Macrocyclic BACE inhibitors: Optimization of a micromolar hit to nanomolar leads

Article abstract of DOI:10.1016/j.bmcl.2010.03.097

We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of Alzheimer's disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin conformation suggesting that constrained macrocycl

Full text of DOI:10.1016/j.bmcl.2010.03.097

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3158–3160
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Macrocyclic BACE inhibitors: Optimization of a micromolar hit
to nanomolar leads
Yifang Huang a, Eric D. Strobel a, Chih Y. Ho a, Charles H. Reynolds a, Kelly A. Conway a, Jennifer A. Piesvaux a,
Douglas E. Brenneman a, George J. Yohrling a, H. Moore Arnold a, Daniel Rosenthal a, Richard S. Alexander a,
Brett A. Tounge a, Marc Mercken b, Marc Vandermeeren b, Michael H. Parker a, Allen B. Reitz a,
Ellen W. Baxter a,
*
a Research and Early Development, Johnson & Johnson Pharmaceutical Research & Development, LLC, Welsh & McKean Roads, PO Box 0776, Spring House, PA 19477-0776, USA
b Research and Early Development, Johnson & Johnson Pharmaceutical Research & Development, LLC, Turnhoutseweg 30, B-2340 Beerse, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of
Alzheimer’s disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin con-
formation suggesting that constrained macrocyclic derivatives may also bind there. Several of the analogs
we prepared were >100ꢀ more potent than 1, such as 7 (5 nM Ki).
Received 8 December 2009
Revised 18 March 2010
Accepted 26 March 2010
Available online 30 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
BACE
BACE inhibitors
Alzheimer’s disease
Alzheimer’s disease (AD) is a chronic degenerative disorder of the
central nervous system resulting in severe cognitive deficits as well
as psychiatric symptoms.1 More than 20 million people suffer from
this condition worldwide, and, with the increasing number of el-
derly, the number of AD patients is expected to increase dramati-
cally. Current therapies are merely palliative and include the
cholinesterase inhibitors, donepezil, galanthamine, and rivastig-
mine, which are used to treat mild and moderate AD and the NMDA
antagonist, memantine, employed for moderate to severe AD. The
discovery of disease-modifying agents for AD is a desperately unmet
need.2 Agents which interfere with the processing of the amyloid
precursor protein (APP) are especially attractive targets. In particu-
lar, the aspartic protease, BACE-1 (b-site amyloid cleaving enzyme,
memapsin2, Asp2)3 catalyzes the initial proteolytic cleavage of
APP to generate the b-amyloid1–40(42) peptides which have a crucial
role in the pathogenesis of AD. The homozygous BACE knockout
mouse has significantly reduced levels of b-amyloid while maintain-
ing a normal phenotype4 although more recent studies may chal-
lenge this claim.5 In addition, BACE-1 gene deletion in the Tg2576
mouse, which overproduces b-amyloid, results in a reduction of
the cognitive deficit.6 BACE-1 inhibitors have been shown to lower
b-amyloid levels in the brains of mice4a,7 and thus show promise in
the treatment of AD.
A high throughput screen of our corporate compound collection
resulted in the identification of 2-amino-3,4-dihydroquinazoline 1
as a BACE-1 inhibitor with modest, but promising, activity (0.9 lM
Ki).8 Unlike previously reported peptide-like BACE-1 inhibitors,
such as OM99-2,9 which bind to the active site in an extended con-
formation, surprisingly, 1 adopted a tight hairpin shape (Fig. 1) by
X-ray crystallography.8 This structural information suggested that
the two ends of the hit compound could be connected to form a
macrocycle which might improve the potency of our series. Utiliz-
ing the structure of 1 as a template, a variety of prospective mac-
* Corresponding author. Tel.: +1 215 885 3648.
E-mail address: EllenBaxter@msn.com (E.W. Baxter).
Figure 1. High throughput screening hit 1 in the active site of BACE-1.8
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.097

