BACE1 inhibitors: A head group scan on a series of amides

Article abstract of DOI:10.1016/j.bmcl.2013.05.003

A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basicity as well as their properties as substrates

Full text of DOI:10.1016/j.bmcl.2013.05.003

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4239–4243
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Bioorganic & Medicinal Chemistry Letters
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BACE1 inhibitors: A head group scan on a series of amides
a
a
a
a
Thomas J. Woltering a, , Wolfgang Wostl , Hans Hilpert , Mark Rogers-Evans , Emmanuel Pinard ,
Alexander Mayweg a, Martin Göbel a, David W. Banner a, Jörg Benz a, Massimiliano Travagli b,
Martina Pollastrini b, Guido Marconi b, , Emanuele Gabellieri b, Wolfgang Guba a, Harald Mauser a,
Matteo Andreini b, Helmut Jacobsen a, Eoin Power b,à, Robert Narquizian a
⇑
a F. Hoffmann-La Roche Ltd., Pharma Research, Grenzacherstr. 124, CH-4070 Basel, Switzerland
b Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, I-53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with
respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basi-
city as well as their properties as substrates of P-glycoprotein revealed that a 2-amino-1,3-oxazine head
group would be a suitable starting point for further development of brain penetrating compounds for
potential Alzheimer’s disease treatment.
Received 22 March 2013
Revised 29 April 2013
Accepted 1 May 2013
Available online 18 May 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
BACE1
Alzheimer’s disease
Inhibitors
pKa
P-gp
The b-site amyloid precursor protein cleaving enzyme
(BACE1) plays a major role in the processing of the amyloid precur-
sor protein (APP). Sequential cleavage of APP by BACE1 and
1
We believed that altering the amidine head group to improve
physicochemical properties, such as pKa, logD, solubility and per-
meability, together with an extension reaching deep into the S3
pocket would result in highly potent and drug-like BACE1 inhibi-
tors.1 The comparison with other BACE1 X-ray structures, which
had been soaked with inhibitors containing S1 and S3 substituents,
had shown that the orientation of the amidine-containing head
groups and the 3D shapes of the binding cavities were quite similar.
Thus, 4bek was considered as a suitable template for interactive 3D
modeling7 in order to explore various options for optimizing the
affinity of the thiazine headgroup. Modeling also revealed that
the S3 pocket could be best reached by meta-substitution on the
S1 phenyl ring. In order to displace bound water molecules in the
S3 pocket, the S1 phenyl ring was meta substituted via an amide
linker (Fig. 2: modeled compound in green). This enables the addi-
tional formation of a hydrogen bond to the backbone carbonyl of
G291 indicated by a dotted line. X-ray analysis of selected com-
pounds out of the series of amides 2–25 revealed that the NH of
the amide bond indeed forms a hydrogen bond to the backbone
carbonyl oxygen of G291 and also leads to a conformationally favor-
able fixation of the two aromatic rings in an almost planar arrange-
ment. For the meta-substitution on the phenyl we used an aromatic
amide and for the aromatic residue in S3 it was found that espe-
cially 2-pyridyl carboxylic acid derivatives are most preferred.5
The 2-pyridyl moiety ensures a planar orientation at the amide lin-
ker mediated by a favorable electrostatic interaction between the
c
-secretase leads to the formation of 40 or 42 amino acid-long
amyloid b-peptides (Ab) which aggregate in the brain of
Alzheimer’s disease (AD) patients. It is suggested that inhibition
of BACE1 would prevent the build-up of Ab and therefore slow
down or stop AD.1–3
A high throughput screen (HTS) revealed the thiazine fragment
1 as a hit (BACE1 enzyme assay: IC50 = 41.2
appealing ligand efficiency (LE) of 0.37 (LE = (
G = ꢁRTlnIC50 and N the number of non-hydrogen atoms) and
l
M; pKa ꢀ 9) with an
D
G)/N, where
D
co-crystallization with BACE1 showed ample opportunities for
optimization. This aminothiazine heterocycle was initially synthe-
sized at Roche4 in the late 1970’s and many examples of this com-
pound class exhibit analgesic activity. It was recently explored by
Shionogi and other research groups as a head group (HG) targeting
the active site of BACE1.5,6 X-ray analysis of 1 co-crystallized with
BACE1 shows that both nitrogens of the protonated amidine moi-
ety form tight hydrogen bonds to the catalytic aspartates D289
and D93 (Fig. 2).
⇑
Corresponding author. Tel.: +41 61 688 0407; fax: +41 61 688 8714.
E-mail address: thomas.woltering@roche.com (T.J. Woltering).
Current address: Autifony S.r.l., Via Fleming 4, 37135 Verona, Italy.
Current address: Warner Chilcott, Xerox Technology Park, Dundalk, Co. Louth,
à
Ireland.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.003

