Discovery and characterization of novel indole and 7-azaindole derivatives as inhibitors of β-amyloid-42 aggregation for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2017.02.001

The aggregation of amyloid-β peptides into cytotoxic oligomeric and fibrillary aggregates is believed to be one of the major pathological events in Alzheimer disease. Here we report the design and synthesis of a novel series of indole and 7-azaindole derivatives containing, nitrile, piperidine and N-methyl-piperidine substituents at the 3-position to prevent the pathological self-assembly of amyloid-β. We have further demonstrated that substitution of the azaindole and indole derivatives at the 3 positions is required to obtain compounds with improved physicochemical properties to allow brain penetration.

Full text of DOI:10.1016/j.bmcl.2017.02.001

Accepted Manuscript
Discovery and characterization of novel indole and 7-azaindole derivatives as
inhibitors of β-amyloid-42 aggregation for the treatment of Alzheimer’s disease
Nampally Sreenivasachary, Heiko Kroth, Pascal Benderitter, Anne Hamel,
Yvan Varisco, David T. Hickman, Wolfgang Froestl, Andrea Pfeifer, Andreas
Muhs
PII:
S0960-894X(17)30121-X
http://dx.doi.org/10.1016/j.bmcl.2017.02.001
BMCL 24673
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 December 2016
30 January 2017
1 February 2017
Please cite this article as: Sreenivasachary, N., Kroth, H., Benderitter, P., Hamel, A., Varisco, Y., Hickman, D.T.,
Froestl, W., Pfeifer, A., Muhs, A., Discovery and characterization of novel indole and 7-azaindole derivatives as
inhibitors of β-amyloid-42 aggregation for the treatment of Alzheimer’s disease, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.001
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Bioorganic & Medicinal Chemistry Letters
Discovery and characterization of novel indole and 7-azaindole derivatives as
inhibitors of β-amyloid-42 aggregation for the treatment of Alzheimer’s disease
Nampally Sreenivasacharya, Heiko Krotha, Pascal Benderittera,b, Anne Hamela, Yvan Variscoa, David T.
Hickmana,Wolfgang Froestla, Andrea Pfeifera, Andreas Muhsa,
a AC Immune SA, EPFL Innovation Park, Building B, 1015 Lausanne, Switzerland
b Present address: Oncodesign, 20, rue Jean Mazen, 21076 Dijon, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The aggregation of amyloid-β peptides into cytotoxic oligomeric and fibrillary aggregates is
believed to be one of the major pathological events in Alzheimer disease. Here we report the design
and synthesis of a novel series of indole and 7-azaindole derivatives containing, nitrile, piperidine
and N-methyl-piperidine substituents at the 3-position to prevent the pathological self-assembly of
amyloid-β. We have further demonstrated that substitution of the azaindole and indole derivatives at
the 3 positions is required to obtain compounds with improved physicochemical properties to allow
brain penetration.
Keywords:
Alzheimer’s disease
Aβ aggregation
2009 Elsevier Ltd. All rights reserved.
Inhibitors
Structure-activity relationship
Indole derivatives
Azaindole derivatives
Alzheimer’s disease (AD) is a form of senile dementia, characterized by a progressive loss of memory and cognitive function1-3
.
It has been hypothesized that formation of β-amyloid (Aβ) plaques is key to the development and progression of the disease. Present
pharmacotherapies with known anti-cholinesterase activity, such as Aricept and Exelon, are only helpful to alleviate some of the
symptoms for a limited time period4. These agents act to stabilize the remaining neuronal networks and prolong neuronal function
until their therapeutic effect diminishes and drug tolerance occurs. The marginal benefits from these therapies emphasize the urgent
need to develop alternative and effective disease-modifying agents5. Several trends have been emerging using small molecules to
target various AD pathological routes such as the amyloidogenic secretases (β/γ- secretase)6-7, amyloid-β aggregation8-14, tau
phosphorylation and fibrillation15-17 and metal-ion redox/reactive oxygen species (ROS)18-22
.
