Discovery of tetrahydroindazoles as a novel class of potent and in vivo efficacious gamma secretase modulators

Article abstract of DOI:10.1016/j.bmc.2018.04.053

The identification and optimization of a novel series of centrally efficacious gamma secretase modulators (GSMs) offering an alternative to the privileged aryl imidazole motif is described. Chiral bicyclic tetrahydroindazolyl amine substituted triazolopyr

Full text of DOI:10.1016/j.bmc.2018.04.053

Accepted Manuscript
Discovery of tetrahydroindazoles as a novel class of potent and in vivo effica-
cious gamma secretase modulators
Kai Gerlach, Scott Hobson, Christian Eickmeier, Ulrike Groß, Clemens Braun,
Peter Sieger, Michel Garneau, Stefan Hoerer, Niklas Heine
PII:
S0968-0896(18)30539-X
https://doi.org/10.1016/j.bmc.2018.04.053
BMC 14336
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
21 March 2018
24 April 2018
26 April 2018
Please cite this article as: Gerlach, K., Hobson, S., Eickmeier, C., Groß, U., Braun, C., Sieger, P., Garneau, M.,
Hoerer, S., Heine, N., Discovery of tetrahydroindazoles as a novel class of potent and in vivo efficacious gamma
secretase modulators, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.053
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Discovery of tetrahydroindazoles as a novel class of
potent and in vivo efficacious gamma secretase
modulators
†
§
†
†
±
Kai Gerlach,* Scott Hobson, Christian Eickmeier,* Ulrike Groß, Clemens Braun, Peter
±
±
†
†
Sieger, Michel Garneau, Stefan Hoerer, and Niklas Heine
†
§
Departments of Medicinal Chemistry , Central Nervous System Diseases Research , Drug
±
Discovery Sciences , Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Straße 65,
8
*
8397 Biberach an der Riss, Germany
Corresponding Authors
Phone: +497351540. Email: kai.gerlach@boehringer-ingelheim.com
Phone: +497351540. Email : christian.eickmeier@boehringer-ingelheim.com
1

ABSTRACT:
The identification and optimization of a novel series of centrally efficacious gamma secretase
modulators (GSMs) offering an alternative to the privileged aryl imidazole motif is described.
Chiral bicyclic tetrahydroindazolyl amine substituted triazolopyridines were identified as
structurally distinct novel series of GSMs. Representative compound BI-1408 ((R)-42) was
demonstrated to be centrally efficacious in rats at a 30 mg/kg oral dose.
OMe
N
N
N
N
N
N
N
H
N
H
N
F
BI-1408
Aβ42 IC50 = 0.04 µM
Key words: Gamma secretase modulators; Tetrahydroindazols; Alzheimer’s disease; Aβ42
reduction;
Abbreviations: AD, Alzheimer’s disease; Aβ, amyloid β pe pp tide; AA PP, amyloid precursor
protein; BACE1, β-amyloid cleavage enzyme 1, β-secretase 1; GSM, gamma secretase
modulator; LHS, left hand side; NSAID, non-steroidal anti-inflammatory drug; SAR, structure
2

activity relationships; HLM, human liver microsomes; RLM, rat liver microsomes; PPB, plasma
protein binding, MDCK cells Madin-Darby canine kidney epithelial cells; pgp,p-glycoprotein;
MDR1, multidrug resistance protein 1; MRT, mean residence time; psi pounds per square inch.
1
.
INTRODUCTION
Alzheimer’s disease (AD) is a chronic and progressive neurodegenerative disease resulting in
cognitive decline and memory loss. One of the hallmarks of this disease is the deposition of
1
extracellular senile plaques comprised of amyloid-beta (Aβ) peptides. The Aβpeptide derives
from the sequential proteolytic cleavage of the amyloid precursor protein (APP) by BACE1,
yielding a short membrane-bound C-terminal fragment (C99). Subsequently, C99 is cleaved via
γ-secretase. While cleavage by γ-secretase yields Aβ peptides ranging from 37-43 amino acids
2
long, the hydrophobic Aβ 42 peptide (Aβ ) appears most prone to aggregation. These
42
3
aggregates form not only the nucleus of the senile plaques, but also soluble oligomers, which are
considered to be the primary neurotoxic agent in AD and have been proposed to play a central
4
role in both the cause and progression of the disease. Supporting this, mutations in presenilin 1
or 2, the catalytic subunits of γ-secretase, are associated with increased production of Aβ and
42
5
result in early onset AD. Therefore, therapeutic approaches aimed at lowering the production of
Aβ represent an attractive pharmacological strategy to slow the progression of the disease.
4
2
Gamma secretase modulators (GSMs), i.e. compounds that do not block gamma secretase
cleavage but specifically lower Aβ production while leaving total Aβlevels unchanged, were
4
2
6
first identified in 2001. Since then, GSMs have been demonstrated to lower Aβ levels in both
4
2
in vitro as well as in vivo studies. Furthermore, GSMs have been demonstrated to lower plaque
7
load as well as rescue cognitive behavior in transgenic mice.
3

