Application of fragment screening by X-ray crystallography to the discovery of aminopyridines as inhibitors of β-secretase

Article abstract of DOI:10.1021/jm061197u

Fragment-based lead discovery has been successfully applied to the aspartyl protease enzyme β-secretase (BACE-1). Fragment hits that contained an aminopyridine motif binding to the two catalytic aspartic acid residues in the active site of the enzyme were the chemical starting points. Structure-based design approaches have led to identification of low micromolar lead compounds that retain these interactions and additionally occupy adjacent hydrophobic pockets of the active site. These leads form two subseries, for which compounds 4 (IC₅₀ = 25 μM) and 6c (IC₅₀ = 24 μM) are representative. In the latter series, further optimization has led to 8a (IC₅₀ = 690 nM).

Full text of DOI:10.1021/jm061197u

1124
J. Med. Chem. 2007, 50, 1124-1132
Application of Fragment Screening by X-ray Crystallography to the Discovery of
Aminopyridines as Inhibitors of â-Secretase§
Miles Congreve,*,† David Aharony,‡ Jeffrey Albert,‡ Owen Callaghan,† James Campbell,‡ Robin A. E. Carr,† Gianni Chessari,†
Suzanna Cowan,† Philip D. Edwards,‡ Martyn Frederickson,† Rachel McMenamin,† Christopher W. Murray,† Sahil Patel,† and
Nicola Wallis†
Astex Therapeutics Ltd., 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, United Kingdom, and Departments of Chemistry
and Neuroscience, AstraZeneca Research and DeVelopment, 1800 Concord Pike, Wilmington, Delaware 19707
ReceiVed October 13, 2006
Fragment-based lead discovery has been successfully applied to the aspartyl protease enzyme â-secretase
(BACE-1). Fragment hits that contained an aminopyridine motif binding to the two catalytic aspartic acid
residues in the active site of the enzyme were the chemical starting points. Structure-based design approaches
have led to identification of low micromolar lead compounds that retain these interactions and additionally
occupy adjacent hydrophobic pockets of the active site. These leads form two subseries, for which compounds
4 (IC50 ) 25 µM) and 6c (IC50 ) 24 µM) are representative. In the latter series, further optimization has led
to 8a (IC50 ) 690 nM).
Introduction
Alzheimer’s disease (ADa) is a neurodegenerative disorder
associated with accumulation of amyloid plaques and neu-
rofibrillary tangles in the brain.1 The major components of these
plaques are â-amyloid peptides (Aâs), which are produced from
amyloid precursor protein (APP) by the activity of â- and
γ-secretases. â-Secretase cleaves APP to reveal the N-terminus
of the Aâ peptides.2 The â-secretase activity has been identified
as the aspartyl protease beta-site APP cleaving enzyme (BACE-
1), and this enzyme is a potential therapeutic target for treatment
of AD.3-7
BACE-1 inhibitors have recently been reviewed.8,9 Most work
has concentrated on the design of peptidomimetic inhibitors
which usually employ a secondary alcohol as a transition state
mimetic to displace the water molecule that sits between the
two catalytic aspartic acids of the enzyme.10-19 A potential
Figure 1. A representation of binding mode of a hydroxyethylamine
inhibitor bound to the active site of BACE-1. Hydrogen bonds to
residues are shown as broken lines.
problem with peptidomimetics is that they tend to be relatively
large and possess multiple hydrogen bond donors which may
number of key hydrogen bonds with the catalytic aspartate
residues, but the recognition in this region is somewhat different
to that seen with many other secondary alcohol-containing
peptide-based inhibitors.28,29 This highlights the importance of
these hydrogen bonds but underlines that there are different ways
that inhibitors can favorably make interactions with the catalytic
residues. The molecule also forms two hydrogen bonds with
the flap region of the active site, and the flap conformation is
essentially identical to the flap-closed conformation adopted in
published structures of the potent peptide-based inhibitor OM99-
2.28 Additionally, the flap residue, Tyr71, forms two edge-face
stacking interactions with the phenyl rings of the inhibitor which
lie within the S1 and S2′ pockets. The inhibitor adopts a
‘collapsed’ conformation in which one of the N-propyl groups
packs against a phenyl ring (see Figure 1). These groups then
form a continuous surface that fills the large hydrophobic S1
and S3 pocket(s) of the enzyme, and this appears to be another
key region for BACE-1 inhibition. This knowledge of how
substrate-like inhibitors bind to the enzyme was an insightful
guide for the structure-based optimization of nonpeptidic
fragment hits described below.
make them less attractive leads for a project which requires
penetration across the blood-brain barrier and oral bioavail-
ability.20,21 There has also been some work on the design of
less peptidic22,23 or nonpeptidic inhibitors24-26 of BACE-1
although in the latter case, there have been no crystal structures
to support the binding modes proposed or to develop SAR.
There is therefore a great deal of interest in the identification
of nonpeptidic inhibitors of BACE-1 and in the experimental
determination of their associated binding modes.
We have previously reported the X-ray crystal structure of
BACE-1 in its apo form and complexed with a peptidomimetic
inhibitor.27 A representation of the inhibitor bound to the active
site of BACE-1 is given in Figure 1. The molecule forms a
§ Coordinates for the â-secretase complexes with compounds 3, 4, 6a,
6b, 7, and 8b have been deposited in the Protein Data Bank under accession
codes 2OHP, 2OHQ, 2OHR, 2OHS, 2OHT and 2OHU, together with the
corresponding structure factor files.
* To whom correspondence should be addressed. Phone: +44 (0)1223
226270; Fax +44 (0)1223 226201; E-mail: m.congreve@astex-
therapeutics.com.
† Astex Therapeutics Ltd.
‡ AstraZeneca Research and Development.
a Abbreviations. AD, Alzheimer’s disease; APP, amyloid precursor
protein; BACE-1, beta-site APP cleaving enzyme; LE, ligand efficiency.