Of those, Aβ is one of the most promising targets for the development of new therapies as the substantial data derived from
genetics, animal modeling, and biochemical studies support the idea that Aβ, the major component of senile plaques, plays a central
role in AD pathophysiology23-24. Thus, we have initiated a program aiming to design novel non dye compounds for the inhibition of
Aβ aggregation for AD therapeutics. We have previously reported small molecule inhibitors of Aβ based on our rational design, i.e.
3-aminopyrazole24 and 2,6-disubstituted pyridine derivatives25 which can interact via a donor-acceptor-donor (DAD) hydrogen bond
pattern complementary to that of the β-sheet of Aβ22-24. However, compounds following this design displayed low metabolic
stability and poor PK properties25. To overcome these issues, we sought to evaluate whether replacing the 2,6-disubstituted pyridine
moiety with 6-amino-substituted indole and 7-azaindole moieties (Fig. 1) would improve metabolic stability and PK properties
while maintaining their inhibition of Aβ aggregation properties.
———
 Corresponding author. Tel: +41 21 345 91 24; fax: +41 21 345 91 20; e-mail: andreas.muhs@acimmune.com

Figure 1. Possible hydrogen bond interactions of 3-aminopyrazole, 2,6-disubstituted pyridine and 6-amino-indole/7-azaindole derivatives with the β-sheet
conformation of Aβ.
In order to synthesize the novel compounds containing indole or 7-azaindole moieties, it was necessary to prepare the required
building blocks.
Thus, suitable 7-azaindole or indole derivatives containing either bromo- or amino substituents were synthesized. The reaction of
commercially available 7-azaindole 1 and 7-azaindole-carbonitrile 2 with m-CPBA in diethyl ether gave the corresponding salts 3
and 4. The suspensions of 3 or 4 in acetonitrile were treated with dimethyl sulfate under reflux followed by quenching with a
concentrated ammonia solution in methanol33 to afford the corresponding amines 5 and 6 (Scheme 1).
Scheme 1. Reagents and conditions: (a) m-CPBA, Et2O, rt, 18 h, 86-96%; (b) (i) dimethylsulfate, CH3CN, 70 °C, 8 h; (ii) 7M NH3/CH3OH, 60 °C, 48 h, 41-
54%; (c) (i) dimethylsulfate, CH3CN, 70 °C, 16 h; (ii) 1-Boc-4-amino-piperidine, CH3OH, 50-60 °C, 3d, 36-45%; (iii) 2M HCl/Et2O, rt,; (d) (i) K2CO3, H2O,
rt, 91%; (ii) HMDS, toluene, benzoyl bromide, rt, 1 h, 65%; (iii) NaOH, CH3OH, rt, 16 h, 60%.
The synthesis of 7 was achieved from 3 as described for 5, employing tert.-butyl 4-aminopiperidine-1-carboxylate followed by
33
acid cleavage of the tert-butyloxycarbonyl (Boc) protecting group The synthesis of the N1-triisoproylsilyl (TIPS) protected 7-
azaindole derivative 8 containing a 6-bromo-substituent was achieved by treating 3 with benzoyl bromide in the presence of
hexamethyldisiliazane (HMDS), followed by saponification of the benzoyl group with sodium hydroxide in methanol, followed by
TIPS-protection of the N1-position of the 7-azaindole moiety to provide 8.
The corresponding 3-substituted indole and azaindole building blocks 12, 13, 14 and 15 containing a 6-bromo-substituent were
prepared according to Scheme 2.