4240
T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4239–4243
amide NH and the pyridyl N. As depicted inFigure 1 the substitution
at the pyridyl residue was found by many research groups to be
Benzoannelation of the HG as in amidine 5 also produced a
moderately active compound, but with much higher basicity
though (pKa = 9.7).
0
0
most favorable in positions marked R3 and R5 where small substit-
0
uents in R3 (e.g., Me and Cl) were preferred.5,12–20 The deep S3
Enlarging the ring size from the aminohydantoin 4 by one CH2
0
channel suggests a para substitution of the S3 pyridyl ring (R5 ) with
group to the 2-aminodihydropyrimidinone 813 resulted in a very
halogens or small linear substituents such as cyano. To evaluate the
active compound (BACE1 enzyme IC50 = 0.015
lM, cellular assay
0
various effects of the different head groups the R5 substituent was
IC50 = 0.001 M), but the basic compound (pKa = 8.4) was also
l
kept to Cl in this study (exception: compound 3; Table 1). The
BACE1 inhibitory activities of the compounds were determined in
a human BACE1 enzyme as well as in a cell-based assay. The pKa
was examined by capillary electrophoresis (cEpKa); exceedingly ba-
sic compounds often suffer from undesired potential liabilities of
phospholipidosis8 and polypharmacology.9,10 The compounds’
properties as a substrate of human P-glycoprotein (P-gp) expressed
as the efflux ratio (ER) were determined. Further in vivo character-
ization of selected compounds for the AD indication was performed
by measuring the reduction of Ab40 in the brain 4 h after adminis-
tration of 30 mg/kg p.o. in our wild type mice model.11 For some
compounds the plasma and brain exposure was also determined
at this time point.
found to be a very good P-gp substrate (hER 18.6) which led to
in vivo inactivity (<10% reduction of Ab40 at 30 mg/kg p.o.; expo-
sure 4 h after 50 mg/kg p.o.: plasma: 2506 426 ng/ml, brain:
586 164 ng/g, brain/plasma ratio: 0.19). Removing the carbonyl
group gave the almost equally potent cyclic guanidine 9, but its ba-
sicity even exceeded the measurable range (pKa >11) and it was
due to very low exposure also inactive in vivo (<15% reduction of
Ab40 at 30 mg/kg p.o.; exposure 4 h after 30 mg/kg p.o.: plasma:
<3 ng/ml, brain: <13 ng/g).
The aminopiperazinone 1014 was much less active than its po-
sition isomer 8 whereas removal of the carbonyl group from HG
in 10 led to the corresponding aminopiperidine 1115 with im-
proved in vitro activity but it is highly basic (pKa = 10.2), a good
P-gp substrate (hER 9.6) and therefore was not tested in vivo.
Enlarging the ring size from the 2-aminooxazoline 2 by one CH2
group to the 2-amino-1,3-oxazine 1216,17 resulted in moderate
The 2-aminooxazoline HG in compound 2 showed a decent
activity (IC50 = 2.02 lM); the corresponding thiazoline 3 was less
0
active also because of the missing Cl in R5 and therefore lacking
the deep penetration into the S3 pocket. Annelation by ring closure
of the methyl group to the central phenyl ring (6 and 7) did not re-
sult in an improvement of activities (both pKa = 8.3).
activity in the enzyme assay (IC50 = 0.137