From a chemistry perspective, GSMs can be divided into two main series: 1) NSAID-derived
lipophilic carboxylic acids and 2) aryl imidazole compounds. In addition, Satori has reported
8
natural product derived triterpenoid GSMs as an independent third chemical class. Several
reviews have been published on that matter and cover work of research teams in the GSM field
9
of an entire decade. The beginnings of aryl imidazole type GSMs can be traced back to an early
1
0
report by Neurogenetics in 2004, claiming compounds of a general structure represented by the
molecule in Figure 1. The compounds show four consecutively arranged (hetero)aromatic rings
1
1
that became known as the A-B-C-D ring motif. Later, Eisai described a similar, more
1
2
13
elaborated compound E-2012 (1), the first aryl imidazole type GSM to reach clinical trials.
OMe
N
N
H
N
N
N
N
N
O
S
A
B
C
D
ABCD Ring sequence
E-2012 (1)
F
Figure 1. Generic structure of aryl imidazole type GSMs.
However, almost all non-NSAID-derived GSMs reported to date - including our own initial
1
4
GSMs - still share the same general characteristics such as a very high degree of aromaticity
and the presence of an aryl imidazole left-hand side (LHS) moiety, which was often incorporated
as aniline substructure into the compounds. While the privileged aryl imidazole A-B ring
sequence seems to be particularly necessary to achieve good potency, we thought that a
structural element lacking (a) the imidazole and (b) the electron rich aniline substructures would
4

be desirable in order to avoid both CYP inhibition and potential formation of reactive
1
5,16
metabolites.
Several efforts have therefore been directed at reducing electron density of the B-ring by the
introduction of electron-withdrawing groups, replacing the central aniline with heteroaromatics
or more recently using benzoic acids and their conjugates as more polar B-C ring
1
7,18,19,20
fragments.
Researchers at Merck reported on benzoazepinones where the imidazole was replaced by other
heterocycles and the B-ring aromatic moity was substituted or replaced by other aromatics or a
2
1
triple bond linker. A small number of patent applications deal with compounds that
demonstrated the successful replacement of the A-Ring imidazole by a nitrile moiety (Figure 2).
2
2
23
First introduced by Roche in 2009 (compound 2), the research team at Bristol-Myers Squibb
highlighted amino benzonitriles (e.g. 3) as a specific structural class of GSMs in two patent
applications in 2012. Additionally, cyano indoles (e.g. 4) were claimed as even further advanced
analogues of the original motif by the research team at Janssen with C-linked connectivity
2
4
towards the C-D fragment. Sekioka et al. recently reported the identification of isoindolinone 5
as replacement for the aryl imidazole motif on an imidazopyridine scaffold as weakly active
2
5
GSM.
5

OMe
NC
S
N
H
N
2
(Roche)
OMe
HN
N
NC
N
H
N
F
3
(Bristol-Myers Squibb)
OMe
NC
N
O
N
H
N
F
4
(Johnson&Johnson)
O
N
N
O
N
5
(Astellas)
Figure 2. Literature-known structural alternatives to the aryl imidazole moiety: benzonitriles and
conjugates.
However, only a handful of reports dealt with finding non-aromatic B ring replacements, which
in turn highlights the difficulties in identifying novel GSMs showing a lower degree of
2
6
aromaticity: methoxyphenyl piperazines (e.g. 6) were reported first by Merck & Co and
6

2
7
subsequently utilized on a different core by the research team from Janssen, whereas
2
8
heteroaromatic piperidinyl amines were published in 2011 and 2012 by Roche (e.g. 7 and 8)
(
Figure 3).
N
N
N
N
N
N
H
N
MeO
N
6
(Merck&Co)
N
N
N
N
N
H
N
7
(Roche)
F
Cl
N
H
N
N
N
N
N
N
S
F
8
(Roche)
F
Figure 3. Literature-known alternatives to the aryl imidazole moiety: non-aromatic B-ring
replacements.
2
9
While there is structural freedom to annellate the B-C ring sequence as well as using bicyclic
core systems as C-ring elements there is yet no evidence in literature that more flexible, partially
3
0
saturated bicyclic amines can be used as A-B ring system.
7