In the preceding article in this journal we outlined how
fragment screening using protein-ligand X-ray crystallography
10.1021/jm061197u CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/22/2007

Aminopyridines as Inhibitors of â-Secretase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1125
Figure 2. The binding mode of 6-substituted 2-aminopyridine inhibitors in the active site of BACE-1. On the left, a schematic representation of
the inhibitors is provided together with available IC50 data. The experimental binding mode appears on the right-hand side with the protein backbone
represented as a cartoon and the protein atoms in thin stick. The ligand and the residues Asp32, Tyr71 and Asp228 are represented in thick stick.
In this orientation the flap is toward the top of the picture and the S1 and S3 pockets are on the right.
had identified a series of ligands that bound to the catalytic
aspartic acid residues in the active site of BACE-1 through an
amidine or aminopyridine motif. This type of charged bidentate
interaction has not previously been described between inhibitor
molecules and aspartyl protease enzymes. Using this finding
as a starting point, virtual screening of related molecules
identified a number of attractive fragments for structure-based
optimization. The syntheses of aminopyridine analogues, the
biological activity, and the protein-ligand interactions made
by the more potent inhibitors are outlined in this article.
The next target selected was the indole-substituted aminopy-
ridine 3 (Figure 2). This compound was designed to better
occupy the S1 pocket by incorporating a larger bicyclic ring
system in the hydrophobic pocket, compared with the phenyl-
substituted precursor 2. The crystal structure of 3 is illustrated
in Figure 2 and clearly indicates how the indole occupies the
S1 region. In addition, a new interaction was observed between
the indole NH and the backbone carbonyl of Gly230 on the
edge of the S3 pocket. The S1 and S3 regions are contiguous in
BACE-1, and the S3 pocket is again rather hydrophobic in
nature. Testing of this analogue in the enzyme assay gave an
IC50 ) 94 µM, indicating that the indole moiety was indeed
forming favorable contacts in the S1-S3 region of the site and
that this was a useful area to target in the next iteration of design.
Results and Discussion
6-Substituted 2-Aminopyridine Inhibitors. The preceding
article describes discovery of 2-aminoquinoline 1 as one ligand
that binds to the active site of BACE-1. The principle interac-
tions are between the charged aminopyridine motif and the two
catalytic aspartates of the enzyme. The region adjacent to this
ligand is the S1 pocket, which is a highly hydrophobic area of
the active site, and was attractive to target. 2-Aminoquinoline
1 had unsuitable vectors to easily target this region of the active
site. Instead, a consideration of the binding mode of fragment
1, in which the fused phenyl ring is adjacent to the S1 pocket,
led us to design 6-substituted phenylethyl-2-aminopyridine 2
(Figure 2). In this design the phenyl group was predicted to
make good hydrophobic contact with the S1 region. A com-
parison of the crystal structures of 1 and 2 indicated that the
aminopyridine moiety retained the bidentate binding to the
catalytic aspartic acid residues (Asp32 and Asp228) and that,
as expected, the phenyl substituent occupied the S1 pocket. The
structure of 2 was of low resolution,30 but this new derivative
still represented a more useful starting point for further
compound design than 2-aminoquinoline itself.
A consideration of the van der Waals contacts between 3 and
the S1-S3 hydrophobic pocket suggested that larger hydrophobic
groups should be tolerated in this region. A small number of
derivatives were designed and synthesized, and one of the more
promising analogues made was 4 in which a 3-methoxy-biaryl
group replaced the indole substituent. Figure 2 shows how this
group makes good contact with the active site. Notably there is
a change in conformation of the flap region of the enzyme, so
that Tyr71 moves and rotates above the aminopyridine ring,
better accommodating the ligand. The twist out of plane of the
second aromatic ring of the biaryl, relative to the indole of 3,
helps improve hydrophobic contact with the pocket. Addition-
ally, the methoxy group pendent to the biaryl moiety comple-
ments a small, more polar area by displacing a water molecule
at the bottom of the S3 pocket and forms an H-bonding
interaction with the backbone NH of Gly13. The potency of 4
is improved, in comparison to 3, to IC50 ) 25 µM, and this
compound represented a good lead molecule for further design

1126 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
CongreVe et al.
Figure 3. The binding mode of 3-substituted 2-aminopyridine inhibitors in the active site of BACE-1. On the left, a schematic representation of
the inhibitors is provided together with available IC50 data. The experimental binding mode appears on the right-hand side with the protein backbone
represented as a cartoon and the protein atoms in thin stick. The ligand and the residues Asp32, Tyr71 and Asp228 are represented in thick stick.
In this orientation the flap is toward the top of the picture and the S1 and S3 pockets are on the right.
and elaboration. In particular, the biaryl motif was suitable for
further substitution and modification, and there were additional
vectors on the aminopyridine ring itself suitable for exploration
of the prime side of the active site.
(Figure 2) the methoxy group had displaced this water molecule.
The potency of 6a was IC50 ) 100 µM, a reasonable improve-
ment over the parent compound 5. Taken together these data
indicated that there were profitable interactions to be made by
occupying the S3 pocket and by targeting the more polar region
at the bottom of this pocket.
3-Substituted 2-Aminopyridine Inhibitors. Virtual screen-
ing of aminopyridine derivatives from commercial sources and
from the Astex compound collection identified 2,3-diaminopy-
ridine 5 as a hit in silico. The subsequent soaking of this
molecule into crystals of the enzyme indicated that binding did
indeed take place (Figure 3). This is described in some detail
in the sister publication. The aminopyridine again forms a
charged bidentate interaction with the catalytic aspartates of the
active site; however, in this case the molecule has changed
orientation relative to the fragment hit 1. This new binding mode
allows an additional interaction to form between the 3-amino
group which donates its hydrogen atom to Asp32. This switch
in orientation of the aminopyridine ring allows the N-benzyl
group of the compound to occupy the S1 pocket, again
demonstrating the enzyme’s preference for hydrophobic binding
to this pocket. This analogue 5 gave an IC50 ) 310 µM in the
enzyme assay, which we considered to be promising potency
for such a small fragment against this very challenging target.