Y. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3158–3160
3159
rocycles were evaluated computationally.10 For example, 2 fit the
model and was synthesized. Much to our delight, this analog had
a 0.06
lM Ki in the BACE-1 inhibition assay.
N
N
O
N
O
O
O
N
N
Me
NH2
NH2
N
2
1
The synthesis of 2 was initiated with 3-(3-hydroxyphenyl)pro-
pionic acid (3) (Fig. 2). Reaction of 3 with benzyl bromide
provided bis-benzylated material. Selective hydrolysis of the
benzyl ester provided the intermediate acid which was converted
to amide 4. The amide was reduced to the corresponding amine
which was then coupled to N-Boc-b-alanine followed by O-deben-
zylation to yield 5. Condensation of 5 with 5-fluoro-2-nitrobenz-
aldehyde with subsequent removal of the Boc group and
intramolecular reductive amination afforded macrocycle 6.
Reduction of the nitro group followed by condensation with cyan-
ogen bromide yielded target 2.
Figure 3. Left: docking of proposed macrocycle 7 in the BACE active site. (Maestro
Version 9, Schrodinger, L.L.C).
O
HO
NH
CN
c, d, e, f
a, b
+
BocHN
While macrocycle
2 had promising BACE inhibition, the
OBn
OBn
compound was only weakly active in a cellular assay measuring
Ab1–40 secretion in CHO cells transfected with the Swedish familial
9
10
8
AD mutant APP (K670N/M671L) with a 0.51
l
M IC50. X-ray crystal-
0
lographic studies of 1 revealed the vacant hydrophobic S1 pocket.
We reasoned that the introduction of a lipophilic substituent that
would fill this pocket might improve the potency of this chemical
series both against BACE and in cellular assays of Ab secretion.
Molecular modeling indicated that a cyclohexyl group would be
O
O
N
N
g, h, i
O
O
O
H
N
0
H2N
an optimal substituent, occupying the S1 pocket (Fig. 3).
NO2
N
NH2
To examine this hypothesis, compound 7 was synthesized
(Fig. 4). Reduction of the cyano group in 8 to the corresponding phen-
ethylamine followed by reductive alkylation with cyclohexanone
11
7
provided 9 whichwas coupledwithc
-aminoacid10(>99%ee).8 Sub-
Figure 4. Reagents and conditions: (a) BH3–THF, THF, reflux, 18 h (71%); (b)
cyclohexanone, NaBH(OAc)3, HOAc, THF, 18 h (92%); (c) 10, DIPEA, HBTU, DMF, 18 h
(100%); (d) H2 (50 psi), 10% Pd/C, EtOH, 6 h (99%); (e) 5-fluoro-2-nitrobenzaldehyde,
K2CO3, DMF, 50 °C, 18 h (97%); (f) TFA, CH2Cl2, 4 h (78%); (g) (i) CH2Cl2, 4A MS,
30 min, (ii) NaBH(OAc)3, 18 h (93%); (h) H2 (50 psi), 10% Pd/C, THF, EtOH, 6 h; (i)
BrCN, EtOH, reflux, 18 h (48%, two steps).
sequent O-debenzylation, condensation with 5-fluoro-2-nitrobenz-
aldehyde, and Boc-group removal provided late stage intermediate
11. Macrocyclization was achieved via an intramolecular reductive
amination. Reduction of the nitro group revealed the penultimate
bis-amine which was reacted with cyanogen bromide to afford
target 7. Macrocycle 7 was a potent BACE inhibitor with a 5 nM Ki
in the enzyme assay and a 17 nM IC50 in the cellular assay.
With this exciting result in hand, we embarked on an SAR study
of our series (Table 1). Expanding the ring size of 7 by one methy-
lene group as in compound 12 resulted in a sixfold loss in enzyme
inhibition. The (S)-enantiomer 12 was six times more potent than
(R)-enantiomer 13 in the BACE assay. Replacement of the cyclo-
hexyl substituent in 7 (Ki = 5 nM) with an iso-propyl (Ki = 8 nM)
as in 14 resulted in equivalent enzymatic activity, but a fivefold de-
crease in cellular activity, suggesting that increased lipophilicity of
the R1 substituent is critical for cell penetrance. Replacement of the
N-cyclohexyl group (compound 7) by N-(4-tetrahydropyranyl)
(compound 15) resulted in an over threefold reduction in both
enzymatic and cellular activity. Moreover, where R2 is 4-tetrahy-
dropyranyl, and R1 is cyclohexyl (compound 15) versus the case
where R1 is iso-propyl (compound 16), the BACE Kis are compara-
ble (17 nM and 22 nM), but the cellular IC50s are vastly different
(73 nM and 685 nM).
O
O
OH
N
H
a, b, c, d
e, f, g
OH
OBn
3
4
N
h, i, j
k, l
O
N
2
O
N
H
O
OH
NO2
BocHN
Introduction of
a 4-carboxycyclohexyl substituent as R2
5
6
resulted in a fourfold loss in enzyme activity and a 18-fold loss
in cellular potency for the cis analog 17 (Ki = 20 nM) while the
corresponding trans isomer 18 was inactive. Additionally, modifi-
cations of the core dihydroquinazoline were explored. Introduction
of a 7-fluoro (analog 19, Ki = 8 nM) or 7-methoxy substituent
(analog 20, Ki = 12 nM) resulted in a slight decrease in BACE activ-
ity, and a twofold reduction in cellular potency. Replacement of the
Figure 2. Reagents and conditions: (a) BnBr, K2CO3, MeCN, reflux, 18 h (100%); (b)
1 N NaOH, MeOH, THF, 3 h (96%); (c) (COCl)2, CH2Cl2, DMF, 4 h (100%); (d)
C6H11NH2, NEt3, CH2Cl2, 1 h (94%); (e) LiAlH4, THF, reflux, 24 h (72%); (f)
BocHNCH2CH2CO2H, 1-methylmorpholine, HBTU, DMF, 18 h (96%); (g) H2 (50 psi),
10% Pd/C, EtOH, 18 h (100%); (h) 5-fluoro-2-nitrobenzaldehyde, K2CO3, DMF, 50 °C,
18 h (68%); (i) TFA, CH2Cl2, 2 h (98%); (j) (i) CH2Cl2, 4A MS, 3 h, (ii) NaBH(OAc)3, 24 h
(78%); (k) SnCl2, EtOH, 18 h; (l) BrCN, EtOH, 24 h (23%, two steps).

