Triazoloamides as potent γ-secretase modulators with reduced hERG liability

Article abstract of DOI:10.1016/j.bmcl.2012.03.054

Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in this communication. Starting from our aryl triazole leads, optimization studies were continued and the series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog in this series, displaying single-digit nanomolar Aβ42 IC ₅₀ in cell-based assays and reduced affinity for the hERG channel.

Full text of DOI:10.1016/j.bmcl.2012.03.054

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3140–3146
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triazoloamides as potent c-secretase modulators with reduced hERG liability
⇑
Christian Fischer , Susan L. Zultanski, Hua Zhou, Joey L. Methot, Sanjiv Shah, Hugh Nuthall,
Bethany L. Hughes, Nadja Smotrov, Armetta Hill, Alexander A. Szewczak, Christopher M. Moxham,
Nathan Bays, Richard E. Middleton, Benito Munoz, Mark S. Shearman
Merck Research Laboratories Boston, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in
this communication. Starting from our aryl triazole leads, optimization studies were continued and the
series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog
in this series, displaying single-digit nanomolar Ab42 IC50 in cell-based assays and reduced affinity for the
hERG channel.
Received 22 February 2012
Revised 8 March 2012
Accepted 13 March 2012
Available online 21 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Gamma secretase modulator
Alzheimer’s
Amyloid beta
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder which in 2010 affected over 5 million patients in the US
alone.1 To date, only symptomatic treatments are available, which
provide short-term stabilization, but have no effect on overall dis-
ease progression. A popular hypothesis for AD describes oligomers
of amyloid b (Ab), and specifically the longer fragment Ab42, as
GSMs such as quinazolinones15 and, more recently, aryl triazoles
(5).16,17
Following the discovery of triazole GSMs (5), we set out to
identify novel triazoles, with the goal to improve their modest cell
potency while maintaining their salient features, for example,
>100-fold Notch selectivity and reduced hERG affinity. We hypoth-
esized that replacing the benzylic substituents with amides would
allow for a convergent optimization, while providing a reasonable
starting point to optimize hERG binding (Fig. 2).
Initial studies aimed at examining whether the hydrophobic
benzyl substituent in 5 could be replaced with amides without loss
in potency. In our initial exploration, we focused on simple alkyl
and aryl amides, 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 reaction
mixture overnight in DMF. Separation of the undesired regioisomer
was achieved through a wash procedure and subsequent purifica-
tion on silica gel.18
Alkynylation of aldehyde 8 with the Bestmann–Ohira reagent
gave the desired alkyne 9 in quantitative yield. A click reaction19
of alkyne 9 with azides 12 in ethanol/water, with copper(II)sulfate
and sodium ascorbate as the reductant, furnished triazoles 13 in
neurotoxic species that are formed by the
c-secretase complex
through proteolytic cleavage of C-99.2–5 Such oligomers have led
to cell death and neurodegeneration in vitro and in vivo, causing
symptoms such as cognitive impairment.6
In parallel to the development of
and others have explored the possibility of modulating
c
-secretase inhibitors (GSIs), we
-cleavage to
c
favor production of shorter fragments, while not affecting total Ab
levels. This was postulated to be a safer approach to a disease-mod-
ifying therapy due to the sparing of Notch processing.7–9
(R)-Flurbiprofen (Tarenflurbil, Flurizan™) (1,Fig. 1)10 is an exam-
ple of the first generation of carboxylic acid c-secretase 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 evolving area of
non-acid GSMs, following the early work from Neurogenetics, Eisai
disclosed aryl imidazoles, which are represented by E-2012 (3).12
In our own program aimed at identifying novel non-carboxylic acids
as modulators of
c-secretase, we previously published our lead
optimization strategy for pyrimidine and purine derivatives (4) as
good to excellent yields. The required
generally prepared from the corresponding
sodium azide in polar aprotic solvents, such as DMF or DMSO, at
room temperature. -Halo amides were either commercially avail-
a
-azido amides 12 were
GSMs.13,14 Additionally, we detailed the discovery of novel non-acid
a
-halo amides 11 and
a
⇑
able or readily prepared from
corresponding amines.
a-chloro acetylchloride (10) and the
Corresponding author.
E-mail address: christian_fischer@merck.com (C. Fischer).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.054

