Synthesis and structure–activity relationship of 2,6-disubstituted pyridine derivatives as inhibitors of β-amyloid-42 aggregation

Article abstract of DOI:10.1016/j.bmcl.2016.05.040

It is assumed that amyloid-β aggregation is a crucial event in the pathogenesis of Alzheimer's disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the β-sheet conformation of Aβ via donor–acceptor–donor hydrogen bond forma

Full text of DOI:10.1016/j.bmcl.2016.05.040

Accepted Manuscript
Synthesis and structure-activity relationship of 2,6-disubstituted pyridine de-
rivatives as inhibitors of β-amyloid-42 aggregation
Heiko Kroth, Nampally Sreenivasachary, Anne Hamel, Pascal Benderitter,
Yvan Varisco, Valérie Giriens, Paolo Paganetti, Wolfgang Froestl, Andrea
Pfeifer, Andreas Muhs
PII:
S0960-894X(16)30525-X
http://dx.doi.org/10.1016/j.bmcl.2016.05.040
BMCL 23899
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 March 2016
10 May 2016
12 May 2016
Please cite this article as: Kroth, H., Sreenivasachary, N., Hamel, A., Benderitter, P., Varisco, Y., Giriens, V.,
Paganetti, P., Froestl, W., Pfeifer, A., Muhs, A., Synthesis and structure-activity relationship of 2,6-disubstituted
pyridine derivatives as inhibitors of β-amyloid-42 aggregation, Bioorganic & Medicinal Chemistry Letters (2016),
doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.040
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Bioorganic & Medicinal Chemistry Letters
Synthesis and structure-activity relationship of 2,6-disubstituted pyridine
derivatives as inhibitors of β-amyloid-42 aggregation
Heiko Krotha, Nampally Sreenivasacharya, Anne Hamela, Pascal Benderittera,b, Yvan Variscoa, Valérie
Giriensa, Paolo Paganettia,c, 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
c Present address: Ente Ospedaliero Cantonale, Viale Officina 3, 6500 Bellinzona, Switzerland
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
It is assumed that amyloid-β aggregation is a crucial event in the pathogenesis of Alzheimer’s
disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the β-sheet
conformation of Aβ via donor-acceptor-donor hydrogen bond formation. A series of pyridine
derivatives were synthesized and tested regarding their potential to inhibit the aggregation of
Aβ. The 2,6-diaminopyridine moiety was identified as a key component to inhibit Aβ
aggregation. Overall, compounds having three 2,6-disubstituted pyridine units separated by at
least one C2- or C3-linker displayed the most potent inhibition of Aβ aggregation.
Keywords:
Alzheimer’s disease
Aβ aggregation
2009 Elsevier Ltd. All rights reserved.
Inhibitors
Structure-activity relationship
Pyridine derivatives
Alzheimer’s disease (AD) is the most common age-related
neurodegenerative disorder affecting an estimated 36 million
patients in 2010.1 Current treatment options are limited to drugs
treating the symptoms and do not slow or reverse disease
progression. Thus, a disease modifying drug would be a major
therapeutic breakthrough towards treatment of the disease.
Although the mechanisms of neurodegeneration in AD are not
yet fully discovered, some major pathological signs, such as
senile plaques (SP) composed of amyloid-β (Aβ) peptides and
neurofibrillary tangles composed of hyperphosphorylated tau
protein, are characterized.2,3 SPs consist predominantly of the 40
to 42 amino acid Aβ peptides, which are derived from the
amyloid precursor protein (APP). The formation of Aβ fibrils, via
aggregation of soluble Aβ in an antiparallel β-sheet
conformation4, and their deposition into neurotoxic amyloid
plaques is considered an initial event in the progression of AD.5
Recent studies on Aβ toxicity suggest that an early stage involves
the aggregation of extracellular soluble Aβ peptide into low
molecular weight soluble oligomers or high molecular weight
prefibrillar intermediates.6-8 A central therapeutic aim in AD is
the removal of toxic β-amyloid deposits9, which can be achieved
by secretase inhibitors (inhibition of Aβ production), drugs
promoting β-amyloid clearance via active or passive
immunotherapy or inhibition of Aβ aggregation and toxicity.
the process is believed to be a purely pathological event and does
not interfere with the physiological role of APP.10
Figure 1. Possible hydrogen bond interactions of 3-aminopyrazole or 2,6-
disubstituted pyridine derivatives with the β-sheet conformation of Aβ and
inhibitor design.