Herein, we report our work leading to a novel class of potent and in vivo efficacious gamma
secretase modulators that comprise tetrahydroindazolyl amines as a replacement of the
prominent aryl imidazole motif. To our best knowledge, this is the first disclosure of highly
potent GSMs with partially saturated bicyclic amines as A-B ring fragments.
2
2
.
Design and Synthesis
.1. Design
3
1
When looking for promising starting points to identify such novel A-B entities we recognized
the marked tolerance of the [1,2,4]triazolo[1,5-a]pyridine core to non-aromatic B ring
modifications (e.g. compound 7, Figure 4). We therefore hypothesized the triazolopyridine core
to be a good probe for the identification of novel A-B structures. To this end, we chose a diverse
set of primary and secondary amines of commercial and internal origin including numerous
bicyclic amines as left-hand side elements for combination with a suitable triazolopyridine
scaffold.
OMe
N
N
N
A
N
:
N
1
N
R
N
N
N
N
N
R
B
2
N
H
N
H
N
F
Series of triazolopyridines
to identify novel A-B unit amines
7
F
Figure 4. Design of an arylimidazole replacement series using the triazolopyridine scaffold.
In the amine selection process, we acknowledged the necessity of keeping a terminal hydrogen
bond acceptor as a key pharmacophore (see Figure 4, pink ellipse) as well as the presence of at
8

least one ring system to limit conformational flexibility. Apart from that, we allowed all kinds of
mono- or bicyclic aromatic, saturated, or partially saturated ring systems.
Applying these hypotheses we selected around 30 amines for synthesis and screened the products
for GSM activity (data not shown). Compounds with an Aβ IC <10 µM were defined as active
4
2
50
from this exercise. Within this subset, we became particularly interested into two compounds:
triazolopyridine 9 with an indazolyl amine moiety as bicyclic A-B ring motif, which was
complemented by identification of compound 10, which can be regarded as its partially
hydrogenated analogue. Both indazolyl and 1-alkyl-4,5,6,7-tetrahydro-1H-indazolyl amines are -
to the best of our knowledge - unprecedented as aryl imidazole replacement options (Figure 5).
1
N
7
4
N
6
5
N
N
N
N
N
2 N
N
N
H
N
N
H
3
9
Aβ42 IC50 = 1.2 µM
F
10
Aβ42 IC50 = 0.38 µM
F
Figure 5. (Tetrahydro)indazolyl amine hits 9 and 10.
While compound 10 is not only more potent than compound 9, it also contained the desired non-
aromatic amine motif. We therefore decided to thoroughly investigate tetrahydroindazole
substituted triazolopyridines as a novel series of GSMs.
2
.2. Synthesis
Preparation of tetrahydroindazolyl amine intermediates: At the time of the discovery of
compound 10 the respective 1-methyl-tetrahydroindazolyl amine building block was available
9

3
2
from one commercial vendor only and no synthesis was described in literature. In order to
explore tetrahydroindazole SAR we needed to find a robust and scalable synthetic approach to
obtain sufficient material and to allow the synthesis of analogues. Initially we intended to
alkylate 5-nitro-1H-indazole 11a in the presence of potassium carbonate (Scheme 1). The
reaction proceeded in high chemical yield, but the regioselectivity of the alkylation was rather
modest with only a 1.7:1 ratio in favor of the desired 1-ethylated product 12a over its 2-ethyl
regioisomer. The desired regioisomer 12a was chromatographically separated from the undesired
isomer and hydrogenated using Nishimura‘s catalyst to give first access to initial quantities of 1-
ethyl-4,5,6,7-tetrahydro-1H-indazol-5-ylamine 13a.
Alkylation regioselectivity was significantly improved upon switching to 3-halo-5-nitro-1H-
indazoles 11b-c as starting materials. 3-Chloro substitution favorably tuned the pyrazole
reactivity in the alkylation reaction in such a way that now a 7:1 ratio of alkylated regioisomers
was obtained. Moreover, the major product 12b was easily separated by recrystallization. Almost
complete regiospecificity was obtained for analogues containing either 3-bromo or 3-chloro
substitution as in 12c-g. Hydrogenation using Nishimura‘s catalyst followed by palladium on
charcoal to completely remove remaining halide provided tetrahydroindazolyl amine 13a in
gram quantities. Chlorinated analogue 13b was obtained as additional intermediate by stopping
the hydrogenation at an earlier timepoint. Intermediates 13c-h were synthesized using the same
alkylation-hydrogenation sequence starting from nitro indazoles 11b-d.
10