C. Fischer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3140–3146
3141
CO2H
Me
O
Me
F
MeO
CO2H
N
N
Ar
N
F
N
R1 R2
Me
FlurizanTM (1)
E-2012 (3)
2
R1
N
R2
N
N
R4
N
R
N
MeO
N
N
H
N
N
R3
N
Me
Me
4
5
Me
MeO
Figure 1. Select gamma secretase modulators.
O
R1
N
N
N
N
N
N
R
N
MeO
N
MeO
R2
N
N
N
5
6
Me
Me
Figure 2. Aryl replacement strategy.
MeO
N
CHO
K2CO3, MeOH
MeO
N
MeO
CHO
Methylimidazole
N
N
K2CO3, DMF, Δ
F
Bestmann reagent
7
8
9
Me
Me
R2
R1
N
H
O
O
O
NaN3
R2
Cl
Cl
R2
N3
N
Cl
N
Hunig's base
THF
R1
DMF or DMSO
R1
10
11
12
R2
O
N
N
N
N
O
MeO
N
MeO
R1
CuSO4
N3
R2
N
R1
Na-ascorbate
EtOH/H2O
N
N
N
9
12
13
Me
Me
Scheme 1. Synthesis of amide GSMs 13.
Cell biochemical data for the inhibition of Ab40/4220 production
and Notch processing21 as well as binding affinity for the hERG
channel22 for our initial set of amides 13 are reported in Table 1.
We were pleased to find no potency loss relative to our earlier
benzylic triazoles,17 as well as some clear SAR trends favoring bicy-
clic and cycloalkyl versus simple aryl substituents (14 vs 16 and 23).
Also consistent with earlier studies, we found hERG binding to be
correlated to the lipophilicity of the sidechains. In our earlier study
on benzylic triazoles, we found that benzoxazole replacements re-
sulted in attenuated hERG binding17; in attempting to translate this
finding to the amide series, we identified compound 24, which in-
deed displayed a modest improvement in hERG binding, albeit at
a loss in potency. We decided to address the hERG liability of this
series in a subsequent optimization where we focused on identify-
ing optimal amide substituents. The trifluoroethyl group (com-
pounds 25–29) was identified as a preferred substituent, and we
were able to drive the cell potency into the double-digit nanomolar
range in combination with previously identified bicyclic pieces. Tri-
azoles 28 and 29 showed the most promising on-target potency,
with a >500-fold window over Notch processing. Hence, it was
decided to invest further into the optimization of this new triazole
series.
To explore the chemical space around the amide moiety and to
address liabilities such as the affinity for the hERG channel, we

3142
C. Fischer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3140–3146
Table 1
Optimization of amide triazoles
O
R1
N
N
N
N
R2
MeO
N
N
Me
Compound
R1
R2
Ab42 IC50
Ab40 IC50
Notch IC50
n.d.
hERG IC50
3.05
14
1.63
>10
15
16
0.62
0.61
8.71
4.85
n.d.
0.80
>10
H
26.7
O
O
F
17
18
H
H
0.45
0.68
2.07
>10
29.6
>50
>10
F
4.03
19
20
0.24
0.37
1.90
4.42
>50
>50
0.25
0.87
F
Me
21
0.23
3.01
>50
0.43
Me
Me
22
23
0.43
0.20
5.17
>10
>50
0.84
1.39
9.00
N
O
24
25
26
0.94
0.27
0.18
>10
>50
>40
24.6
10.3
2.87
1.49
CF3
2.43
1.25
CF3
CF3
O
O
F
27
28
0.16
0.08
1.18
0.66
38.2
48.5
1.92
0.63
F
CF3
CF3
29
0.09
0.41
45.1
0.80
All compounds were tested at least twice in independent experiments (at least once for hERG). For a description of assay conditions, see Refs. 21–23. All data is reported in
M.
l
began to explore constrained amides, and particularly, lactams.
Synthesis of triazololactams proceeded as outlined in Scheme 2.
Owing to the facile click chemistry approach, we focused on
an efficient synthesis of the corresponding azido lactams 35,
which were prepared from the corresponding chloro-, bromo-, or
iodolactams 32–34. -Halogenation of lactams 31 was accom-
plished using the standard methods described in Scheme 2; lac-
a
tams 31 were either commercially available or prepared using