Scheme 2. Reagents and conditions: (a) 25% NaOCH3/CH3OH, 80°C, 48 h, 52-91%; (b) (i) H2, PtO2, TEA, CH3OH, rt, 18 h, 63%; NaH, THF, TIPS-Cl, rt,
30 min, 91%; (c) (i) NaH, THF, TIPS-Cl, rt, 30 min, 65%; (ii) H2, PtO2, TEA, EtOAc, rt, 18 h, 85%; (d) NaH, THF, TIPS-Cl, 30 min, rt, 99%; (e) H2, PtO2,
TEA, CH3OH, rt, 18 h, 63%; (ii) NaH, THF/CH3I, 30 min, 94%
The synthesis of 14 was achieved from 9 by protecting the N1-position of 6-bromo-indole 9 with TIPS-Cl in the presence of
sodium hydride as base to afford 14. The condensation reaction of 6-bromo-indole 9 with tert.-butyl 4-oxopiperidine-1-carboxylate
or 1-methylpiperidin-4-one in the presence of sodium methoxide in methanol yielded the corresponding 3-substituted 6-bromo-
indole derivatives 10 and 11. Reduction of the double in 10 with platinum(IV) oxide, followed by TIPS-protection of the N1-
position provided the corresponding piperidine-derivative 12. TIPS-protection of the N1–position of compound 11 followed by
double bond reduction yielded 13. N1-methylation of 10 with methyl iodide followed by reduction of double bond afforded
compound 15.
To test positional effects on indole and 7-azaindole core structures, the indole building blocks 19 and 20 containing a 5-bromo-
substituent were prepared (Scheme 3).

Scheme 3. Reagents and conditions: (a) 25% NaOCH3/CH3OH, 80°C, 48 h, 73-86%; (b) (i) NaH, THF, rt, 10 min; (ii) H2, PtO2, TEA, CH3OH, rt, 16 h, 30-
50%. (iii) TIPS-Cl, rt, 1-2 h, 65-70%
Commercially available 5-bromo-indole was protected with TIPS to yield 16. The 5-bromo-indole was reacted with tert.-butyl 4-
oxopiperidine-1-carboxylate or 1-methylpiperidin-4-one as described to afford the corresponding 3-substituted 5-bromo-indole
derivatives 17 and 18. Reduction of the 1,2,3,6-tetrahydropyridin moiety in 17 and 18 with platinum(IV) oxide, followed by TIPS-
protection of the N1-position provided the corresponding piperidine-derivatives 19 and 20.
The corresponding 6-bromo-7-azaindole building block containing a piperidine substituent at the 3-position was prepared from 1
(Scheme 4).
Scheme 4. (a) 25% NaOCH3/CH3OH, 80°C, 2 h 30 min, 67%; (b) (i) H2, Pd/C, CH3OH, rt, 48 h, 91%; (c) m-CPBA, CH2Cl2, rt, 12 h, 74%; (d) (i) HMDS,
toluene, benzoyl bromide, rt, 12 h; (ii) NaOH, CH3OH, rt, 2 h, 40%; (e) (i) NaH, DMF, rt, 1 h; (ii) TIPSCl, rt, 19 h, 71%.
The condensation reaction of 1 with tert.-butyl 4-oxopiperidine-1-carboxylate as described yielded the corresponding 3-
substituted 7-azaindole derivative 21. Reduction of the double bond was carried out with palladium on carbon, under the hydrogen
gas pressure to give corresponding reduced product 22. The N-oxide derivative 23 was prepared by treatment with m-CPBA.
Treatment of 23 with benzoyl bromide in the presence of HMDS, followed by saponification of the benzoyl group provided 24.
TIPS-protection of the N1-position of 24 afforded the desired building block 25.
The corresponding 6-amino-indole building blocks containing a piperidine substituent at the 3-position were prepared from 6-
nitro indole 26 (Scheme 5). Treatment of 26 with tert.-butyl 4-oxopiperidine-1-carboxylate or 1-methylpiperidine-4-one as
described yielded the corresponding 3-substituted 7-indole derivatives 27 and 28. TIPS-protection of 27 and 27 followed by
hydrogenation with palladium on carbon afforded building blocks 29 and 30.
Scheme 5. Reagents and conditions: (a) 25% NaOCH3/CH3OH, 80°C, 16 h, 56-72%; (b) (i) NaH, THF, rt, 10 min; (ii) TIPSCl, rt, 1 h, 46-50%; (iii) H2,
Pd/C, EtOAc, rt, 16 h, 36-38%.