We had already prepared small molecule inhibitors of Aβ
aggregation based on the connection of two substituted 3-
aminopyrazole moieties via a linker to enable donor-acceptor-
donor hydrogen bond interactions complementary to that of the
Preventing Aβ aggregation is therapeutically attractive because
β-sheet
conformation
of Aβ
and
additional
π-π
———
Corresponding author. Tel: +41 21 693 91 24; fax: +41 21 693 91 20; e-mail: andreas.muhs@acimmune.com

stacking/hydrophobic interactions with amino acid residues of
Aβ.11 This previous series of Aβ aggregation inhibitors based on
the 3-aminopyrazole moiety, however, displayed low solubility at
physiological pH (< 10 μM) and low metabolic stability leading
to poor bioavailability. The aim of the novel 2,6-disubstituted
pyridine series was to maintain potency but to increase solubility
as well as metabolic stability. Overall, the novel inhibitors should
contain up to 3 hydrogen bond donors and 3 hydrogen bond
acceptors. The individual 2,6-disubstituted pyridine moieties
should be connected via C1- to C3-linker units to identify the
optimal linker length.
To synthesize the novel Aβ aggregation inhibitors containing
two or three 2,6-disubstituted pyridine moieties, it was necessary
to prepare suitable building blocks. The synthesis of monomeric
2,6-disubstituted pyridine building blocks is shown in Schemes 1
and 2. The starting material, compound 1, to synthesize
compounds 2 to 9 was prepared as described.12 Building blocks 5,
7 and 9 were prepared via 3-5 steps. First, compound 1 was
alkylated with methyliodide using sodium hydride.12 Next
compound 2 was treated with lithiumdiisopropylamine (LDA) at
–78°C, followed by the addition of dimethylformamide to obtain
the C1-elongated aldehyde intermediate. Reduction with sodium
tetrahydroborohydride at –78°C in the presence of acetic acid and
methanol yielded the corresponding alcohol 3.13Activation of the
alcohol with methanesulfonylchloride followed by displacement
with sodium azide under neutral conditions yielded 4.14
Reduction of the azide under Staudinger conditions afforded the
amine building block 5 containing a C2-linker.14 Compound 6
was prepared from 1 using the same conditions employed for the
synthesis of 3. The azide building block 7 containing a C2-linker
was prepared in the same manner as described for 4. NBS-
bromination of 2 as described15 afforded 8, which was converted
to the C1-linker amine building block 9 as described for 5.
phosphine)palladium[0] (Pd[P(Ph)3]4) utilizing an intermediate
formed by the reaction of allylalcohol with 9-borabicyclo[3.3.1]-
nonane (9-BBN) to afford the corresponding alcohol 11.17
Mitsunobu reaction of 11 using diethyl azodicarboxylate
(DEAD), triphenylphosphine (TPP) and phthalimide followed by
the cleavage of the protecting group with hydrazine hydrate
afforded the C3-linker amine building block 12.18
The preparation of the required dimeric building blocks 14,
16, 17 and 19 via 1-2 steps synthesis is shown in Scheme 3.
Starting material 13 was prepared as described19 and treated with
9-BBN to form a boron intermediate. This intermediate was then
reacted with commercially available 2,6-dibromopyridine as
described for 11 to afford the dimeric building block 14
containing a C3-linker. The corresponding building block 16
containing a C1-linker was prepared from 15 using commercially
available Boc-2-amino-pyridine and the alkylation conditions
employed for the preparation of 2. Starting material 15 was
synthesized as described.20 Attempts to use the methanesulfonate
derivative of 3 under the basic conditions described for the
preparation of 16 resulted in the formation of the corresponding
vinylpyridine derivative of 3.