Using the insight we had gained from the work described
above in the 6-substituted 2-aminopyridine series, a small
number of biaryl substituted 2,3-diaminopyridine targets were
designed to better occupy the S1-S3 hydrophobic pocket.
Introduction of a 3-substituted 3-pyridyl group was found to
be particularly useful because these analogues gave excellent
quality protein-ligand crystal structures (Figure 3), probably
due to the 3-pyridyl phenyl group forming favorable contacts
with the pocket and because the pyridyl group improved aqueous
solubility of these ligands. Interestingly, the pyridyl nitrogen
forms an H-bond with a water molecule at the bottom of the S3
pocket in compound 6a. In the complex of 4 described earlier
The next analogues identified contained either a methoxy
group (6b) or a propyloxy group (6c) in addition to the pyridyl
nitrogen atom at the meta-positions of the ring system and were
designed in order to understand whether the best interactions
involved displacement of the water molecule in this region, or
binding to it. Figure 3 shows the complex of 6c. For this ligand,
as well as in 6b, the oxygen atom of the ligand displaces the
water molecule that had been targeted, and the alkyl group is
positioned within the narrow opening at the bottom of the S3
pocket. The methoxy derivative 6b had an IC50 ) 40 µM and
the propyloxy compound 6c an IC50 ) 24 µM, indicating that
the addition of the propyloxy substituent increased affinity
compared with 6a (IC50 ) 100 µM). At this point we considered
analogue 6c to be a good quality lead molecule, particularly as
analogues in the 2,3-diaminopyridine series benefited from a
short and convenient synthesis (described later). This series was
therefore prioritized over the 6-substituted 2-aminopyridines
outlined earlier for the next round of compound design.
Identification of a Sub-micromolar Inhibitor. Having
successfully optimized the 3-substituted 2-aminopyridine series
to low micromolar potency, higher molecular weight inhibitors
were designed, first to better fill the S3 pocket, and second to
target substituents beneath the ‘flap’ region of the active site.
The objective of this work was to establish if it was possible to
identify nonpeptidic inhibitors with sub-micromolar affinity for
the BACE-1 enzyme. It was rationalized that modification of
the substituted pyridyl ring to a bicyclic heterocycle might better
occupy the S3 pocket, and 6-indolyl was one of the modifications

Aminopyridines as Inhibitors of â-Secretase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1127
LE ) - ∆G/HAC ≈ -RT ln(IC50)/HAC
This compares favorably with published peptide-based inhibi-
tors28,29 that have low LE (<0.2). A ligand having a value >0.3
is consistent with identifying a compound with a molecular
weight <500 Da and potency in the 10 nM range. The LE values
for the compounds identified during the work described above
have been added to Figures 2-4. It can be seen that the most
potent compounds have LE generally in the range 0.25-0.29,
just below the target value of 0.3. These values are significantly
superior to known peptide-based inhibitors and would suggest
that lead optimization could result in identification of potent
inhibitors with an acceptable molecular weight range.
Conclusions
Two series of micromolar potency nonpeptidic inhibitors of
BACE-1 have been successfully identified and crystallized in
the enzyme active site. These two novel compound classes
include a binding motif not previously observed in aspartic
protease inhibitors.36 Further studies in which use of this
pharmacophore has allowed identification of nanomolar inhibi-
tors will be the subject of future publications.
Figure 4. The optimization of 3-substituted 2-aminopyridine ligands
in the active site of BACE-1. On the left, a schematic representation
of the inhibitors is provided together with available IC50 data. The
experimental binding mode appears on the right-hand side with the
protein backbone represented as a cartoon and the protein atoms in
thin stick. The ligand and the residues Asp32, Tyr71 and Asp228 are
represented in thick stick. In this orientation the flap is toward the top
of the picture and the S1 and S3 pockets are on the right.
Experimental Section
Crystallography. Recombinant human â-secretase 1 (BACE-
1), residues 14-453, was produced in bacteria as inclusion bodies
and refolded using a method described previously.27 Crystals of
apo BACE-1 suitable for ligand soaking were obtained by a
procedure described by Patel et al.27 In brief, BACE-1 was buffer-
exchanged into 20 mM Tris (pH 8.2), 150 mM NaCl, and 1 mM
DTT and concentrated to 8 mg/mL. DMSO (3% v/v) was added to
the protein prior to crystallization. Apo BACE-1 crystals were
grown using the hanging drop vapor diffusion method at 20 °C.
Protein was mixed with an equal volume of mother liquor
containing 20-22.5% (w/v) PEG 5000 monomethylethyl (MME),
200 mM sodium citrate (pH 6.6), and 200 mM ammonium iodide.
Crystals were cryoprotected for data collection by brief immersion
in 30% PEG 5000 MME, 100 mM sodium citrate (pH 6.6), 200
mM ammonium iodide, and 20% (v/v) glycerol. For inhibitor
soaking, 0.5-0.1 M DMSO stock solutions were made. A tenfold
dilution of the compound stock solution in a stabilization solution
(33% (w/v) PEG 5000 monomethylethyl (MME), 110 mM sodium
citrate (pH 6.6), and 220 mM ammonium iodide) was made.
Crystals were added to the soaking solution for up to 6 h prior to
flash freezing and data collection. Data were collected at a number
of synchrotron beam lines at the European Synchrotron Radiation
Facility, Grenoble, France. All data sets were processed with
MOSFLM37 and scaled using SCALA.38 Structure solution and
initial refinement of the protein-ligand complexes were carried
out by our automated scripts using a modified apo BACE-1 structure
(PDB id code 1w50) as a starting model. Bound ligands were
automatically identified and fitted into Fo - Fc electron density using
AutoSolve39 and further refined using automated scripts followed
by rounds of rebuilding in AstexViewer240 and refinement using
Refmac.41 Data collection and refinement statistics for crystal
structures are presented in Table 1.
chosen to probe this question. Figure 4 illustrates the binding
mode of this derivative 7 to BACE-1. Compared with the biaryl-
containing inhibitors 6a-c, the phenyl ring of this ligand sits
higher in the S1 pocket, and there is a small rotation of the 2,3-
diaminopyridine moiety, but without causing any disruption of
its interactions with the two catalytic aspartates. The indolyl
group sits in S3 as expected and forms a new interaction between
the NH of the heterocycle and the backbone carbonyl of Gly230.