A small library of compounds having a 7-azaindole moiety linked to a substituted phenyl or pyridine moiety via an amine linker
(Scheme 6) was prepared first to get an idea about the structural motifs necessary to retain some of the inhibition of aggregation
properties described for our previous compounds24, 25. We employed Buchwald reaction conditions for C-N bond formation. A

highly active palladium(0) catalyst was generated from palladium(II) acetate (Pd(OAc)2) and 2-dicyclohexyl-phosphino-2′,4′,6′-
triisopropyl-biphenyl (XPhos)29-31 to enable the coupling of the bromo- and amino building blocks (Scheme 6).
Scheme 6. Reagents and conditions: (a) Pd(OAc)2, XPhos, NaOtBu, 2-bromopyridine or 1-bromo-4-fluorobenzene, dioxane, 110°C, 2 h, 39-94%. (b) (i)
Pd(OAc)2, XPhos, 3,5-difluoroaniline, NaOtBu, dioxane, 110°C, 2 h, 50% or Pd(OAc)2, XPhos, 3,4-difluoroaniline or 3,5-difluoroaniline or 6-
(trifluoromethyl)pyridin-3-amine, K2CO3, t-BuOH, 110°C, 3 h, 77-91%; (ii) 1 M TBAF/THF, CH3CN, rt, 1 h; (iii) 1 M HCl/H2O, 66-81%.
The reaction of 6-amino-azaindole 5 with 2-bromo-pyridine or 1-bromo-4-fluorobenzene afforded the final compounds 32 and
33. TIPS-protected building blocks 8 and commercially available 6-bromo-1-(triisopropylsilyl)-1H-pyrrolo[3,2-b]pyridine 31 were
reacted under Buchwald conditions with aniline derivatives to form the intermediate palladium coupling products, which were
treated with tetra-butylammonium fluoride (TBAF) to cleave the TIPS-protecting group. The final compounds 32-37 were isolated
after acid treatment as HCl-salts.
In our screening cascade of small molecule inhibitors of Aβ peptide, the thioflavin-T (ThT) fluorescence assay27 using crude Aβ1-
42 peptide film at 33 μM and compounds at 330 μM was employed as a primary screen. The data in Figure 2 indicated compounds
32-37 of library 1 displayed inhibition of Aβ aggregation in the range of 52% (33) to 71% (32). Exchange of a phenyl or pyridine
ring in 32-37 with piperdine to obtain additional hydrogen bond donor interactions, did not result in an improved compound as 7
displayed only 48% inhibition of Aβ aggregation. However, this could be explained by the lack of additional aromatic -
interactions of compound 7 with the Aβ peptide, Compounds 35 and 37 showed inhibition of Aβ aggregation of 60% and 52%,
respectively. This indicated shifting the hydrogen bond acceptor from 7-azaindole-core (35) to a 4-azaindole-core (37) had little
effect on inhibition Aβ aggregation. Due to their small size, compounds 32-37 appeared not to be able to pick-up additional π-π
interactions to compensate for the reduction in DAD interactions. Thus, we identified a new scaffold containing an azaindole/indole
core, but the inhibition of Aβ aggregation was lower compared to the best compounds containing the 3-aminopyrazole24 or 2,6-
disbstituted pyridine moieties25 we reported earlier.
In order to increase the inhibition of Aβ aggregation properties and further diversify the scaffold, we envisaged inhibition of Aβ
aggregation would be improved by dimeric compounds (38-44), which can offer additional hydrogen bond donor and aromatic π-π
interactions with the Aβ peptide. The synthesis of dimeric compounds 38-44 (Scheme 7) was achieved by Buchwald Pd-coupling
reaction conditions31 of amino building block 5 and commercially available 6-aminoindole with bromo building blocks 8, 14, 16,
31, and commercially available 5-bromo-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine, as the first step. Cleavage of the TIPS-
protecting group with TBAF followed by acid treatment afforded dimeric compounds 38-44. The synthesis of 38 was achieved by
pd-coupling reaction of 7-aminoazaindole 5 and commercially available corresponding bromo building block 31. Testing the
inhibition of Aβ aggregation properties of 38 (98%) in ThT, displayed pronounced activity when compared to compound 32. The
encouraging results of 38 from ThT assay, we then expanded the library of dimeric compounds 38-44.