Scheme 3. Reagents and conditions: (a) (i) 9-BBN, THF 0 °C, 4 h; (ii) THF,
NaOH, Pd[P(Ph)3]4, DMA, 2,6-dibromopyridine, 95 °C, 90 min, 69%; (b)
NaH, DMF, Boc-2-amino-pyridine, 65 °C, 3 h, 82%; (c) NaH, DMF, 8, 60
°C, 2 h, 72%; (d) TPP, THF, H2O, rt, 24 h, 68%; (e) NaH, DMF, 15, 60 °C, 2
h, 82%; (f) H2, Pd/C, EtOH, TEA, rt, 90%.
The dimeric building blocks 17 and 19 (Scheme 3) were both
prepared from compound 7. Alkylation of 7 with 8 followed by
Staudinger reduction as described for 5 afforded the dimeric
building block 17 containing a C1- and a C2-linker. Alkylation of
compound 7 with 15 afforded compound 18. Removal of the
bromo-substituent and reduction of the azide was accomplished
by catalytic hydrogenation with palladium on carbon to afford
the dimeric building block 19 containing a C1- and a C2-linker.
The starting material 20 for the preparation of dimeric
building blocks 23 and 25 (Scheme 4) was prepared as
described.15 Protection of the alcohol with the triisopropylsilyl
(TIPS) moiety gave 21. Compound 21 was then reacted with 5
using tris(dibenzylideneacetone)dipalladium (Pd2(dba)3), 2,2-Bis-
(diphenylphosphino)-1,1-napthalene (BINAP) and sodium tert.-
butoxide under Buchwald conditions.21 The use of 10 mol%
palladium catalyst and a short reaction time (45 min) were
critical for good conversion and yield. Extended reaction times
lead to significant decomposition of 22 in the presence of the
strong base sodium tert.-butoxide. No formation of dimeric
Scheme 1. Reagents and conditions: (a) NaH, DMF, CH3I, 0 °C to rt, 16 h,
74%; (b) (i) LDA, THF, -78 °C; (ii) DMF, -78 °C; (iii) HOAc, MeOH,
NaBH4, -78 °C to rt, 35-44%; (c) CH3SO2Cl, TEA, DCM, 0 °C to rt, 1 h; 65-
90% (d) NaN3, DMA, 75 °C, 16 h, 89-95%; (e) TPP, THF, H2O, rt, 24 h, 82-
90%; (f) NBS, AIBN, CCl4, 100 °C, 5 h, 29%.
Scheme 2. Reagents and conditions: (a) (i) allylalcohol, 9-BBN, THF 0 °C, 4
h; (ii) Pd[P(Ph)3]4, THF, NaOH, DMA, 95 °C, 90 min, 79%; (b) phthalimide,
TPP, THF, DEAD, rt, 16 h, 86%; (c) N2H4 x H2O, MeOH, rt, 16 h, 60%.
Building block 12 was prepared via 3 steps (Scheme 2).
Compound 10 was prepared as described.16 The C3-linker was
introduced via Suzuki coupling using tetrakis(triphenyl-

coupling products was observed under the reaction conditions.
The free NH-group in 22 was Boc-protected at elevated
temperature. The best results were obtained by dissolving 22 and
di-tert-butyl dicarbonate (Boc2O) in dichloromethane and
evaporation of the solvent followed by heating of the oily
reaction mixture. After cleavage of the TIPS-group with tetra-
butylammoniumfluoride, the free OH-group was converted via 3
additional steps to the amine as described for 5 to afford the
dimeric building block 23 containing two C2-linkers. The
synthesis of the dimeric building block 25 containing two C2-
linkers was performed as described for 23 except that
commercially available 2(2-aminoethyl)-pyridine was used for
the Pd-coupling reaction.