b
N
N
N
NH2
12d
1
R
1
R
H
N
3
R
3
R
3
R
a
b
N
N
N
N
NO2
2
NO2
NH2
2
R
2
R
R
2
3
1
2
3
1
2
3
1
1
1
1
1a (R =H, R =H)
12a (R =Et, R =H, R =H)
1
13a (R =Et, R =H, R =H)
3
2
3
2
3
1
2
1b (R =Cl, R =H)
12b (R =Et, R =Cl, R =H)
1
13b (R =Et, R =Cl, R =H)
1
2
3
2
3
2
3
1c (R =Br, R =H)
1d (R =H, R =OEt)
12c (R =n-Pr, R =Cl, R =H)
1
13c (R =n-Pr, R =H, R =H)
2
2
3
2
3
1
3
12e (R =2-methyl-allyl, R =Cl, R =H)
13d (R =i-Pr, R =H, R =H)
1
1
2
3
2
3
1
1
1
2f (R =1-but-2-ynyl, R =Br, R =H)
3
13e (R =i-Bu, R =H, R =H)
1
1
2
2
3
2g (R =1-pent-2-ynyl, R =Br, R =H)
1
13f (R =n-Bu, R =H, R =H)
1
2
3
2
3
2h (R =Et, R =H, R =OEt)
13g (R =n-Pentyl, R =H, R =H)
3
1
2
1
3h (R =Et, R =H, R =OEt)
a
Scheme 1. Synthesis of tetrahydroindazolyl amine building blocks 13a-h
a
Reagents and conditions: (a) 12a: ethyl iodide, K CO , DMF, 70°C, 57%; 12b: ethyl iodide,
2
3
K CO , DMF, 70°C, 83%; 12c: n-propyl iodide, K CO , DMF, 70°C, 79%; 12e: 3-bromo-2-
2
3
2
3
methyl-propene, K CO , DMF, 70°C, 90%; 12f: 1-bromo-but-2-yne, K CO , DMF, 70°C, 96%;
2
3
2
3
1
2g: 1-bromo-pent-2-yne, K CO , DMF, 70°C, 93%; 12h: ethyl iodide, K CO , DMF, 70°C,
2
3
2
3
5
6%; (b) H , 3 bar, Nishimura’s catalyst, MeOH, 11-99%.
2
Introduction of a methyl group at position 7 warranted a different synthetic approach (Scheme
): commercially available 5-bromo-7-methyl-1H-indazole 14 was alkylated (with poor
2
regioselectivity of 1:1.6 in favor of the undesired 2-alkylated isomer) using sodium hydride as
base and ethyl iodide to give intermediate 15 which was converted into the indazolyl amine 16
by a palladium-catalyzed amination with benzophenone imine as nitrogen source and subsequent
acidic hydrolysis of the intermediate imine. The indazole amine 16 was hydrogenated to give the
7
-methylated precursor 17 as mixture of diastereomers.
11

H
N
a
b
c
N
N
N
N
N
N
N
Br
Br
NH2
NH2
1
4
15
16
17
a
Scheme 2. Synthesis of 7-methyl substituted tetrahydroindazolyl amine 17
a
Reagents and conditions: (a) ethyl iodide, NaH, DMF, 0°C, 31%; (b) (i) benzophenone imine,
t
Xantphos, Pd (dba) , NaOBu, toluene, 130°C, (ii) TFA/water, 39% over two steps; (c) H , 3 bar,
2
3
2
Nishimura’s catalyst, MeOH, 94%.
Regioisomeric tetrahydroindazol-6-yl amines 21a-b were obtained using 3-bromo-1-methyl-1H-
indazole 18 and 1-methyl-indazol-6-yl amine 20 as starting materials. In order to introduce an
ethyl group at position 3 Suzuki-Miyaura cross coupling of 18 with vinyl boronic acid pinacole
ester followed by hydrogenation of intermediate 3-vinyl-6-nitroindazole 19 gave the desired 3-
ethyl analogue 21a in a two-step sequence. Indazole amine 20 was directly hydrogenated to give
2
1b.
Br
N
a
b
N
N
N
NO2
N
N
NO2
NH2
18
19
21a
c
N
N
N
N
NH2
NH2
2
0
21b
12

a
Scheme 3. Synthesis of regiosomeric tetrahydroindazolyl amines 21a and 21b
a
Reagents and conditions: (a) vinyl boronic acid pinacol ester, Pd(Ph P) Cl , aq. Na CO (2M),
3
2
2
2
3
1
,4-dioxane/MeOH, 90°C, 81%; (b) H , 3ꢀ50 bar, Nishimura’s catalyst, MeOH, 50°C, 38%; (c)
2
H , 50 bar, Pd/C, MeOH, conc. aq. HCl, 50°C, 29%.
2
Scheme 4 outlines the preparation of additional analogues to the tetrahydroindazole core: the
introduction of an extra nitrogen atom in the aromatic part gives rise to three possible types of
bicyclic triazole building blocks: tetrahydro-1H-benzotriazol-5-ylamine 24, tetrahydro-
[
1,2,4]triazolo[4,3-a]pyridin-7-ylamine 28 and tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-6-
ylamine 32.
F
a
b
N
N
N
N
N
N
H N
NO2
2
NO2
NH2
22
23
24
N
O
c
d
e
N
H N
N
N
2
N
H
NO2
HN
N
N
N
H
NO2
N
N
NO2
NH2
25
26
27
28
Tol-SO2
N
N
H
NC
f, g
h
i
N
N
N
N
NH2
N
N
N
N
NH2
NH2
NH2
29
30
31
32
a
Scheme 4: Preparation of variations of the original tetrahydroindazole core
13