Scheme 7. Reagents and conditions: (a) (i) Pd(OAc)2, XPhos, 8, 14, 16, 31, 5-bromo-1-(tri-isopropylsilyl)-1H-pyrrolo[2,3-b]pyridine, NaOtBu, dioxane,
110°C, 2 h, 6-30%; (ii) 1M TBAF/THF, CH3CN, rt, 1 h; (iii) 1 M HCl/H2O, 48-84%.
Testing the inhibition of Aβ aggregation properties of compound 38-44 of library 2 in the ThT-assay (Fig. 2) showed dimeric
compounds displayed inhibition of Aβ aggregation between 75-98%. Whereas 39 and 40 showed only a minor improvement over
32, i.e. 71% vs. 75% inhibition of Aβ aggregation, compounds 44 (86%), 41 (96%), 42 (94%) and 43 (97%) displayed considerably
better inhibition of Aβ aggregation as 32. These data suggested that dimeric compounds like 39 and 40 containing all hydrogen
bond donors and acceptors on one side of the molecule were inferior to compounds where at least one hydrogen bond donor (42-44)
or acceptor (38) was on the opposite site. Interestingly, compounds 41 and 42 containing only hydrogen bond donors showed 96%
and 94% inhibition of Aβ aggregation, respectively.
Inthibition of A 1-42 aggregation in primary screen ThT assay
library-2
library-1
100
50
0
32 33 34 35 36 37
7
38 39 40 41 42 43 44
Compounds
Figure 2: Aβ1-42 lyophilized powder was reconstituted in hexafluoroisopropanol to 1 mM to prepare the Aβ1-42 peptide film. Aβ1-42 inhibition of aggregation
measured by a standard ThT assay at 1:10 Aβ1-42 to compound molar ratio (33:330 M). The data are expressed as mean of two independent experiments.
Data are expressed as percentage (mean ± standard deviation) of control conditions: Aβ1-42 aggregation with DMSO only.
Taken together the results of library 1 & 2, aromatic - interactions and orientation hydrogen bond donor and acceptors are
quite important. In line with previous results of our 2,6-disubstitued pyridine derivatives25, 3 hydrogen bond donors in the correct
arrangement were required to achieve >80% inhibition of Aβ aggregation in the ThT-assay. In contrast to the 2,6-disubstitued
pyridine derivatives, ThT-data for compounds 38-44 suggested the hydrogen bond acceptors were not required for inhibition of Aβ
aggregation. Compound 38 was selected as representative molecule having a dimeric structure to assess its in vitro stability.
However, 38 displayed poor metabolic stability in human liver microsomes with a half-life of only 11 minutes. It was also found
that indole-3 position was susceptible for oxidation and causing for poor metabolic stability in a metabolic identification study (data
not shown).
In order to block the potential metabolic weak spot at the 3 position of the indole/7-azaindole moieties, nitrile, piperidine and N-
methyl piperidine substituents were introduced with the aim to maintain the inhibition of Aβ aggregation properties of 38-44 but to
improve metabolic stability and solubility. The synthesis of dimeric compounds 45-51 having nitrile, piperidine and N-methyl
piperidine substituents was achieved by Buchwald Pd-coupling reaction conditions29-31 of amino building blocks 6 , 29 and 30 with
bromo building blocks 12, 13, 19, 20 and 25 as the first step (Scheme 8) to yield corresponding TIPS protected Pd-coupling
derivatives. The cleavage of the TIPS-protecting group with TBAF or polymers supported fluorine followed by acid treatment
afforded compounds 45-51 of library 3 (Scheme 8).