Subsequent Boc-protection and cleavage of the TIPS-group as
described for 23 afforded 27. Conversion of the OH-group to the
corresponding amine was performed as described for 12 to obtain
the dimeric building block 28 containing two C3-linkers.
Treatment of 2,6-dibromopyridine with 2(2-aminoethyl)pyridine
at elevated temperature in the presence of potassium
hydrogencarbonate afforded 29, which was Boc-protected as
described for 23 to obtain the corresponding dimeric building
block 30 containing a C2-linker.
The synthesis of Aβ aggregation inhibitors containing either
two or three 2,6-disubstitued pyridines and different linkers is
shown in Schemes 6, 7 and 8. The inhibitors 31-34 (Scheme 6)
were prepared by using building blocks 5, 9, 12, 14, and 16. First,
the building blocks were combined via Buchwald coupling21 at
85 °C for 45 minutes to yield the corresponding Boc-protected
inhibitors. Under the reaction conditions no di-substituted
coupling products were observed. Cleavage of the Boc-group
under acidic conditions afforded the desired inhibitors as
hygroscopic HCl-salts. The best way to obtain the inhibitors was
to remove the organic solvents by syringe and to dissolve the
precipitated HCl-salt in water. Lyophilization of the aqueous
solution then afforded the Aβ aggregation inhibitors as pale
yellow/orange powders. Thus, 31 was prepared from 9 and 16, 32
was prepared from 5 and 16, 33 was prepared from 5 and 14, and
34 was prepared from 12 and 14 (Scheme 6).
Scheme 4. Reagents and conditions: (a) TIPS-Cl, imidazole, DMF, rt, 16 h,
87%; (b) Pd2(dba)3, BINAP, NaOtBu, toluene, 5, 85 °C, 45 min, 78%; (c)
Pd2(dba)3, BINAP, NaOtBu, toluene, 2-(2-aminoethyl)pyridine, 85 °C, 45
min, 81%; (d) Boc2O, 70 °C, 16 h, 93%; (e) TBAF, THF, CH3CN, rt, 16 h,
88-92%; (f) CH3SO2Cl, TEA, DCM, 0 °C to rt,1 h, 85-90%; (g) NaN3, DMA,
75 °C, 16 h, 65-92%; (h) TPP, THF, H2O, rt, 24 h, 85-93%.
The preparation of the dimeric building blocks 28 and 30 via
2-6 steps is shown in Scheme 5. Commercially available 2,6-
dibromopyridine was converted to 26 via Suzuki coupling with
the reaction product of 9-BBN and allylalcohol followed by
TIPS-protection of the coupling product as described for 11.
Compound 26 was then coupled under Buchwald conditions with
3-(pyridin-2-yl)propylamine, which was prepared as described.18
Scheme 6. Reagents and conditions: (a) Pd2(dba)3, BINAP, NaOtBu, toluene,
85 °C, 45 min, 50-82%; (b) 2 M HCl, Et2O, CHCl3, rt, 16 h, 69-85%.
The inhibitors 35-38 were prepared from 17 and 23 via
coupling with 2-bromopyridine under Buchwald conditions
(Scheme 7). Congruent to literature data21, the use of 2-
bromopyridine yielded a mixture (~1:1) of mono- and di-
substituted coupling products, which could be separated by
preparative thin layer chromatography. The faster moving band
was always the di-substituted coupling product, i.e. Boc-
protected 37 or 38. Thus, the Boc-protected derivatives of 35 and
37 were obtained from 17 and 2-bromopyridine, whereas the
Boc-protected derivatives of 36 and 38 were obtained from 23
and 2-bromopyridine (Scheme 7).
Scheme 5. Reagents and conditions: (a) (i) allylalcohol, 9-BBN, THF 0 °C, 4
h; (ii) Pd[P(Ph)3]4, THF, NaOH, DMA, 95 °C, 90 min, 49%; (b) TIPS-Cl,
imidazole, DMF, rt, 16 h, 79%; (c) Pd2(dba)3, BINAP, NaOtBu, 3-(pyridin-2-
yl)propylamine, toluene, 85 °C, 45 min, 83%;(d) Boc2O, 70 °C, 16 h, 53-
92%; (e) TBAF, THF,CH3CN, rt, 16 h, 94%; (f) (i) TPP, phthalimide, THF,
DEAD, rt, 16 h; (ii) N2H4 x H2O, MeOH, rt, 16 h, 62%; (g) KHCO3, DMA, 2-
(2-aminoethyl)pyridine, 110 °C, 5 h, 21%.
