Pyridinyl aminohydantoins as small molecule BACE1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.136

A novel class of pyridinyl aminohydantoins was designed and prepared as highly potent BACE1 inhibitors. Compound (S)-4g showed excellent potency with IC₅₀ of 20 nM for BACE1. X-ray crystallography indicated that the interaction between pyridine

Full text of DOI:10.1016/j.bmcl.2010.01.136

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2326–2329
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridinyl aminohydantoins as small molecule BACE1 inhibitors
a
a
a
c
c
b
Ping Zhou a, , Yanfang Li , Yi Fan , Zheng Wang , Rajiv Chopra , Andrea Olland , Yun Hu ,
Ronald L. Magolda a, Menelas Pangalos b, Peter H. Reinhart b, M. James Turner b, Jonathan Bard b,
Michael S. Malamas a, Albert J. Robichaud a
*
a Department of Chemical Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, United States
b Neuroscience, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, United States
c Department of Chemical Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of pyridinyl aminohydantoins was designed and prepared as highly potent BACE1 inhibitors.
Compound (S)-4g showed excellent potency with IC50 of 20 nM for BACE1. X-ray crystallography indi-
cated that the interaction between pyridine nitrogen and the tryptophan Trp76 was a key feature in
the S20 region of the enzyme that contributed to increased potency.
Received 18 December 2009
Revised 26 January 2010
Accepted 28 January 2010
Available online 12 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
b-Secretase
BACE1
Pyridinyl aminohydantoins
Alzheimer’s disease (AD) is the most common cause of demen-
tia and is rapidly becoming the most prevalent problem in the
aging human population. Although the cause of AD is still unclear,
deposition of b-amyloid peptide (Ab) in the brain is one of the few
recognized hallmarks of AD pathogenesis.1 It has been well estab-
lished that Ab is produced from membrane-bound b-amyloid pre-
cursor protein (APP) by the sequential proteolytic cleavage of two
trend would hold true for the pyridine analog 3, and examined
whether additional interactions between the ligand and protein
backbone of the S3 pocket, by functionalization of the phenyl ring,
could enhance the potency of derivatives of 3.
Here, we report the design and synthesis of pyridinyl amin-
ohydantoins 4 as small molecule BACE1 inhibitors. Particular fo-
cus was on exploration of the S3 pocket by introduction of
substituents at the 3-position of the phenyl ring in 3. In addition,
through the use of an X-ray crystal structure, we observed a key
interaction between the pyridine nitrogen in derivatives of 4 with
Trp76 in the S20 pocket, and we chose to utilize this in our design
strategy.
aspartyl proteases, b- and
c-secretase. b-Secretase (BACE1, also
called Asp2 or memapsin2), has been identified as the enzyme
responsible for the initial processing of APP.2,3 Hence, the inhibi-
tion of this enzyme represents a strategy for the development of
disease-modifying therapeutics for the treatment of AD.4
In a program to design and develop novel small molecule BACE1
Two approaches were used to prepare the target molecules 4:
(I) direct aminohydantoin formation from diketone intermediate
5, and (II) derivatization of pyridinyl aminohydantoin intermediate
6 (Scheme 2).
inhibitors, a weak BACE1 inhibitor (IC50 = 38 lM), aminoimidazole
1 was discovered via high-through screening (Scheme 1).5 Initial
SAR around 1 quickly led to aminohydantoin 2 which showed a
10-fold improvement in BACE1 potency (IC50 = 3.4
l
M).6 Replace-
As shown in Scheme 3, the diketone intermediates 5 used in
strategy (I) were obtained via oxidation of 4-(3-arylphenylethy-
nyl)pyridine 9 which was prepared starting with 3-aryl ethynyl-
benzene 7. Sonogashira coupling of 7 with 4-bromopyridine 8
gave diaryl substituted ethyne 9 in good to excellent yield.7 Alter-
natively, 9 could be prepared by Sonogashira coupling of commer-
cially available 3-bromoiodobenzene 10 and 4-ethynylpyridine 11
followed by Suzuki coupling of the resulting 4-(3-bromophenyle-
thynyl)pyridine 12 with aryl boronic acids or by Stille coupling of
12 with aryl stannanes.8 Oxidation of 9 with KMnO4 afforded dike-
tone 5 in good to excellent yield.9 Treatment of 5 with N-methyl
ment of one of the phenyl groups in 2 with a pyridine gave pyrid-
inyl aminohydantoin 3 which maintained the ligand’s affinity for
the BACE1 enzyme (IC50 = 2.7 lM).
We have previously reported that introduction of a 3-substitu-
ent on the phenyl group (projected in the S1 pocket) of compound
2 resulted in improvement in BACE1 activity through enhanced S3
pocket interactions.6 We have now investigated whether the same
* Corresponding author. Tel.: +1 732 274 4661; fax: +1 732 274 4505.
E-mail addresses: zhoupb31@gmail.com, zhoup3@wyeth.com (P. Zhou).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.136

P. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2326–2329
2327
H2N
N
H2N
H2N
N
H2N
N
N
N
N
N
N
O
O
O
N
Ar
N
N
1
3
2
4
BACE1 IC50 = 2.7 µM
Pyridinyl aminohydantoin
BACE1 IC50 = 3.6 µM
Aminohydantoin
BACE1 IC50 = 38 µM
HTS Hit
Scheme 1. From HTS hit to pyridinyl amino hydantoin.
N
O
N
O
Br
Ar
Br
a
N
(I)
H2N
CH3
O
O
O
5
N
12
13
N
H2N
CH3
O
Ar
H2N
H2N
CH3
CH3
N
N
N
N
N
(II)
Suzuki Coupling
4
N
N
b
O
O
c
Br
Br
Ar
N
N
N
6
6
4
Scheme 2. Strategy for the synthesis of pyridinyl aminohydantoins 4.
Scheme 4. Reagents and conditions: (a) KMnO4/NaHCO3/MgSO4/H2O, rt, 75%; (b)
N-methylguanidine/Na2CO3/EtOH/H2O, reflux, 61%; (c) ArB(OH)2/tetrakis(triphen-
ylphosphine)palladium/Na2CO3/DME, reflux, 51–95%.
guanidine in the presence of Na2CO3 produced 3-arylphenyl pyrid-
inyl aminohydantoin 4.10
Furthermore, 3-heteroayl analogs (4i–m) provided derivatives
with improved potency. For example, the thienyl and furyl deriva-
tives showed a 4–11-fold improvement in BACE1 potency (4i and
4j vs 3), while the 2-pyrazinyl (4k) had 38-fold increase in ligand
potency, giving a compound with an IC50 of 70 nM at BACE1. The
3-pyridinylphenyl and 2-fluoropyridin-3-ylphenyl groups were
previously studied in related aminohydantoins with comparable
results.6 Similar trends were also found when these two groups
were incorporated in pyridinyl aminohydantoins 4. Both com-
pounds 4l and 4m showed 45- and 89-fold BACE1 potency
improvement compared to the parent derivative 3.
The majority of the prepared analogs were equally potent in
both BACE1 and BACE2 activity, with the exception of compound
4m, which demonstrated 33-fold selectivity versus BACE2. On
the other hand, most of the tested compounds demonstrated high
selectivity (>100-fold) for BACE1 versus cathepsin D.
The precursor 3-bromophenyl pyridinyl aminohydantoin 6 used
in the strategy (II) for Suzuki coupling, was prepared from 4-(3-
bromophenylethynyl)pyridine 12 (Scheme 4). Oxidation of 12 with
KMnO4 under similar reaction conditions as 9 afforded bromo
diketone 13 in good yield. Treatment of 13 with N-methyl guani-
dine as described above produced 3-bromophenyl pyridinyl amin-
ohydantoin 6, the precursor for Suzuki coupling. Standard Suzuki
coupling of 6 with boronic acids furnished final products 4.11
BACE1 and BACE2 affinities of the target ligands are summa-
rized in Table 1. As an additional measure of selectivity, inhibition
of cathepsin D was determined for selected compounds exhibiting
potent BACE1 activity. The data show that introduction of a 3-aryl
group to the benzene ring of 3 was beneficial for BACE1 affinity.
Simple 3-phenyl analog gave 20-fold improvement in potency at
BACE1 (4a vs 3), which confirmed our previous findings6 that
extending in the S3 region improves potency. Replacement of phe-
nyl with 3-methoxyphenyl (4b), 3-cyanophenyl (4d) or 3-fluoro-
phenyl (4e) provided analogs with further improved BACE1
potency (54–89-fold), with the exception of the trifluoromethoxy-
phenyl derivative (4c) that resulted in only an 11-fold increase in
ligand potency. Difluorophenyl derivatives (4f, 4g, 4h) also showed
improved BACE1 activity up to 67-fold.
Racemic pyridinyl aminohydantoin 4g was separated into enan-
tiomers (S)-4g and (R)-4g, with the (S)-enantiomer showing higher
affinity than the (R)-enantiomer. The (S)-configuration of enantio-
mer 4g is supported by X-ray studies. As shown in Figure 1, the
amino group of hydantoin 4g interacts with both aspartic acids
(Asp32 and Asp238) of the enzyme backbone and the N3 nitrogen
Ar
Ar
N
O
5
a
d
Ar
+
N
Br
N
O
7
9
8
e
c
H2N
CH3
O
N
Br
Br
N
b
I
Ar
N
+
N
N
11
12
10
4
Scheme 3. Reagents and conditions: (a) Pd(PPh3)2Cl2/CuI/TEA/DMF, 65 °C, 59–88%; (b) Pd(PPh3)2Cl2/CuI/TEA/DMF, 65 °C, 87%; (c) ArB(OH)2 or ArSnBu3/tetrakis(triphen-
ylphosphine)palladium/Na2CO3/DME or toluene, reflux, 40–77%; (d) KMnO4/NaHCO3/MgSO4/H2O, rt, 49–96%; (e) N-methylguanidine/Na2CO3/EtOH/H2O, reflux, 37–52%.