a
Reagents and conditions: (a) (i) ethyl amine (2M THF solution), DMSO, 120°C, sealed tube, (ii)
AcOH, NaNO (2M aq. solution), 47% over two steps; (b) H , 3 bar, Nishimura’s catalyst,
2
2
MeOH, 66%; (c) propionyl chloride, DIPEA, THF, quant.; (d) Burgess’ reagent, THF, 65°C,
1%; (e) H , 3 bar, Nishimura’s catalyst, MeOH, 54%; (f) (i) ethyl magnesium bromide (3M in
6
2
Et O), Et O, refluxꢀrt, (ii) aq. HCl, rtꢀ40°C, 16%; (g) para-toluene sulfonyl hydrazide, MeOH,
2
2
quant. (mixture of isomers); (h) (Z)-30, morpholine, 100°C, 69%; (i) H , 0.5 bar, Pd/C 10%,
2
MeOH, 23%.
The benzotriazole scaffold was synthesized in a two-step sequence starting from 2-fluoro-5-nitro
aniline. Ethyl amine was introduced by nucleophilic aromatic substitution to give an intermediate
phenylene diamine, which was cyclized with sodium nitrite in aqueous acetic acid to directly
3
3
afford 1-ethyl-5-nitro-1H-benzotriazole 23. Subsequent hydrogenation using Nishimura’s
catalyst provided desired tetrahydrobenzotriazol-5-ylamine 24.
The synthesis of isomeric 3-ethyl-tetrahydro-[1,2,4]triazolo[4,3-a]pyridin-7-yl amine 28
commenced with the propionylation of 4-nitropyridin-2-ylhydrazine 25. The acylated product 26
3
4
was condensed with the aid of Burgess reagent to form the corresponding triazolopyridine 27,
which was hydrogenated to give the desired amine precursor 28.
Addition of ethyl magnesium bromide to cyano pyridine 29 converted the nitrile moiety into
intermediate ethyl ketone, which was condensed with tosyl hydrazide to furnish the
corresponding tosyl hydrazones 30 as a mixture of E- and Z-isomers. The Z-isomer of 30 was
3
5
taken forward and heated in morpholine to construct [1,2,3]triazolo[1,5-a]pyridine amine 31,
which was finally hydrogenated to yield building block amine 32.
14

The core framework of triazolopyridines was constructed starting from commercially available
2
-amino 3-halo pyridines 33a-b (scheme 5).
O
S
N
N
O
NH
N
N
N
a
b
H N
2
N
H
H N
2
X
X
X
3
3
3a (X=Br)
3b (X=Cl)
34a (X=Br)
34b (X=Cl)
35a (X=Br)
35b (X=Cl)
N
N
N
N
N
Br
H N
2
N
e
d
c
1
2
1
2
40-55
R
R
3
R
3
R
R
R
F
F
1
2
3
1
2
3
3
3
7a (R =H, R =H, R =H)
1
36a (R =H, R =H, R =H)
1
2
3
2
3
7b (R =CH , R =H, R =H)
3
36b (R =CH , R =H, R =H)
3
3
3
1
2
1
2
3
7c (R =H, R =F, R =F)
36c (R =H, R =F, R =F)
N
N
h
g
f
I
N
N
N
56-73
N
N
N
H
N
X
X
3
3
9a (X=Br)
9b (X=Cl)
38a (X=Br)
38b (X=Cl)
a
Scheme 5: Syntheses of central intermediates 37a-c, 39a-b and final compounds 40-73
a
Reagents and conditions: (a) ethoxycarbonyl isothiocyanate, DCM, rt, 87-96%; (b)
hydroxylamine hydrochloride, DIPEA, EtOH/MeOH, reflux, 74-75%; (c) aryl boronic acid,
t
Pd(dppf)Cl , aq. Na CO (2M), 1,4-dioxane, 110°C, 45-80%; (d) 37a: BuNO , CuBr , 60°C,
2
2
3
2
2
5
9%, 37b: aq. HBr (47%), NaNO , CuBr, 0°C, 53%, 37c: aq. HBr (47%), NaNO , 0°C, 42%;
2
2
15

(
e) (i) amine, Pd (dba) , Johnphos, NaOtBu, 1,4-dioxane, 80°C, or (ii) amine, CsF, DMSO,
2
3
1
60°C, microwave irradiation; (f) 38a: HI, NaNO , DMSO, 50°C, 57%, 38b: KI, p-toluene
2
sulfonic acid monohydrate, NaNO , MeCN/water, 50°C, 85%; (g) 39a: 13a, CsF, DMSO, 140°C,
2
t
microwave irradiation, 29%; 39b: 13a, Pd-PEPPSI-IPent Cl, NaOBu, 1,4-dioxane, 100°C, 51%;
(
h) (i) (hetero)aryl boronic acid or pinacole ester, SPhos palladacycle, K PO , toluene, 80°C or
3
4
THF/water, 120°C, sealed tube or (ii) aryl boronic acid, Pd(Ph P) Cl , aq. Na CO (2M), 1,4-
3
2
2
2
3
dioxane/MeOH, 90°C or (iii) (hetero)aryl boronic acid or pinacole ester, Pd(dppf) Cl *DCM
2
2
complex, 1,4-dioxane/MeOH, 70°C.
Treatment of 33a-b with ethoxycarbonyl isothiocyanate provided the corresponding thiourea
derivatives 34a-b, which were readily cyclized upon heating with hydroxylamine hydrochloride
3
6
in presence of a base to form the halogenated amino triazolopyridines 35a-b. Intermediates
3
5a-b enabled flexible synthesis of building blocks 39a-c and 37a-b. These two types of
functionalized building blocks would set the stage to either explore the nature of the distant aryl
ring or the LHS amine’s SAR.
Amino triazolopyridines 35a-b were converted into iodo triazolopyridines 38a-b using
Sandmeyer conditions, which were reacted with tetrahydroindazolyl amine 13a by heating in
presence of cesium fluoride or using chemoselective palladium-catalyzed Buchwald-Hartwig
3
7
conditions with Organ’s catalyst system to give precursors 39a-b ready for aryl ring
exploration. Finally, Suzuki-Miyaura cross coupling reactions with bromo amino
triazolopyridine 35a and the appropriate phenyl boronic acids provided intermediates 36a-c,
16