Scheme 7. Reagents and conditions: (a) Pd2(dba)3, BINAP, NaOtBu, toluene,
85 °C, 45 min, 7-22%; (b) 2 M HCl, Et2O, CHCl3, rt, 16 h, 57-97%.

After acidic cleavage of the Boc-protection groups, the desired
inhibitors 35-38 were obtained as hygroscopic solids.
distance between acceptor and donor should be 2.6-2.9 Å.23
Superiority of the C2-linker in this series was also evident from
the stronger inhibition of Aβ1-42 aggregation of 38 compared to
37.
The Boc-protected inhibitors 39-41 were prepared via
palladium catalyzed coupling of 19, 25 and 28 with 10 at 110 °C
for 45 minutes (Scheme 8). Since the coupling resulted in the
formation of a 2,6-diaminopyridine moiety, a higher reaction
temperature was required.22 The Boc-protected dimeric inhibitors
42 and 43 were prepared in the same manner by coupling 2-(2-
aminoethyl)pyridine and 5 with 10. The Boc-protected inhibitor
44 was prepared by coupling of 30 with 5. Unlike the reactions
with 2-bromopyridine, no di-substituted palladium coupling
products were observed. The synthesis of inhibitors 39-44 was
completed by cleavage of the Boc-protection group with acid
(Scheme 8).
Figure 2. In vitro screening assays using Aβ1-42 peptide film. The
concentration of Aβ1-42 peptide film was 33 μM. The test concentration for
compounds 31-44 was 330 μM and the incubation time was 24 h. Data are
expressed as percentage (mean
± S.D.) of control conditions: Aβ1-42
aggregation with DMSO only. Freshly prepared Aβ1-42 peptide film (4 μg)
was analyzed by SDS-PAGE to confirm the presence of oligomeric Aβ1-42
present (a, molecular weight marker; b, Aβ1-42 peptide film).
In order to evaluate the effect of different distribution patterns
of the three hydrogen bond donors and acceptors, compounds 39-
44 were prepared and their inhibition of Aβ1-42 aggregation
properties tested (Figure 2). Taken the results for 31-38 into
account, the C2-linker was preferentially incorporated into
compounds 39-44. In contrast to 31-38, each of compounds 39-
Scheme 8. Reagents and conditions: (a) Pd2(dba)3, BINAP, NaOtBu, toluene,
10, 110 °C, 45 min, 22-89%; (b) 2 M HCl, Et2O, CHCl3, rt, 16 h, 72-84%.(c)
Pd2(dba)3, BINAP, NaOtBu, toluene, 5, 110 °C, 45 min, 94%
44 contained
a
2,6-diaminopyridine moiety. The 2,6-
The inhibition of Aβ aggregation in the presence of 31-44 was
determined using a thioflavin T (ThT) fluorescence assay and
crude Aβ1-42 peptide film at 33 μM as described.11 First, 31-38
were screened and their capability to reduce the ThT fluorescence
signal of the control reaction (Aβ1-42 aggregation in DMSO) at
330 μM was determined (Figure 2). The data in Figure 2 showed
that 31 containing two C1-linkers, 32 having a C1-linker to the
left and C2-linker to the right of the central pyridine, and 35
having a C2-linker to the left and a C1-linker to the right of the
central pyridine displayed rather low inhibition of Aβ1-42
aggregation properties (>60% of the control value). Thus,
compounds with one or two short C1-linkers most likely could
not form strong donor-acceptor-donor interactions with the β-
sheet conformation of Aβ1-42. In contrast, 37 containing a bis-
pyridyl moiety, 33 having a C3-linker to the left and a C2-linker
to the right, and 34 containing two C3-linkers displayed better
inhibition of Aβ1-42 aggregation properties (23-29% of the control
value). This indicated a preference for compounds with either
longer and more flexible linkers (33, 34) or the capability to form
additional π-π interactions (37). For all compounds containing
three 2-aminopyridine moieties separated by different linkers, 36
and 38 displayed the best inhibition of Aβ1-42 aggregation
properties (16% of the control value). In contrast to 35,
compound 36 containing two C2-linkers displayed a similar
potency as 38 containing a bis-pyridyl moiety. This suggests that
proper hydrogen bond donor interactions of each of the three 2-
aminopyridyl moieties in 36 have a similar effect than additional
π-π interactions for 37 and 38. The distance between donor and
acceptor should be ideally in the range of 3.5-4.0 Å and the
diaminopyridine moiety in 39-43 is located at a terminal right
position, whereas in 44 the position is central. To get an idea on
how many hydrogen donors and acceptors are required for
efficient inhibition of Aβ1-42 aggregation, the truncated
compounds 42 and 43 were prepared as well.