This compound had an affinity of IC50 ) 9.1 µM.31 The position
of the phenyl ring suggested that the ortho-position, adjacent
to the methylene linking the ring to the 2,3-diaminopyridine,
might be a useful vector to explore substitution under the ‘flap’
toward the S2′ region. It has been observed that piperidine-based
inhibitors of renin are capable of binding groups in a similar
region, whereupon a significant protein conformational change
occurs, opening a large hydrophobic pocket.32-34 To this end,
two compounds were synthesized in which a benzyloxy (8a)
or a 2-pyridinylmethyloxy group (8b) was added (Figure 4).
These compounds had affinities of IC50 ) 0.69 µM (8a) and
4.2 µM (8b), respectively. The benzyloxy compound was not
sufficiently soluble to afford a high quality crystal structure;
however, the pyridyl-containing 8b did give a useful complex.
The new substituent does not effect a significant protein
conformational change and occupies the S2′ pocket of the
enzyme as expected, making hydrophobic contact and forming
a hydrogen bond between the side chain NH of Trp76 and the
pyridyl nitrogen atom. The 2,3-diaminopyridine and phenylin-
dolyl groups bind in an identical fashion to compound 7. It is
likely that the sub-micromolar inhibitor 8a adopts a similar
overall binding mode.
BACE-1 Assays. Activity of BACE-1 was measured using the
peptide R-E(EDANS)-E-V-N-L-*D-A-E-F-K(DABCYL)-R-OH from
Bachem. Assays were carried out in 50 mM sodium acetate, pH 5,
10% DMSO, in 96-well black, flat bottomed Cliniplates in a final
assay volume of 100 µL. Compounds were preincubated with in-
house produced BACE-1, and the reaction was initiated by adding
10 µM peptide substrate. The reaction rate was monitored at room
temperature on a Fluoroskan Ascent platereader with excitation and
emission wavelengths of 355 and 530 nm, respectively. Initial
reaction rates were measured, and IC50s were calculated from
replicate curves using GraphPad Prizm software.
Ligand Efficiency. In the previous publication in this issue
the ligand efficiencies (LE) of the aminopyridine-containing
fragments discovered using high throughput crystallography
were described. Aminoquinoline 1 has an estimated LE ) 0.33
according to Hopkins’s formula:35

1128 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Table 1. Crystallographic Data Collection and Refinement Statisticsa
CongreVe et al.
3
4
6a
6b
7
8b
Data Collection
X-ray source
ESRF ID14.1,
λ ) 0.934 Å
2.2
ESRF ID14.3,
λ ) 0.931 Å
2.1
ESRF ID14.3,
λ ) 0.931 Å
2.2
ESRF ID14.3,
λ ) 0.931 Å
2.5
ESRF ID14.3,
λ ) 0.931 Å
2.3
in house,
λ ) 1.54 Å
2.4
resolution (Å)
no. unique reflections
completeness (%)b
average multiplicity
25567
99.8(100)
2.6
31079
99.0(96.0)
2.5
24903
98.7(99.4)
2.5
18681
95.8(87.3)
2.3
22857
99.4(99.4)
2.5
21939
98.8(98.0)
2.6
b
Rmerge
9.1(36.1)
6.9(31.0)
10.5(39.6)
11.8(43.0)
10.5(37.3)
8.1(36.6)
Refinement
Rfree
Rcryst
28.1
23.2
0.013
1.4
44.7
55.3
37.4
27.6
21.8
0.013
1.4
42.8
54.6
46.1
21.4
17.0
0.013
1.4
31.6
26.5
37.6
25.1
18.2
0.013
1.5
30.8
29.6
32.1
26.9
21.2
0.013
1.4
42.4
47.0
36.7
24.8
19.5
0.013
1.4
44.2
49.0
40.3
rmsd bond lengths (Å)
rmsd bond angles (deg )
average B-factor protein (Å2)
average B-factor ligand (Å2)
average B-factor solvent (Å2)
a Rmerge ) ΣhΣi|I(h,i) - (h)| ΣhSi (h); I(h,i) is the scaled intensity of the ith observation of reflection h and (h) is the mean value. Summation is over all
measurements. Rcryst ) Σhkl,work||Fobs| - k|Fcalc||/Σhkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, k is a weighting factor and
work denotes the working set of 95% of the reflections used in the refinement. Rfree )Σhkl,test|Fobs| - k|Fcalc||/Σhkl|Fobs|, where Fobs and Fcalc are the observed
and calculated structure factors, k is a weighting factor and test denotes the test set of 5% of the reflections used in cross validation of the refinement. λ refers
to wavelength, rmsd to root-mean-square deviations. b Numbers in parentheses indicate the highest shell values.
Scheme 1. Synthesis of 6-Substituted 2-Aminopyridine
Compounds that fluoresced under the conditions described above
were assayed in an alternative assay. BACE-1 activity was measured
Inhibitorsa
using a FRET-based substrate supplied by PanVera (kit P2985).
Assays were carried out in 50 mM sodium acetate, pH 4.5 (provided
with kit), 5% DMSO in 96-well black, flat-bottomed 1/2 area Costar
plates in a final assay volume of 50 µL. Compounds were
preincubated as above for 5 min with in-house-produced BACE-1,
and the reaction was initiated by adding 0.25 µM peptide substrate.
The reaction rate was monitored at room temperature on a
SpectraMax Gemini XS platereader (Molecular Devices) with
excitation and emission wavelengths of 545 and 595 nm, respec-
tively. Initial reaction rates were measured, and IC50s were
calculated as described above.