which were converted employing Sandmeyer conditions to yield bromo triazolopyridine building
blocks 37a-c.
The final compounds were synthesized by the two different approaches described already above
depending on SAR purposes (Scheme 5): i) to evaluate the LHS area, 2-bromo triazolopyridine
building blocks 37a-c were reacted with the required amines 13a-h, 17, 21a-b, 24 and 32 under
conditions similar (vide supra) to those that were used to prepare intermediates 39a-b to give the
final compounds 40-54 (see Table 1) and 56 (see Table 2); ii) to explore SAR of the phenyl
substituent, Suzuki-Miyaura cross coupling reactions with boronic acids and building blocks
3
9a-b were used to successfully introduce various phenyl and pyridinyl substituents furnishing
final compounds 55 and 57-73 (see Table 2).
3
3
.
Results and discussion
.1. Left-hand side SAR analysis
Excited by the already submicromolar activity of the initial hit 10 (IC = 0.38 µM), we explored
50
the SAR of this novel LHS systematically (Table 1).
N
N
R
N
H
N
F
a
Aβ42 IC50 [µM]
Cpd.
R
17

N
N
10
40
41
42
43
44
45
0.38 (5)
>30 (1)
4.57 (2)
0.07 (5)
0.06 (2)
0.43 (2)
3.40 (1)
N
N
N
H
N
N
N
N
N
N
N
N
N
18

N
4
4
4
6
7
8
2.77 (2)
>30 (2)
0.75 (1)
0.95 (3)
21.70 (1)
2.20 (1)
0.74 (2)
N
N
N
N
N
Cl
N
a
4
5
9
0
N
N
N
N
O
N
b
N
N
5
5
1
2
N
N
19

N
5
3
4
N
1.39 (1)
3.13 (1)
N
N
5
N
a
For multiple determinations, values are reported as geometric mean. Number of experiments is
b
given in parentheses. Compound was prepared as 3,4,5-trifluoro-phenyl derivative.
Table 1. LHS SAR Analysis
The regioisomeric 6-amino tetrahydroindazolyl derivative 40 did not modulate gamma secretase
up to a concentration of 30 µM and served to illustrate the importance of exactly positioning the
acceptor atom in the pyrazole unit relative to the attachment point to the core. Also demethylated
tetrahydroindazole derivative 41 (IC = 4.57 µM) was significantly less active, which may in
5
0
part be attributed to tautomerism of the pyrazole unit leading to a less confined electron density.
Next, we turned our attention to the pyrazole substituent itself and attached alkyl chains of
varying length and steric bulk (Table 1). Extension of the original N-methyl group to an ethyl
(
compound 42 (IC = 0.07 µM)) or an n-propyl group (compound 43(IC = 0.06 µM)) improved
50
50
activity 4-5-fold compared to the parent compound 10 and lead to derivatives with Aβ IC
4
2
50
<100 nM. The significant increase in activity achieved by simply switching from methyl to ethyl
substitution can be seen as the SAR key finding in retrospect and spurred our efforts in further
optimizing this series of GSMs. Branching of the alkyl chain as in compound 44 (IC = 0.43
50
µM)reduced potency slightly. Further substitutions of increasing size and chain length with
isobutyl, n-butyl and n-pentyl groups (derivatives 45-47) revealed very steep, but reliable and
20