The ThT-assay data of compounds 45-51 of library 3 in Figure 3 indicated >90% inhibition of Aβ aggregation for all
compounds. Thus, no preference was observed for either of the nitrile, piperidine and N-methyl piperidine substituents or the
orientation of the indole/7-azaindole core structures, i.e. 6,6-substitution (45, 47, 48, 51) versus 5,6 substitution (46, 49, 50). The
>90% inhibition of Aβ aggregation of compounds 45-51 could be rationalized by an additional hydrogen bond donors. To test our
hypothesis for improved metabolic stability in human liver microsomes, compounds 45-51 were characterized in vitro to assess their
solubility, metabolic stability, permeability and if they are P-gp substrates (Table 1). The solubility of 45, 46 and 48 in PBS (pH
7.4) was between 153-196 µM and we assumed compounds 47, 49-51 would show a similar behavior. Incubation with human liver
microsomes showed that compounds 45-51, were quite stable (89-100% parent remaining after 1 hour), which was a marked
improvement compared to compound 38.

Scheme 8. Reagents and conditions: (a) (i) Pd(OAc)2, XPhos, NaOtBu, dioxane, 110°C, 2 h, or Pd(OAc)2, XPhos, K2CO3, t-BuOH, 110°C, 3 h, 6, 29, 30, 12,
13, 25, 19, 20, 30-79%; (ii) 1M TBAF/THF, CH3CN, rt, 1 h or polymer-supported fluorine, THF, rt, 16 h, 60-92%; (iii) 2 M HCl/Et2O, rt, 16 h, 56-99%.
Inhibition of aggregation in primary screen ThT assay
Library-3
Library-4
100
50
0
45
46
47
48
49
50
51
52
53
54
55
56
57
Compounds
Figure 3: Aβ1-42 lyophilized powder was reconstituted in hexafluoroisopropanol to 1 mM to prepare the Aβ1-42 peptide film. Aβ1-42 inhibition of aggregation
measured by a standard ThT assay at 1:10 Aβ1-42 to compound molar ratio (33:330 M).The data are expressed as mean of two independent experiments
Data are expressed as percentage (mean ± standard deviation) of control conditions: Aβ1-42 aggregation with DMSO only.
However, permeability assessment u sing a sub-clone of the Caco-2 cell line showed rather low apical-to-basolateral (A->B)
permeability (0.02-0.5 x 10-6 cm/s) for compounds 45-51, most likely caused by the multiple hydrogen bond donors (three to five)
and the presence of two aliphatic amine moieties. In addition, compounds 45, 50 and 51 were P-gp substrates with efflux ratios of
21-100. Thus, no plasma or brain uptake of 46 after oral administration of 10 mg/kg to male Swiss mice could be detected (data not
shown).
Thus, it was necessary to reduce the number of hydrogen bond donors and aliphatic amine moieties. The aim was to obtain
compounds able to inhibit Aβ aggregation comparable to 45-51, with improved permeability and PK properties. As there was no
marked difference in inhibition of Aβ aggregation of library 2 (38-41) and library 3 (45-51) compounds, we envisaged combining
the right-hand side scaffold of library 3 (45-51) i.e. 3-piperidine-substituted indole- or 7-azaindole moiety with the substituted
phenyl-derivatives used for library 1 (32-37).

Scheme 9. Reagents and conditions: (a) (i) Pd(OAc)2, XPhos, NaOtBu, dioxane, 110°C, 2 h, or Pd(OAc)2, XPhos, K2CO3, t-BuOH, 110°C, 3 h, 3,4-
difluroaniline, 3, 4-dimethoxyaniline, 6-(trifluoromethyl)pyridin-3-amine, 12, 13, 19, 25, 47-75%; (ii) 1 M TBAF/THF, CH3CN, rt, 1 h or polymer-
supported fluorine, THF, rt, 16 h, 68-80%; (iii) 2 M HCl/Et2O, rt, 16 h, 78-83%.