Modeling. Docking experiments were used to predict the binding
mode of designed aminopyridines and to select compounds for
synthesis. The aminoquinoline and aminoisoquinoline BACE-1
structures described in the previous paper were selected to dock
the ligands. For each structure, a number of binding sites were
constructed. Each of them contained the catalytic region (Asp32
and Asp228) with both Asp residues in the ionized state, the flap
residues potentially involved in the binding of the ligands (Val69,
Pro70, Tyr71, Thr72, and Trp76), and finally one of the following
combinations of adjacent pockets: S1, S1 plus S3, S2′ plus S1 and
a large binding site constituted by S1, S1′, S2′, and S3. All dockings
were run on a Linux cluster using Goldscore scoring functions
within the Astex web-based docking facilities.42 Methods and
settings have been previously described by Verdonk et al.43
Chemistry. Reagents and solvents were obtained from com-
mercial suppliers and used without further purification. Thin layer
chromatography (TLC) analytical separations were conducted with
E. Merck silica gel F-254 plates of 0.25 mm thickness and were
visualized with UV light (254 nM) or I2. Flash chromatography
was performed using E. Merck silica gel. 1H nuclear magnetic
resonance (NMR) spectra were recorded in the deuterated solvents
specified on a Bruker Avance 400 spectrometer operating at 400
MHz. Chemical shifts are reported in parts per million (δ) from
the tetramethylsilane resonance in the indicated solvent (TMS: 0.0
ppm). Data are reported as follows: chemical shift, multiplicity
(br ) broad, s ) singlet, d ) doublet, t ) triplet, m ) multiplet),
coupling constants (Hz), integration. Compound purity and mass
spectra were determined by a Waters Fractionlynx/Micromass ZQ
LC/MS platform using the positive electrospray ionization technique
(+ES) using a mobile phase of acetonitrile/water with 0.1% formic
acid. Compound 5, N3-benzyl-pyridine-2,3-diamine was prepared
as per Khanna et al.44
a Reagents: (a) Phthalic anhydride, 190 °C; (b) NBS, AIBN, C6H6, reflux;
(c) PPh3, MeCN, reflux; (d) ArCHO, KOtBu, THF, reflux; (e) H2, 10% Pd
on carbon, MeOH; (f) N2H4 hydrate, EtOH, reflux; (g) 3-MeO-PhB(OH)2,
K2CO3, Pd(PtBu3)2, PhMe, H2O, EtOH, MeOH, microwave irradiation, 135
°C.
anhydride (29.6 g, 0.2 mol) were stirred and held at 190 °C for 1
h and then cooled to room temperature to afford 10a as a pale
1
yellow solid (45.5 g, 95%). H NMR (400 MHz, DMSO-d6): δ
2.52 (s, 3H), 7.37 (d, J ) 7, 1H), 7.40 (d, J ) 7, 1H), 7.90-7.95
(m, 3H), 7.97-8.03 (m, 2H). MS (+ES): m/e 239 (MH+).
[6-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)pyridin-2-ylmethyl]-
triphenylphosphonium Bromide (10c). A mixture of the 2-(6-
methylpyridin-2-yl)isoindole-1,3-dione 10a (23.8 g, 0.1 mol),
N-bromosuccinimide (19.0 g, 0.11 mol), and AIBN (1.2 g) in
anhydrous benzene (400 mL) was stirred and held at reflux for 5
2-(6-Methylpyridin-2-yl)isoindole-1,3-dione (10a). A mixture
of 2-amino-6-methylpyridine 9 (21.6 g, 0.2 mol) and phthalic

Aminopyridines as Inhibitors of â-Secretase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1129
Scheme 2. Synthesis of 3-Substituted 2-Aminopyridine
mixture was stirred and held at reflux for 6 h. Upon cooling to
room temperature, the solvent was removed in Vacuo and the
mixture partitioned between dichloromethane and water. The
organic layer was separated, the solvent removed in Vacuo, and
the residue subjected to column chromatography on silica. Elution
with diethyl ether afforded 11a as a pale yellow solid (420 mg,
Inhibitorsa
1
65%). H NMR (400 MHz, DMSO-d6): δ 7.33-7.46 (m, 5H),
7.65-7.76 (m, 4H), 7.95-8.02 (m, 2H), 8.03-8.08 (m, 3H). MS
(+ES): m/e 327 (MH+).
Compounds 11b and 11c were prepared in a similar fashion to
that outlined above for 11a. For NMR and MS data of these
compounds, see Supporting Information.
2-(6-Phenethylpyridin-2-yl)isoindole-1,3-dione (12a). 10% Pal-
ladium on carbon (80 mg) was added to 11a (260 mg, 0.80 mmol)
in methanol (10 mL) and ethyl acetate (5 mL), and the mixture
was stirred at room temperature under a hydrogen atmosphere for
16 h. The catalyst was removed by filtration, and the solvent was
removed in Vacuo to afford crude 12a as a colorless oil (250 mg,
96%) which was used without further purification. 1H NMR
(400 MHz, DMSO-d6): δ 3.02 (m, 2H), 3.08 (m, 2H), 7.18
(m, 1H), 7.25 (m, 4H), 7.37 (t, J ) 7, 2H), 7.95 (m, 3H), 8.02
(m, 2H). MS (+ES): m/e 329 (MH+).
a Reagents: (a) Aryl aldehyde, AcOH, NaBH(OAc)3, CH2Cl2; (b) aryl alde-
hyde, Et3N, NaBH(OAc)3, 4 Å sieves, CH2Cl2; (c) 15a, 5-methoxypyridyl-
3-boronic acid, K2CO3, Pd(PtBu3)2, EtOH, MeOH, toluene, water, 135 °C,
microwave; (d) 15b, 6-bromoindole, K3PO4,Pd(PPh3)4, DMF, 100 °C.
Compound 12b was prepared in a similar fashion to that outlined
above for 12a. For NMR and MS data of this compound, see
Supporting Information.