3160
Y. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3158–3160
Table 1
Structure–activity relationships
R2
N
N
O
n
*
O
R1
N
X
NH2
Compd
Configuration
R1
R2
n
X
BACE Ki (nM)
Cellular Ab1–40 IC50 (nM)
7
12
13
14
15
16
17
18
19
20
21
(S)
(S)
(R)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
Cyclohexyl
Cyclohexyl
Cyclohexyl
iso-Propyl
Cyclohexyl
iso-Propyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
4-Tetrahydropyranyl
4-Tetrahydropyranyl
cis-4-Carboxycyclohexyl
trans-4-Carboxycyclohexyl
Cyclohexyl
1
2
2
1
1
1
1
1
1
1
1
CH
CH
CH
CH
CH
CH
CH
CH
CF
5
17
34
660
90
73
685
310
>1000
30
36
7
31
186
8
17
22
20
>1000
8
Cyclohexyl
Cyclohexyl
C(OMe)
N
12
5
Schwake, M.; D’Hooge, R.; Bach, P.; Kalinke, U.; Moechars, D.; Alzheimer, C.;
Reiss, K.; Saftig, P.; De Strooper, B. J. Biol. Chem. 2005, 280, 30797.
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Vassar, R.; Disterhoft, J. F. Neuron 2004, 41, 27.
7-carbon atom with nitrogen (analog 21) maintained BACE activity
(Ki = 5 nM), but improved cellular activity to an IC50 of 7 nM. A
number of these macrocyclic analogs were tested in vivo, and b-
amyloid lowering was detected in plasma.11 However, upon intra-
venous, intraperitoneal, or oral administration, only trace levels of
compound were detected in the brain, and no lowering of b-amy-
loid in the brain was observed. Preliminary experiments suggest
that P-glycoprotein efflux may prevent brain penetration of these
compounds. In conclusion, structural biology is a powerful tool in
the design of potent, non-peptide BACE inhibitors, but a continuing
challenge is the discovery of compounds which reduce b-amyloid
levels in the brain upon oral administration.
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Acknowledgments
We thank Chris Teleha, Scott Ballentine, Craig Diamond, and
Laura Reany for the large scale synthesis of several key inter-
mediates.
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7
was dosed orally in non-transgenic mice,
a
statistically significant effect on Ab lowering in plasma was observed:
23%, 22%, and 13%, at 50 mg/kg, 25 mg/kg, and 10 mg/kg dose,
respectively, in 10% solutol.