robust SAR: just a simple one carbon atom chain extension from the most potent n-propyl
residue to its higher homologue (compound 46 (IC = 2.77 µM)) lead to a significant drop in
5
0
activity, which was completely abolished upon further extension to the n-pentyl homologue 47
IC > 30 µM).
(
5
0
SAR on other positions of the bicycle appears to be even more limited: 3-substitution at the
pyrazole ring with chlorine (compound 48 (IC = 0.75 µM)) lead to reduction of activity.
5
0
Taking into consideration that methylation at position 7 (compound 49 (IC = 0.95 µM)) or
50
ethoxylation at position 6 (compound 50 (IC = 21.7 µM)) of the saturated part of the bicycle
5
0
installed a second stereogenic center and thus gave rise to diastereomeric mixtures of micromolar
activity, none of the single isomers – if there was a eutomer - could potentially provide a superior
compound over parent derivative 42 (IC = 0.07 µM). Therefore no attempts were undertaken to
5
0
synthesize the pure diastereomers.
We investigated other closely related heterobicyclic systems as isosteric replacement of the
tetrahydroindazole element. However, all of the variations synthesized were significantly less
potent (Table 1, compounds 51-54). Interestingly, attaching an ethyl group at position 3 of
inactive regioisomeric tetrahydroindazol-6-yl amine derivative 40 (IC > 30 µM) restored
50
activity to some degree. Therefore, although being a tetrahydroindazole derivative, 54 (IC =
50
3
.13 µM) may technically also be envisioned as additional isostere.
3
.2. Aryl head group SAR
Next, we turned towards exploration of the distal aryl group (Table 2). In order to interpret the
individual contribution of the aryl substituents of our original hit to compound activity, we
synthesized mono-substituted o-CH - and p-fluoro analogues 55 (IC = 0.18 µM) and 56 (IC =
3
50
50
21

0
.16 µM). Interestingly, both derivatives appeared to be less active compared to the parent
molecule, however in comparison to the unsubstituted phenyl analogue 57 (IC = 0.07 µM) the
50
individual activity spread was rather small.
N
N
N
N
N
H
N
R
a
Aβ42 IC50 [µM]
Cpd.
R
42
55
56
57
0.07 (5)
F
0.18 (2)
0.16 (2)
0.07 (2)
F
22

Cl
58
59
60
61
62
63
64
0.06 (2)
0.04 (2)
0.06 (2)
0.02 (3)
0.03 (2)
0.35 (2)
0.73 (1)
Cl
Cl
Cl
F
F
F
F
N
N
23

65
66
67
68
69
70
71
0.68 (1)
0.20 (1)
0.67 (2)
0.12 (2)
0.50 (2)
0.20 (1)
0.09 (1)
N
N
N
OMe
N
MeO
N
N
OMe
N
OMe
24

7
2
3
0.05 (1)
0.04 (1)
N
N
7
N
Cl
a
For multiple determinations, values are reported as geometric mean. Number of experiments is
given in parentheses.
Table 2. Aryl head group SAR
We followed up by synthesizing all three mono-chloro analogues (compounds 58-60). Within
this subset of compounds the meta-substituted derivative 59 (IC = 0.04 µM) appeared to be
5
0
more active than its regioisomers. An additional fluorine substituent at the para position
compound 61 (IC = 0.02 µM)) helped to further increase potency by a factor of two and
(
50
resulted in preparation of the most potent racemic compound within this structural class. This
effect was further substantiated by the 3,4,5-trifluoro derivative 62 (IC = 0.03 µM), which was
5
0
also more potent than its 4-fluoro analogue 55 (IC = 0.18 µM).
50
Since the individual substituent effect on potency was rather limited, the increase in activity
presumably is mainly lipophilicity-driven. Therefore, in order not to compromise the beneficial
contribution of our tetrahydroindazole residue to the overall molecular polarity, we incorporated
3
0
-pyridines as replacement for the phenyl group. When comparing parent compounds 57 (IC =
5
0
.07 µM) and 63 (IC = 0.35 µM), this bioisosteric replacement initially seemed to result in
50
activity reduction, which is in line with the increased polarity of the basic pyridyl group.
25

We revisited the substituent pattern by scanning with a neutral methyl substituent in order to
retain the pyridine basicity (entries 64-67): Addition of a methyl group in position 2 and 4 as
well as in the 6-position of the pyridine lead to a reduction of activity, whereas the 5-methyl
substitution in 66 (IC = 0.20 µM) increased activity relative to the other methyl analogues, but
50
also in comparison to parent pyridine derivative 63 (IC = 0.35 µM), confirming the previously
50
observed phenyl group substituent effect pattern.
We hypothesized that modulating pyridine basicity could influence activity. To this end, we
prepared methoxy analogues 68-71. Alkoxy substitution in the alpha position relative to a
3
8
pyridine nitrogen atom is known to significantly reduce basicity . Indeed, 6-methoxy pyridine
3
9
7
6
1 (IC = 0.09 µM) (pKa = 4.55) showed improved activity, but not its 2-methoxy analogue
50
calc.
9 (IC = 0.50 µM) (pKa = 4.55), indicating a potential negative interaction with the GS
50
calc.
binding site in the case of 69. Interestingly, the 4-methoxy analogue 68 (IC = 0.12 µM, pKa
=
5
0
calc.
7
.16), which should be more basic than its regioisomer 69 (IC = 0.50 µM) (pKa = 4.55) is
50
calc.
about 4-fold more potent. Also, 6-dimethylamino pyridine 72 (IC = 0.05 µM) is surprisingly
50
potent in light of its high calculated pKa value (pKacalc. = 6.67). For the latter two compounds, 68
and 72, basicity is increased in a way that could potentially provide a favorable balance between
solubility at low pH and permeability at physiological pH. Overall, there was no clear correlation
of basicity alone with potency of the pyridine derivatives.
The most potent compound of the pyridine subseries was the more lipophilic 5-chloro pyridine
7
3 with an Aβ IC of 37 nM, which is equal to the activity of 3-chloro phenyl derivative 59
42
50
(
IC = 0.04 µM), although it is more polar.
5
0
3
.3. Influence of stereochemistry on biological activity
26