6-Phenethylpyridin-2-ylamine (2). Hydrazine hydrate (103 mg,
2.06 mmol) was added to a solution of 12a (210 mg, 0.64 mmol)
in ethanol (8 mL), and the mixture was stirred and held at reflux
for 3 h. Upon cooling, the solvent was removed in Vacuo and the
residue partitioned between dichloromethane and water. The organic
layer was separated, the solvent removed in Vacuo, and the residue
subjected to column chromatography on silica. Elution with diethyl
ether afforded 2 as a pale yellow oil (95 mg, 75%). 1H NMR (400
MHz, DMSO-d6): δ 2.77 (t, J ) 7, 2H), 2.90 (t, J ) 7, 2H), 5.82
(br s, 2H), 6.26 (d, J ) 8, 1H), 6.34 (d, J ) 8, 1H), 7.18 (t, J ) 8,
1H), 7.15-7.30 (m, 5H). MS (+ES): m/e 199 (MH+).
Scheme 3. Synthesis of 3-Substituted 2-Aminopyridine
Inhibitorsa
Compounds 3 and 13a were prepared in a similar fashion to that
outlined above for 2. For NMR and MS data of these compounds,
see Supporting Information.
6-[2-(3′-Methoxybiphenyl-3-yl)ethyl]pyridin-2-ylamine (4). A
mixture of 13a (110 mg, 0.4 mmol), 3-methoxyphenylboronic acid
(92 mg, 0.6 mmol), anhydrous potassium carbonate (276 mg, 2.0
mmol), and bis(tri-tert-butylphosphine)palladium(0) (5 mg) in
methanol (1 mL), ethanol (1 mL), water (1 mL), and toluene
(1 mL) was subjected to microwave irradiation (CEM microwave)
at 135 °C for 30 min. The mixture was filtered, the organic solvent
evaporated in Vacuo, and the residue partitioned between dichlo-
romethane and 1 M sodium hydroxide. The organic layer was
separated and the solvent removed in Vacuo to afford crude 13b.
Methanol (15 mL) and 10% palladium on carbon (60 mg) were
added, and the mixture was stirred under an atmosphere of hydrogen
for 5 h. The catalyst was removed by filtration, the solvent was
removed in Vacuo, and the residue was subjected to column
chromatography on silica. Elution with ethyl acetate afforded 4 as
a Reagents: (a) Benzylic halide, K2CO3, THF, reflux; (b) 2,3-diami-
nopyridine, AcOH, NaBH(OAc)3, CH2Cl2; (c) indole-6-boronic acid, K2CO3,
Pd(PtBu3)2, PhMe, H2O, EtOH, MeOH, microwave irradiation, 135 °C.
1
a colorless oil (85 mg, 70%). H NMR (400 MHz, DMSO-d6): δ
2.83 (t, 2H, J ) 7), 3.00 (t, 2H, J ) 7), 3.83 (s, 3H), 5.80 (br s,
2H), 6.27 (d, 1H, J ) 8), 6.37 (d, 1H, J ) 7), 6.93 (dm, J ) 8,
1H), 7.14 (t, J ) 1.5, 1H), 7.18-7.30 (m, 3H), 7.33-7.39 (m, 2H),
7.47 (m, 2H). MS (+ES): m/e 305 (MH+).
h. The mixture was cooled to room temperature and then chilled
in an ice bath for 10 min with vigorous stirring. The insoluble
material was removed by filtration and the solvent removed in
Vacuo. The resulting solid crude benzylic bromide 10b was
dissolved in acetonitrile (400 mL), triphenylphosphine (26.2 g, 0.1
mol) was added, and the mixture was stirred and held at reflux for
16 h. Upon cooling, the solid material was collected by suction
filtration, washed with acetonitrile, and sucked dry to afford 10c
as a colorless solid (36.75 g, 63%).1H NMR (400 MHz, DMSO-
d6): δ 5.54 (d, J ) 18, 2H), 7.43 (m, 2H), 7.62-7.68 (m, 6H),
7.78-7.85 (m, 9H), 7.95-8.02 (m, 5H). MS (+ES): m/e 499 (M+).
2-[6-((E)-Styryl)pyridin-2-yl]isoindole-1,3-dione (11a). Potas-
sium tert-butoxide (260 mg, 2.32 mmol) was added to a stirred
suspension of 10c (1.16 g, 2.0 mmol) in anhydrous tetrahydrofuran
(30 mL), and the mixture was stirred at room temperature for 15
min. Benzaldehyde (224 mg, 2.11 mmol) was added, and the
N3-(3-Pyridin-3-ylbenzyl)pyridine-2,3-diamine (6a). A mixture
of 2,3-diaminopyridine (327 mg, 3.0 mmol), 3-pyridin-3-ylbenzal-
dehyde (3.0 mmol), and acetic acid (8-10 drops) in dichlo-
romethane (15 mL) was stirred at room temperature for 30 min.
Sodium triacetoxyborohydride (1.91 g, 9.0 mmol) was added and
the mixture stirred at room temperature overnight. The mixture was
washed with an equal volume of 10% aqueous potassium carbonate
solution, the organic layer separated, the solvent removed in Vacuo,
and the residue purified by column chromatography on silica.
Elution with mixtures of ethyl acetate and methanol afforded the
1
product. H NMR (400 MHz, CDCl3): δ 3.65 (br s, 1H), 4.23
(br s, 2H), 4.38 (d, J ) 4.8, 2H), 6.79 (dd as t, J ) 6.0, 1H), 6.84

1130 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
CongreVe et al.
(d, J ) 7.5, 1H), 7.37 (dd, J ) 7.9, 4.2, 1H), 7.43 (d, J ) 7.3, 1H),
7.47 (d, J ) 7.6, 1H), 7.51 (t, J ) 6.5, 1H), 7.59 (s, 1H), 7.63
(d, J ) 5.0, 1H), 7.86 (d, J ) 7.8, 1H), 8.60 (d, J ) 4.8, 1H), 8.84
(s, 1H). MS (+ES): m/e 277 (MH+).
the conditions described above in the preparation of compound 6a
to give the title compound after purification by preparative HPLC/
1
MS as its formic acid salt (38 mg, 37%). H NMR (400 MHz,
MeOH-d4): δ 1.10 (t, J ) 7.4, 3H), 1.87 (sextet, J ) 6.3, 2H),
4.11 (t, J ) 6.4, 2H), 4.53 (s, 2H), 6.73 (dd, J ) 7.9, 5.9, 1H),
6.93 (d, J ) 7.8, 1H), 7.27 (dd, J ) 1.3, 5.8, 1H), 7.46-7.54
(m, 2H), 7.58-7.63 (m, 2H), 7.71 (s, 1H), 8.23 (d, J ) 2.8, 1H),
8.28 (d, J ) 1.7, 1H). MS (+ES): m/e 335 (MH+).