The synthesis of compounds 52-57 having a lower molecular weight, two to three hydrogen bond donors and only one aliphatic
amine was achieved by Buchwald Pd-coupling reaction conditions32-34 of 3,4-difluro- 3, 4-dimethoxyaniline and 6-
(trifluoromethyl)pyridin-3-amine with bromo building blocks 12, 13, 19, and 25 as the first step (Scheme 9, and see compound 57
synthesis detail in supplementary information). Cleavage of the TIPS-protecting group with TBAF followed by acid treatment
afforded compounds 52-57 of library 4. Compounds 52-57 displayed inhibition of Aβ aggregation of 81-93% in the ThT-assay (Fig.
3). Thus, it was possible to reduce the number of hydrogen bond donors to two or three and aliphatic amines to one but maintaining
inhibition of Aβ aggregation similar to compounds 52-57.
As compounds 45-57 displayed >80% inhibition of Aβ aggregation in the ThT-assay under screening conditions, it was
necessary to determine IC50 values of selected compounds to see differences. Subsequently, we assessed the IC50 values of
compounds 38, 45, 46, 48, 50, 52, 53, 54, 55, 56, 57 in the ThT assay with Congo Red and Curcumin as reference standards.
Compounds 38, 45, 46, 48, 50, 52, 53, 54 and 56 were more potent than 55 and 57 (Fig. 4). Thus, reducing the number of hydrogen
bond donors from three to two (55, 57) resulted in a drop of potency. Compounds 45 and 50 (library 3) displayed inhibition of Aβ
aggregation properties comparable to Curcumin with IC50 of 17.8 M and 15.3 M, respectively. Though compound 56 contained
only two hydrogen bond donors, inhibition of Aβ aggregation was comparable to compounds 38, 46, 48, 52 and 54, i.e. IC50< 30
M, by introducing the 3,4-dimethoxy-phenyl moiety. Congo Red remained the most potent compound in the ThT-assay.
In vitro characterization (Table 1) showed compounds 52-57 have a solubility in PBS (pH 7.4) of 154-200 µM, are metabolically
stable when incubated with human (74-95% parent remaining after 1 hour) and mouse liver microsomes (70-85% parent remaining
after 1 hour). Only compound 55 was less stable in mouse liver microsomes (30% parent remaining after 1 hour). Compounds 52-57
displayed good apical-to-basolateral (A->B) permeability (3-24 x 10-6 cm/s) and were not P-gp substrates (efflux ratio <1), except
compound 54 (efflux ratio of 33). Thus, compounds 52-54, and 56 combined inhibition of Aβ aggregation properties of IC50< 30
M, comparable to potent 2,6-disubstituted pyridine derivatives25, with improved physicochemical properties.
Inhibition of Aß42 Aggregation - Peptide Film
50
40
30
20
10
0
d
in
m
38
45
46
48
50
52
53
54
55
56
57
re
o
compounds
Curcu
Cong
Figure 4. IC50 determination by ThT assay using Aβ1-42 peptide film. The concentration of Aβ1-42 peptide film was 33 M. The test concentration for
compounds 38, 45, 46, 48, 50, 52, 53, 54, 55, 56, 57, Congo Red and Curcumin were 330 M, 82.5 M, 20.63 M, 5.16 M, 1.29 M, 0.32 M and 0.08
M. The IC50 values were determined from the fluorescence values obtained. The IC50-data (M) are expressed as mean ± standard deviation and indicated
on top of each graph.
Table 1: Summary of ADME results of compounds

Solubility Metabolic
Metabolic
stability
mouse
[% parent]b
85
A->B
Efflux
in PBS
stability
human
[% parent]b
100
92
permeability ratio
[x 10-6
[M]a
cm/sec]
196
153
n.d.c
166
n.d.c
n.d.c
n.d.c
178
176
n.d.c
200
200
169
0.1
0.1
0.01
0.4
0.02
0.5
0.3
7.1
14.5
1.2
10.4
24
>100
1
0
45
46
47
48
49
50
51
52
53
54
55
56
57
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
85
89
95
95
0.5
n.d.c
32
100
100
89
95
90
21
0.51
0.64
33
0.18
0.96
0.53
81
n.d.c
35
91
92
77
70
83
3
aDetermined at pH 7.4 in phosphate buffered saline (PBS)
bRemaining parent compound after 1 hour of incubation at 37°C.
cNot determined (n.d.).