C. Fischer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3140–3146
3143
O
O
NH
NaN3/HCl (37% aq.)
OR: 1) NH2OH.HCl, THF
2) PPA, 100oC
R3
30
R3
31
O
PCl5, 100oC
Benzene
Cl
NH
32
R3
O
O
O
1) PCl5, cat. I2
2) Bromine
Br
NH
NH
N3
NH
NaN3
DMF or DMSO
CHCl3
R3
O
31
33
35
R3
R3
1) TMEDA, TMSI
2) Iodine
I
NH
CHCl3
34
R3
O
H
N
N
N
O
N
MeO
MeO
CuSO4
N3
NH
N
R3
O
Na-ascorbate
EtOH/H2O
N
N
N
9
R3 35
36
Me
Me
R4
N
O
H
N
N
N
N
N
N
N
R4OTf, CsCO3, THF, reflux
OR: R4Br, KHMDS, THF
MeO
N
MeO
N
R3
R3
N
N
36
37
Me
Me
Scheme 2. Synthesis of triazololactams.
conventional ring-expansion chemistry from commercially avail-
able cyclic ketones 30. We found the Schmidt reaction to provide
lactams in reproducibly high yields; alternatively a Beckmann
rearrangement strategy was employed if the Schmidt reaction
proved difficult due to solubility limitations. Regioisomeric mix-
tures, in the case of unsymmetrical ketones, were readily separable
on silica gel. Installation of a second N-substituent on the lactam
was generally accomplished through alkylation chemistry at any
step in the sequence, but preferably as the final step.
With robust chemistry in hand, we proceeded to explore a vari-
ety of lactams to evaluate the effect of ring size and substitution
pattern to select the optimal scaffold for further optimization
(Table 2).
A survey of various ring sizes identified the piperidinone and
azepinone rings as optimal (only selected examples are shown).
Gratifyingly, we observed no drop in potency relative to the simple
amide derivatives in Table 1, and we were delighted to find that
binding to the hERG channel was generally attenuated, while excel-
lent Notch selectivity was maintained. Additional substitution in
the 5-position of the ring with hydrophobic substituents led to a
consistent increase in intrinsic potency, while maintaining good
Notch selectivity. Substitution on other positions on the ring was
also tolerated, for example, in the 7-position (45) or 6,7-fused bicyl-
ic rings (43). We proceeded to further optimize triazoloazepinones,
but we also continued to test other ring sizes for key compounds. A
systematic exploration of the lactam N-substituent as well as addi-
tional substitutions around the ring is reported in Table 3.
We initially embarked upon finding the optimal substituent on
the lactam nitrogen, and found hydrophobic groups to be preferred
with benzyl and trifluoroethyl being optimal. This was consistent
with our initial observations with acyclic amides; polar functional-
ity was not tolerated (data not shown).23 To reassess the effect of
substituents in the 5-position and the azepinone ring system rela-
tive to smaller ring size lactams, we prepared triazoles 54 and 55
and found them to be significantly less potent. For all compounds
in this series, we saw a preference for one enantiomer, with the
active enantiomer being 5- to 15-fold more potent than its less
potent congener.24 Ultimately, we identified triazole 57 to be the
optimal combination in terms of potency and selectivity over
Ab40, Notch and hERG.
In summary, we have reported the discovery and SAR of novel
amides and lactams as modulators of
c-secretase. Starting from
simple amides as replacements of the benzylic groups in triazoles
5, we were able to optimize potency while attenuating binding to
the hERG channel. A novel series of potent and selective lactam
GSMs was subsequently discovered, and through SAR studies
within this series, we identified lactam 57, which is one of the most
potent GSMs we have discovered during our program. To the best
of our knowledge, this is one of the first structural disclosures of
GSMs with single-digit nanomolar cell activity. Although the

3144
C. Fischer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3140–3146
Table 2
Modulation of Ab40/42 processing by lactams 38–46
N
N
N
R
MeO
N
N
Me
Compound
R
Ab42 IC50
Ab40 IC50
Notch IC50
>50
hERG IC50
43.9
O
O
O
NH
NH
NH
38
4.42
>10
39
40
1.09
1.27
9.87
>50
>50
5.35
>30
9.10
6.16
F3C
O
NH
41
1.11
>50
>30
CF3
O
NH
42
43
1.31
0.62
7.42
3.86
>50
>50
20.8
20.8
Me
Me
O
O
NH
NH
44
45
46
0.33
0.48
0.16
1.23
5.12
0.66
45.3
>50
6.27
44.2
3.68
TMS
O
NH
NH
t-Bu
O
17.0
c-Hex
All compounds are racemic and were tested at least twice in independent experiments (at least once for hERG). For a description of assay conditions, see Refs. 20–22. All data
is reported in M.
l
selectivity of Ab42 over Ab40 was moderate (ꢀ5- to 25-fold),
the compounds in this series behaved as GSMs, and the selectivity
over Notch processing was generally very high (>100- to 1000-
fold). Interestingly, the selectivity over Notch processing appears
to be independent of Ab 42/40 ratios and compound potencies;
even for our most potent compounds, the selectivity over Notch
processing was not diminished. Selected compounds in this series
were profiled in vivo, and while we found the PK to be acceptable,
the brain to plasma ratio was generally poor.25 Future directions
in this series that focus on improving central exposure
while maintaining the intrinsic potency will be reported in due
course.