N3-(3-Bromobenzyl)pyridine-2,3-diamine (15a). To a stirred
solution of 2,3-diaminopyridine (5 g, 45.8 mmol) in DCM
(200 mL) were added 3-bromobenzaldehyde (5.37 mL, 45.8 mmol),
triethylamine (19.2 mL, 137 mmol), and 4 Å molecular sieves
followed by sodium triacetoxyborohydride (38.8 g, 183 mmol). The
reaction was allowed to stir at RT for 16 h. The reaction was
monitored by TLC, and further equivalents of sodium triacetoxy-
borohydride were added as required. The mixture was filtered and
then diluted with DCM, washed with H2O, and dried over MgSO4
and the solvent removed in Vacuo. The residue was purified by
column chromatography eluting with 5% MeOH in DCM to give
N3-[3-(1H-Indol-6-yl)benzyl]pyridine-2,3-diamine (7). To a
mixture of N3-(3-benzylboronic acid)pyridine-2,3-diamine 15b
(150 mg, 0.62 mmol) in DMF (3 mL) was added tetrakis(triph-
enylphosphine)palladium(0) (5 mg) followed by 6-bromoindole
(120 mg, 0.62 mmol). Potassium phosphate (2 N, 1 mL) was then
added, and the reaction was heated to 100 °C for 12 h. The mixture
was cooled to room temperature and the solvent removed in Vacuo.
The resulting material was partitioned between DCM and H2O,
dried over MgSO4, and evaporated to dryness. Material purified
by preparative HPLC to give the title compound as a pale yellow
1
the title compound as a pale yellow oil (4.33 g, 34%). H NMR
(400 MHz, MeOH-d4): δ 4.40 (s, 2H), 6.62 (dd, J ) 7.8, 5.5, 1H),
6.73 (dd, J ) 7.6, 1.2, 1H), 7.24-7.33 (m, 2H), 7.38 (d, J ) 7.6,
1H), 7.43 (br d, J ) 8.0, 1H), 7.58 (s,1H). MS (+ES): m/e 278,
280 (MH+).
Compound 15b was prepared in a similar fashion to that outlined
above for 15a, but in this case no aqueous work up procedure was
used. For NMR and MS data of this compound, see Supporting
Information.
N3-[3-(5-Methoxypyridin-3-yl)benzyl]pyridine-2,3-diamine (6b).
To a degassed solution of N3-(3-bromobenzyl)pyridine-2,3-diamine
(100 mg, 0.36 mmol) in toluene (1.5 mL) was added bis(tri-tert-
butylphosphine)palladium(0) (5 mg) followed by 5-methoxypyridyl-
3-boronic acid (202 mg, 0.86 mmol) as a solution in ethanol (1.5
mL). Potassium carbonate (299 mg, 2.16 mmol) was then added
as a solution in H2O (2 mL) followed by methanol (2 mL). The
reaction was heated to 135 °C for 35 min in a CEM microwave.
The mixture was cooled to room temperature and the solvent
removed in Vacuo. The resulting material was partitioned between
DCM and H2O, dried over MgSO4, and evaporated to dryness.
Material purified by preparative HPLC to give the title compound
as a pale yellow solid (41 mg, 37%). 1H NMR (400 MHz, MeOH-
d4): δ 3.92 (s, 3H), 4.44 (s, 2H), 6.53 (dd, J ) 7.5, 5.4, 1H), 6.72
(dd, J ) 7.9, 1.2, 1H), 7.33 (dd, J ) 5.1, 1.4, 1H), 7.45 (d, J )
5.1, 1H), 7.51-7.56 (m, 2H), 7.66 (s, 1H), 8.20 (d, J ) 2.7, 1H),
8.36 (d, J ) 1.8, 1H). MS (+ES): m/e 307 (MH+).
1
solid. H NMR (400 MHz, MeOH-d4): δ 4.45 (s, 2H), 6.46 (d, J
) 3.3, 1H), 6.58 (dd, J ) 7.8, 5.3, 1H), 6.79 (d, J ) 9.0, 1H), 7.27
(d, J ) 3.0, 1H), 7.30-7.34 (m, 3H), 7.40 (t, J ) 7.5, 1H), 7.56
(d, J ) 7.6, 1H), 7.59-7.61 (m, 2H), 7.7 (s, 1H). MS (+ES): m/e
315 (MH+).
2-Benzyloxy-5-bromobenzaldehyde (17a). Benzyl bromide
(6.16 g, 36.0 mmol) was added to a stirred mixture of 5-bromo-
2-hydroxybenzaldehyde 16 (6.03 g, 30.0 mmol) and anhydrous
potassium carbonate (8.28 g, 60.0 mmol) in tetrahydrofuran
(80 mL), and the mixture was stirred and held at reflux for 16 h.
Upon cooling, the solvent was removed in Vacuo, the mixture
partitioned between dichloromethane and water, the organic layer
separated, and the solvent removed in Vacuo. The residue was
triturated with 33% diethyl ether in petroleum ether, and the
resulting solids were collected by suction filtration and sucked dry
under reduced pressure to afford 7.65 g of 17a as a colorless solid
1
(88%). H NMR (400 MHz, DMSO-d6): δ 5.31 (s, 2H), 7.30-
7.37 (m, 2H), 7.42 (t, J ) 7, 2H), 7.51 (d, J ) 7, 2H), 7.78 (d, J
) 2, 1H), 7.83 (d, J ) 8, 2, 1H), 10.34 (s, 1H). MS (+ES): m/e
292, 294 (MH+).
Compound 17b was prepared in a similar fashion to that outlined
above for 17a. For NMR and MS data of this compound, see
Supporting Information.