The results for 39-41 and 44 clearly showed that all
compounds containing the 2,6-diaminopyridine unit displayed
efficient inhibition of Aβ1-42 aggregation (12-16% of the control
value). The position of the 2,6-diaminopyridine moiety, i.e.
terminal right (39-41) or central (44), had no significant impact
on their inhibition of Aβ1-42 aggregation properties in the
screening assay. The same applied for the use of C2-linkers (40)
or C3-linkers (41). Interestingly, compound 39 was equally
potent (14% of the control value) when compared to 40 and 41.
This was in sharp contrast to compound 32 (53% of the control
value), although the linker composition and number of hydrogen
bond donors and acceptors were identical. This suggests the 2,6-
diaminopyridine moiety is a preferred unit to enable donor-
acceptor interactions with the β-sheet conformation of Aβ1-42
.
Further evidence came from the ‘’truncated’’ inhibitors 42 and 43
containing a 2,6-diaminopyridine unit. Compound 42 displayed a
higher inhibition of Aβ1-42 aggregation (36% of the control value)
with just 2 hydrogen bond donors and acceptors when compared
to 31, 32 and 35 having 3 hydrogen bond donors and acceptors
(Figure 2). Compound 43 having 3 hydrogen bond donors and 2
hydrogen bond acceptors was equally potent as 33 and 34,
indicating the importance of hydrogen bond donors for inhibition
of Aβ1-42 aggregation.

In order to better compare inhibitors displaying >80%
inhibition of the control value in the ThT-screening assay, the
IC50 for 36, 38, 39, 40, 41 and 44 was determined using the ThT
fluorescence assay. To compare our inhibitors with Aβ
aggregation inhibitors from literature, the IC50 of Congo Red and
Curcumin were determined as well (Figure 3).
Inhibition of Aß42 Aggregation - Peptide Film
1000
280 M 240
M
100
10
1
13
M
25
M
17
M
35
M
1.3 M
9
M
Figure 4. Brain and plasma levels of 40 after i.v. administration of 2 mg/kg
Table 1
Pharmacokinetic properties in male Swiss micea
40 (plasma)b
40 (brain)b
5.23 ± 2.71
863 ± 195
t1/2 (h)
0.63 ± 0.18
36
38
39
40
41
44
Red
o
AUC (ng/mL* h) 150 ± 13
Curcumin
Cong
Vd (L/kg)
Cmax (ng/mL)
B/Pc
11.99 ± 3.24
18.94 ± 12.93
1845 ± 484
Compounds
Figure 3. IC50 determination assay using Aβ1-42 peptide film. The
concentration of Aβ1-42 peptide film was 33 μM. The test concentration for
compounds 36, 38, 39, 40, 41, 44, Congo Red and Curcumin were 330 μM,
82.5 μM, 20.63 μM, 5.16 μM, 1.29 μM, 0.32 μM and 0.08 μM with an
incubation time of 24 h. The IC50 values were determined from the
fluorescence values obtained. The ThT IC50-data (μM) are expressed as mean
± standard deviation and indicated on top of each graph.