C. Fischer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3140–3146
3145
Table 3
Modulation of Ab40/42 processing by triazoles 47–57
N
N
N
R
MeO
N
N
Me
Compound
R
Ab42 IC50
Ab40 IC50
Notch IC50
hERG IC50
O
N
N
N
47
0.24
1.33
>50
0.87
2.10
t-Bu
O
t-Bu
Ph
48
0.13
0.84
20.0
t-Bu
O
49
50
51
0.08
0.08
0.08
0.42
0.51
0.42
40.1
11.1
40.8
n.d.
t-Bu
O
Ph
Ph
N
0.20
5.48
Ph
O
N
CF3
O
Ph
N
52
53
0.07
0.02
0.44
0.21
10.2
>50
40.8
31.9
TMS
O
CF3
N
CF3
O
N
CF3
54
55
56
0.29
0.30
0.02
1.91
2.21
0.15
>50
>50
>50
13.1
21.9
CF3
O
CF3
CF3
N
O
N
2.22
Ph
(continued on next page)

3146
C. Fischer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3140–3146
Table 3 (continued)
Compound
R
Ab42 IC50
Ab40 IC50
Notch IC50
24.3
hERG IC50
O
CF3
N
57
0.009
0.17
5.4
t-Bu
All compounds are enantiopure (the more active enantiomer is shown) and were tested at least twice in independent experiments (at least once for hERG). For a description of
assay conditions, see Refs. 20–22. All data is reported in lM.
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.
References and notes
1. Alzheimer’s Association: 2010 Alzheimer’s disease facts and figures http://
www.alz.org/national/documents/report_alzfactsfigures2010.pdf.
2. C-99 is the C-terminal fragment of APP, which is formed from APP by BACE1
mediated proteolysis.
15. 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.
16. Fischer, C.; Munoz, B.; Zultanski, S.; Methot, J.; Zhou, H.; Brown, W. C.
WO2008156580.
17. 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.
18. Careful work-up and purification was necessary to remove the unwanted
regioisomer. See Ref. 16 for detailed synthetic procedures.
19. 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.
20. 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.
3. Thinakara, G.; Koo, E. H. J. Biol. Chem. 2008, 283, 29615.
4. (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.
5. 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.
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.
7. For a recent review on secretase biology and modes of c-secretase inhibition/
Consistent with the profile of
were constant; selected GSMs were confirmed in
c
-secretase modulators, total Ab peptide levels
SELDI experiment
modulation, see: De Strooper, B.; Vassar, R.; Golde, T. Nat. Rev. Neurol. 2010, 6,
99.
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.
9. For a recent comprehensive review of the GSM chemical space, see: Oehlrich,
D.; Berthelot, D. J.-C.; Gijsen, H. J. M. J. Med. Chem. 2011, 54, 669.
10. Flurizan is the COX-inactive (COX1 & 2) enantiomer of (S)-flurbiprofen.
11. (a) For patents in this area, see: Hannam, J. C.; Hartmann, S.; Andrew; R.M.;
Mark P. WO2007110667.; (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.
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. (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.
14. (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.
a
confirming the 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. Pharm. 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.
21. 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-
J
j.
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.
22. 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.
23. The fluoroethyl and difluoroethyl groups were found to be less potent (data not
shown) while affording no improvements in terms of selectivity.
24. We were unable to obtain a single crystal X-ray for compounds in Table 3;
however, for a structurally related compound which will be the subject of a
forthcoming publication, the absolute stereochemistry was established as 3S.
25. Upon oral dosing of 50 mpk in SD rats or APP YAC mice, compounds in this
series generally displayed brain to plasma ratios of <0.1, and consequently did
not show significant brain exposure upon oral dosing. Owing to the low brain
exposures, we were unable to observe consistent Ab lowering at 50 mpk.