N3-(2-Benzyloxy-5-bromobenzyl)pyridine-2,3-diamine (18a).
A mixture of 2,3-diaminopyridine 14 (55 mg, 0.50 mmol), 17a (192
mg, 0.50 mmol), and acetic acid (4 drops) in dichloromethane
(3 mL) was stirred at room temperature for 10 min. Sodium
triacetoxyborohydride (300 mg, 1.50 mmol) was added, and the
mixture was stirred at room temperature overnight. The mixture
was washed with an equal volume of 2 M sodium hydroxide
solution, the organic layer separated, the solvent removed in Vacuo,
and the residue subjected to column chromatography on silica.
Elution with 5% methanol in ethyl acetate afforded 82 mg of 18a
N3-[3-(5-Propoxypyridin-3-yl)benzyl]pyridine-2,3-diamine (6c).
To a solution of 5-bromo-3-pyridinol (629 mg, 3.61 mmol) in DMF
(10 mL) was added potassium carbonate (500 mg, 3.62 mmol),
and the reaction stirred at room temperature. 1-Bromopropane
(660 mg, 5.4 mmol) was then added and the reaction stirred for 18
h. The solvent was then removed in Vacuo. The resulting material
was partitioned between diethyl ether and H2O, and the organics
were washed with water, dried over MgSO4, and evaporated to
dryness. The material did not require further purification. The
product was 3-bromo-5-propoxypyridine as a pale yellow liquid
1
1
as a yellow oil (43%). H NMR (400 MHz, DMSO-d6): δ 4.29
(660 mg, 85%). H NMR (400 MHz, MeOH-d4): δ 1.07 (t, J )
(d, J ) 5.5, 2H), 5.21 (s, 2H), 5.35 (t, J ) 5.5, 1H), 5.52 (br s,
2H), 6.37 (d, J ) 3.5, 2H), 7.08 (d, J ) 9, 1H), 7.29 (t, J ) 3.5,
1H), 7.33-7.42 (m, 5H), 7.50 (d, J ) 8.5, 2H). MS (+ES): m/e
384, 386 (MH+).
Compound 18b was prepared in a similar fashion to that outlined
above for 18a. For NMR and MS data of this compound, see
Supporting Information.
7.4, 3H), 1.79-1.88 (m, 2H), 4.04 (t, J ) 6.4, 2H), 7.63 (t, J )
2.2, 1H), 8.22 (m, 2H). MS (+ES): m/e 216, 218 (MH+). To
3-benzaldehydeboronic acid (300 mg, 2 mmol) were added
3-bromo-5-propoxypyridine (216 mg, 1 mmol) and potassium
carbonate (828 mg, 6 mmol). Toluene (2 mL), ethanol (2 mL), and
water (2 mL) were then added. Bis(tri-tert-butylphosphine)-
palladium(0) (30 mg) was then added and the mixture vigorously
stirred under nitrogen until all the reagents dissolved. The reaction
was then heated to 130 °C for 30 min in a CEM microwave. The
mixture was cooled to room temperature and then partitioned
between DCM and H2O, dried over MgSO4, and evaporated to
dryness. The crude material was purified by flash chromatography
on silica to give 3-(5-propoxypyridin-3-yl)benzaldehyde (150 mg,
62%). 1H NMR (400 MHz, CDCl3): δ 1.02 (t, J ) 7.7, 3H), 1.80
(sextet, J ) 7.2, 2H), 4.00 (t, J ) 6.6, 2H), 7.41 (m, 1H), 7.60
(t, J ) 8.0, 1H), 7.76-7.80 (m, 1H), 7.86 (dt, J ) 7.6, 1.2, 1H),
8.02 (d, J ) 2.2, 1H), 8.26 (d, J ) 2.9, 1H), 8.41 (d, J ) 1.6, 1H),
10.03 (s, 1H). MS (+ES): m/e 242 (MH+). 3-(5-Propoxypyridin-
3-yl)benzaldehyde and 2,3-diaminopyridine were then treated under
N3-[2-Benzyloxy-5-(1H-indol-6-yl)benzyl]pyridine-2,3-di-
amine (8a). A mixture of 18a (77 mg, 0.2 mmol), indole-6-boronic
acid (77 mg, 0.48 mmol), anhydrous potassium carbonate (166 mg,
1.20 mmol), and bis(tri-tert-butylphosphine)palladium(0) (4 mg)
in methanol (0.7 mL), ethanol (0.7 mL), water (0.7 mL), and toluene
(0.7 mL) was subjected to microwave irradiation (CEM microwave)
at 135 °C for 15 min. The mixture was filtered, the organic solvent
evaporated in Vacuo, and the residue partitioned between dichlo-
romethane and 2 M sodium hydroxide. The organic layer was
separated, the solvent removed in Vacuo, and the residue subjected
to column chromatography on silica. Elution with 5% methanol in
ethyl acetate afforded 8a as a pale brown foam (40 mg, 48%).1H

Aminopyridines as Inhibitors of â-Secretase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1131
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NMR (400 MHz, DMSO-d6): δ 4.38 (d, J ) 5.5, 2H), 5.26 (s,
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6.52 (d, J ) 7.5, 1H), 7.18 (d, J ) 8.5, 2H), 7.27 (dd, J ) 5, 1.5,
1H), 7.32-7.36 (m, 2H), 7.41 (t, J ) 7, 2H), 7.50-7.59 (m, 6H),
11.10 (br s, 1H). MS (+ES): m/e 421 (MH+).
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Acknowledgment. The authors thank Harren Jhoti, Anne
Cleasby, and Glyn Williams for their helpful advice and
suggestions during the course of this research. We acknowledge
the European Synchrotron Radiation Facility, Grenoble, and the
Synchrotron Radiation Source, Daresbury, for access to beam-
time.
Supporting Information Available: Additional spectral data
for compounds 3, 8b, 11b, 11c, 12b, 13a, 15b, 17b, and 18b and
information on compound purity. This material is available free of
charge via the Internet at http://pubs.acs.org.
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