Since the tetrahydroindazole compounds were obtained as racemates we sought to obtain their
pure enantiomers and investigate potential enantiomer effects with representatives (Figure 6).
chiral HPLC
separation
N
ent2-13a
ent1-13a
+
N
NH2
a
a
13a
N
N
N
N
N
N
N
N
N
H
N
Br
N
H
N
Br
ent1-39a
ent2-39a
b
b
N
N
N
N
N
N
Structure of ent1-61
solved by
X-ray analysis
N
N
N
H
N
N
H
N
Cl
Cl
ent1-61
F
ent2-61
F
a
Figure 6: Determination of the absolute stereochemistry of tetrahydroindazole GSMs
a
Reagents and conditions: (a) 38a, CsF, DMSO, 130°C, microwave irradiation, ent1-39a: 46%,
ent2-39a: 62%; (b) 3-chloro-4-fluoro-phenylboronic acid, aq. K CO (2M), Pd(dppf) Cl *DCM
2
3
2
2
complex, 1,4-dioxane/MeOH, 90°C, ent1-61: 57%, ent2-61: 71%.
In order to determine the absolute stereochemistry of the enantiomers the racemic amine building
block 13a was resolved into its enantiomers by chiral HPLC and served to prepare chiral
intermediates ent1-39a and ent2-39a (with at this stage still undetermined absolute
27

stereochemistry). We then synthesized chloride containing analogues to determine the absolute
stereochemistry by analysis of anomalous scattering in X-ray analysis of single-crystals. From
intermediate ent1-39a we synthesized chiral tetrahydroindazole ent1-61, one enantiomer of our
most potent racemic compound 61 (Aβ IC = 0.02 µM). The corresponding enantiomer ent2-61
42
50
was prepared from the antipode building block ent2-39a.
We were delighted to discover a pronounced enantiomer effect for our tetrahydroindazolyl amine
moiety: ent1-61 (Aβ IC = 0.009 µM) was approximately two-fold more active than racemic 61
42
50
and over 20-fold more active compared to its corresponding enantiomer ent2-61 (Aβ IC =
42
50
0
.194 µM) when measured within the same assay run. A crystalline batch of ent1-61, obtained by
recrystallization from methanol, was used in X-ray analysis (for details see Supporting
Information) to unambiguously determine the absolute stereochemistry of the more active
tetrahydroindazole enantiomer to be (5R).
With this information in hand, we now readily matched the stereochemistry to both chiral
batches of 13a and prepared additional chiral tetrahydroindazole derivatives. In particular, based
on its overall profile, we chose to synthesize the more active ingredient of compound 42
(
Scheme 6).
N
N
N
a
N
ent1-13a
N
H
N
BI-1408 ((R)-42)
F
a
Scheme 6: Preparation of chiral tetrahydroindazole BI-1408 ((R)-42)
28

a
Reagents and conditions: (a) 37b, CsF, DMSO, 160°C, microwave irradiation, 80%.
3
.4. In vitro and rat in vivo profile of BI-1408
While most of the described compounds suffer from poor metabolic stability in rodents, the
overall profile (Table 3-1 and 3-2) of BI-1408 ((R)-42) led us to the conclusion that this
compound might be an appropriate tool to asses in vivo activity of the series.
In accordance with our expectations, tetrahydroindazole (R)-42 was also approximately two-fold
more active compared to the racemate 42. Furthermore, (R)-42 was equipotent in rat primary
cortical neurons, but affected neither total Aβ concentration (ratio Aβtotal / Aβ = 254) nor Notch
42
receptor processing, which is in line with the proposed mode of action for gamma secretase
modulation.
In vitro profile
BI-1408 ((R)-42)
0.04 (6)
254 (6)
0.04
Aβ IC [µM] (no. of experiments)
4
2
50
Ratio Aβtotal / Aβ (no. of experiments)
42
Rat primary cortical neurons Aβ IC [µM]
42
50
Notch IC [µM]
>30
5
0
HLM / RLM [mL/min*kg]
PPB rat (%)
12.6/57
98.8
-
6
MDCK P [10 cm/s] (efflux ratio)
78 (0.7)
8/39
a-b
hERG inhibition [% inhibition at 1 and 10 µM]
29