308 ± 7
5.98 ± 1.51
aAdministration at 2 mg/kg i.v.
biv formulation (100 % DMSO).
cB/P = brain to plasma ratio, 5 minutes post i.v dosing.
In summary, we have identified the 2,6-diaminopyridine
moiety as a key component in the design of potent inhibitors of
Aβ1-42 aggregation with good aqueous solubility. The most
balanced profile between inhibition of Aβ1-42 aggregation and
aqueous solubility was achieved by separating the 2,6-
diaminopyridine unit via a C2-linker from the adjacent 2,6-
disubstituted pyridine(s). At least three 2,6-disubstituted pyridine
moieties containing 3 hydrogen bond donors and 3 hydrogen
bond acceptors in the correct arrangement, i.e. compounds 39-41,
44, were required for the most potent inhibition of Aβ
aggregation. Further efforts describing additional compounds
with improved metabolic stability and pharmacokinetic
properties are in progress.
The IC50 determination for 36, 38, 39, 40, 41 and 44 clearly
showed that compounds containing a 2,6-diaminopyridine moiety
(39-44) displayed a better inhibition of Aβ1-42 aggregation (13-35
μM) when compared to 36 and 38 (240-280 μM) having a 2-
aminopyridine moiety (Figure 3). Compound 44 bearing a central
2,6-diaminopyridine was slightly less active (35 μM) than 39, 40
and 41 containing a terminal right 2,6-diaminopyridine moiety.
Compounds 39-41 displayed comparable Aβ1-42 aggregation
properties with IC50 values of 13 μM, 25 μM, and 17 μM,
respectively. The control compound Curcumin inhibited Aβ1-42
aggregation in the same range (9 μM). Congo Red was the most
potent compound (1.3 μM) in this assay.
The aqueous solubility of selected compounds 31, 40, 41, 43
and 44 at physiological pH (PBS, pH 7.4) were determined as 18
μM, >200 μM, 103 μM, 195 μM and > 200 μM, respectively.
Compared to the previous 3-aminopyrazole class of Aβ1-42
aggregation inhibitors11, the introduction of 2,6-disubstituted
pyridines moieties allowed to improve the aqueous solubility of
Aβ1-42 aggregation inhibitors from <10 μM (3-aminopyrazoles) to
>200 μM (40 and 44) while maintaining similar potency.
Overall, compound 40 appeared to be a good compromise
between inhibition of Aβ1-42 aggregation and high aqueous
solubility and was further profiled in vitro. Though the metabolic
stability of 40 in human liver S9 fraction was low (t1/2 = 10 min;
0.07 mL/min/mg), 40 displayed very high permeability (Papp A to
B = 49.2 x 10-6 cm/sec), was not a P-gp substrate (Efflux ratio =
0.78) and displayed a favorable LogD (2.96). Thus, the
pharmacokinetic properties of compound 40 were tested in male
Swiss mice (Figure 4, Table 1).24 After intravenous application at
2 mg/kg, compound 40 rapidly entered the brain (1845 ng/g after
5 minutes) in line with the excellent in vitro permeability.
However, the poor metabolic stability was most likely the reason
for the rapid clearance of 40 from the brain.
Supplementary data
Supplementary (experimental procedures for the preparation
of compounds 2-9, 11, 12, 14, 16, 17-19, 21-25, 26-44, in vitro
fluorescence assay) data associated with this article can be found,
in the online version.
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Synthesis and structure-activity relationship of
2,6-disubstituted pyridine derivatives as
inhibitors of β-amyloid-42 aggregation
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Heiko Krotha, Nampally Sreenivasacharya, Anne Hamela, Pascal Benderittera,b, Yvan Variscoa, Valérie Giriensa,
Paolo Paganettia,c, Wolfgang Froestla, Andrea Pfeifera, Andreas Muhsa*