However, compounds 52, 53 did not display good brain exposure after oral administration at 10 mg/kg to male Swiss mice (data
not shown) despite having good permeability (>4 x 10-6 cm/s) and showing good metabolic stability (human and mice liver
microsomes). Thus, it appeared compound 52, 53 were still too polar with three hydrogen bond donors including one secondary
amine. Reducing the number of hydrogen bond donors by N1-alkalytion did improve brain uptake for 57 after oral administration at
10 mg/kg to male Swiss mice (data not shown). However, only a small amount of 57 (~200 ng/g) in the brain of male Swiss mice
could be detected after 2 hours. In contrast, compounds 55 and 56 displayed significant brain uptake in a four time-point PK-
assessment after oral administration at 10 mg/kg to male Swiss mice (Fig. 5) with a brain-to-plasma (B/P) exposure ratio >1 (Table
2). Reducing the number of hydrogen bond donors to two, and conversion of the secondary amine (piperidine moiety) to a tertiary
amine (N-methyl piperidine moiety) was most likely the reason for the improved brain uptake. Interestingly, 55 exhibited high brain
exposure despite of its reduced metabolic stability in mice liver microsomes
Table 2: PK parameter of 55 and 56 in male Swiss micea
55 (plasma)b 55
56
56 (brain)a
(brain)a
2.74
(plasma)a
1.85
t1/2 (h)
2.66
711
ND
AUC
39584
2495
4040
(ng.h/mL or
ng.h/g)0-last
Cmax (ng/mL 107
or ng/g)
5833
549
2.1
642
B/Pb
55.5
aAdministration at 10 mg/kg p.o.
bB/P = brain to plasma ratio, at Cmax at 4h post p.o. dosing.
PK parameters
55 Plasma (ng/ml)
55 Brain (ng/g)
10000
1000
100
10
56 Plasma (ng/ml)
56 Brain (ng/g)
1
0
3
6
9
12 15 18 21 24
Time-point (h)
Figure 5: Brain and plasma levels of 55 and 56 after oral administration of 10 mg/kg.
In summary, we have identified a novel series of indole and 7-azaindole derivatives containing nitrile, piperidine and N-methyl
piperidine substituents at the 3-position as inhibitors of Aβ aggregation (45-57) leading to improved metabolic stability in human
and mice liver microsomes. The study described above indicated it was required to abandon the dimeric compound design of

libraries 2 and 3 to improve permeability and to avoid compounds being P-gp substrates. The introduction of the N-methyl
piperidine moiety, together with the reduction of the hydrogen bond donors to two in library 4 was essential for good brain exposure
in Swiss male mice after oral administration of compounds 55 and 56. In contrast to compounds containing the 3-aminopyrazole24
or 2,6-diaminopyridine moieties25, it was possible to eliminate all nitrogen atoms acting as hydrogen bond acceptors for compounds
52, 53, 55-57 of library 4. Taken together, these results showed 56 to be the best compound in terms of inhibition of Aβ aggregation
properties and brain uptake after oral administration.
Supplementary data
Supplementary (experimental procedures for the preparation of compounds 5-8, 10-25, 27-30, 32-57, in vitro fluorescence assay)
data associated with this article can be found, in the online version.
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Discovery and characterization of novel indole
and 7-azaindole derivatives as inhibitors of β-
amyloid-42 aggregation for the treatment of
Alzheimer’s disease
Leave this area blank for abstract info.
Nampally Sreenivasacharya, Heiko Krotha, Pascal Benderittera,b, Anne Hamela, Yvan Variscoa, David T.
Hickmana,Wolfgang Froestla, Andrea Pfeifera, Andreas Muhsa,