton, R. E.;
Szewczak, A. A.; Munoz, B.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2011, 21,
773.
17. Fischer, C.; Munoz, B.; Zultanski, S.; Methot, J.; Zhou, H.; Brown, W. C. WO
2008156580.
Targeting c-secretase: Effects of enzyme inhibition vs. modulation of cleavage
specificity on CSF and plasma Ab in conscious rhesus monkeys. Presented at ICAD
Madrid, Spain, 2006.
18. (a) Fischer, C.; Zultanski, S. L.; Zhou, H.; Methot, J. L.; Brown, W. C.; Mampreian,
D. M.; Schell, A. J.; Shah, S.; Nuthall, H.; Hughes, B. L.; Smotrov, N.; Kenific, C.
M.; Cruz, J. C.; Walker, D.; Bouthillette, M.; Nikov, G. N.; Savage, D. F.;
Jeliazkova-Mecheva, V. V.; Diaz, D.; Szewczak, A. A.; Bays, N.; Middleton, R. E.;
Munoz, B.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2011, 21, 4087; (b) Fischer,
C.; Zultanski, S. L.; Zhou, H.; Methot, J. L.; Shah, S.; Nuthall, H.; Hughes, B. L.;
Smotrov, N.; Hill, A.; Szewczak, A. A.; Moxham, C. M.; Bays, N.; Middleton, R. E.;
Munoz, B.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2012, 22, 3140.
32. Serotonin re-uptake was the most significant off-target activity in a set of
enzymes tested at MDS Pharma and was confirmed in-house.
33. Methot, J. L.; Fischer, C.; Li, C.; Rivkin, A.; Ahern, S. P.; Brown, W. C.; Kattar, S.;
Kelley, E.; Mampreian, D. M.; Schell, A. J.; Rosenau, A.; Zhou, H.; Basll, R.;
Deshmukh, S. V.; Jeliazkova-Mecheva, V. V.; Diaz, D.; Moy, L. Y.; Kenific, C. M.;
Moxham, C.; Shah, S.; Nuthall, H.; Szewczak, A. A.; Hill, A.; Hughes, B.; Smotrov,
N.; Munoz, B.; Miller, T. A.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2015, 25,
3495.
19. Part of this work has previously been presented as
a poster: Fischer, C;
Zultanski, S. L.; Zhou, H.; Methot, J. L.; Shah, S.; Hughes, B. L.; Moxham, C. M.;