2328
P. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2326–2329
Table 1
Biological activities of pyridinyl aminohydantoinsa
H2N
N
N
O
Ar
N
4
Compound
Ar
BACE1 IC50
(lM)
BACE2 IC50
(
lM)
Selectivity BACE2/BACE1
Cathepsin D IC50 (lM)
3
4a
4b
4e
4d
4e
4f
4g
(S)-4g
(R)-4g
4h
4i
H
Ph
3-MeOPh
3-CF3OPh
3-NCPh
2.68
0.13
0.03
0.25
0.04
0.05
0.04
0.05
0.02
3.81
0.06
0.24
0.63
0.07
0.06
0.03
2.01
0.14
0.02
0.14
0.04
0.08
0.13
0.12
0.10
0.70
0.06
0.19
0.57
1.01
0.48
1.00
0.8
1.1
0.7
0.6
1.0
1.6
3.3
2.4
5.0
0.2
1.0
0.8
0.9
14
ND
30.1
12.2
14.1
24.2
20.8
13.1
9.4
3-FPh
2,3-diFPh
2,5-diFPh
2,5-diFPh
2,5-diFPh
3,5-diFPh
3-Thienyl
3-Furyl
2-Pyrazinyl
3-Pyridinyl
2-Fluoropyrindin-3-yl
5.5
26.4
34.1
31.6
46.8
ND
4j
4k
4l
8.0
33
ND
17.4
4m
ND = not determined.
a
A homogenous, continuous fluorescence resonance energy transfer (FRET) was used to assess compound inhibition for BACE1, BACE2, Cathepsin D, Pepsin, and Renin
activities. The BACE1 and BACE2 activities were based on the cleavage of peptide substrate Abz-SEVNLDAEFR-Dpa (Swedish substrate), while peptide substrate MOCAc-
GKPILFFRLK (Dnp)-D-R-NH2 was used for Cathepsin D. Kinetic rates were calculated and IC50 values were determined by fitting the % inhibition, as a function of compound
concentration, to the Hill equation (y = ((B * Kn) + (100 * xn))/(Kn + xn).
significantly more potent than the R-isomer, in line with the X-
ray analysis. These potent BACE1 leads can be optimized further
and may prove to be useful tools for biological studies.
Acknowledgements
The authors acknowledge the members of the Wyeth Discovery
Analytical Chemistry group for analytical and spectral determina-
tions, and Dr. David P. Rotella for helpful discussions.
References and notes
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Figure 1. Crystal structure of BACE1 complexed with (S)-4g (PDB ID code: 3LHG)
highlighting the key hydrogen-bonding interactions in yellow dashed lines between
the catalytic aspartic acids Asp32 and Asp228 and the aminohydantoin ring, as well
as between pyridine nitrogen and Trp76 at S20.
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of the imidazole ring interacts with Asp32 in a coplanar fashion. In
addition, the pyridine nitrogen at S20 interacts with the tryptophan
Trp76. The difluoro phenyl moiety orients itself into the S3 pocket
and enhances binding affinity for the BACE1 enzyme.
In summary, a novel class of pyridinyl aminohydantoins 4 was
explored to develop a better understanding of BACE1 SAR in the
S3 pocket pf the enzyme. This work revealed that more potent
BACE1 inhibitors could be prepared taking advantage of several
key interactions with the enzyme. X-ray crystallography indicated
that the interaction between pyridine nitrogen and Trp76 was a
key feature in the S20 region of the enzyme that contributed to in-
creased potency. When enantiomers of the hydantoin were re-
solved, for example, (S)-4g with IC50 of 20 nM, they proved to be
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J.; Hu, Y.; Erik Wagner, E.; Fan, K.; Olland, A.; Bard, J.; Robichaud, A. J. J. Med.
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2329
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4-one12 (104 mg, 0.3 mmol) in DME (5 mL) is added 2,5-difluorophenylboronic
acid (96 mg, 0.6 mmol), tetrakis(tripheny1phosphine)palladium (35 mg,
0.03 mmol) and 2.0 M aqueous sodium carbonate (0.6 mL, 1.2 mmol) at room
temperature. The reaction mixture is refluxed for 1 h and cooled. After
evaporation of the solvent, the crude mixture is purified by chromatography
(silica gel, EtOAc/2 M methanolic NH3: 92/8) to give the title compound
(95 mg, 84%) as a solid. Mp: 200–202 °C.; MS(+) ES: 379 (M+H)+.
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4-yl-3,5-dihydro-imidazole-4-one, 4g, typical procedure: to
a solution of 2-