lM) but high activity
in the cellular assay (IC50 = 0.010 M). Surprisingly, the quite basic
l
compound (pKa = 9.8) showed at least marginal activity of 29%
reduction of Ab40 at 30 mg/kg p.o. despite being a very good P-
gp substrate (hER 10.7). The same experiment in mice revealed a
weak brain penetration with high variability (exposure 4 h after
30 mg/kg p.o.: plasma: 2702 3987 ng/ml, brain: 363 494 ng/g,
brain/plasma ratio 0.13). Although 12 and 13 have quite similar
permeability (PAMPA assay: 12: peff = 6.12; 13: peff = 6.77) and sol-
ubility (LYSA assay: 12: 360 mg/l; 13: 370 mg/l) the more lipo-
The less basic aminohydantoin HG in compound 4 (pKa = 7.3)
has been described as a part of a potent BACE1 inhibitor12 but
when decorated as in 4, it proved to be only moderately active.
H2N
S
H2N
R5'
philic 2-amino-1,3-oxazine 13 (logD: 13: 0.68; 12: 0.26) bearing
0
N
H
N
an extra chlorine substituent in position R3 was found to be even
N
HG
R
N
more potent in vitro (BACE1 enzyme IC50 = 0.039
lM, cellular assay
R3'
IC50 = 0.001 M) and also in vivo (38% reduction of Ab40 at 30 mg/
l
O
MeO
kg p.o.; exposure 4 h after 30 mg/kg p.o.: plasma: 190 54 ng/ml,
brain: 306 44 ng/g, brain/plasma ratio: 0.99) – again despite
being a very good P-gp substrate (hER 14.5). A single dose PK in
mice for compound 13 (9.76 mg/kg p.o.) showed the following pro-
1
compounds 2-25
BACE1, enzyme
IC50 = 41.2 µM
file: tmax = 0.5 h, Cmax = 1835 ng/ml, t = 1.32 h, AUC = 4582 h ng/
½
Figure 1. HTS hit 1 and general structure of compounds described in this study.
ml, F = 111%, brain/plasma ratio 0.14. Determination of the protein
binding in mice revealed an 11% fraction of unbound compound.
This quite promising profile was accompanied by undesired activ-
ities, like, example, cytochrome P450 2D6 isoenzyme inhibition
(IC50 = 0.9
lM) and hERG inhibition (IC20 = 0.9 lM); both effects
were largely attributed to the high basicity of 13.
Another way of enlarging the ring size of the 2-aminooxazoline
2 by one CH2 group led to 3-amino-1,4-oxazine 1418 which was
S1
highly active in vitro (BACE1 enzyme IC50 = 0.031
lM, cellular as-
say IC50 = 0.003 M) but its extremely good P-gp substrate proper-
l
ties (hER 37.2) together with low exposure led to in vivo inactivity
(<10% reduction of Ab40 at 30 mg/kg p.o.; exposure 4 h after
30 mg/kg p.o.: plasma: 106 41 ng/ml, brain: 51 4 ng/g, brain/
D93
plasma ratio: 0.76). Addition of an extra chlorine substituent in
0
R3 gave compound 15, but no improvement of neither in vitro
S3
nor in vivo activity was observed.
A variation of the thiazine fragment by annelation of a cyclopro-
pyl ring led to compound 16 which was not a P-gp substrate (hER
1.1), but only showed moderate in vitro activity.
G291
We also prepared plain 6-ring amidines, but with attached
cyclopropyl groups to hopefully lower the pKa, which resulted in
spiro-compound 17 and bicycle 18 (both pKa >11). Both com-
pounds showed good in vitro activity but neither of them displayed
any in vivo effect (17: <5% reduction of Ab40 at 30 mg/kg p.o.;
Figure 2. HTS hit 1 (yellow) bound to the BACE1 binding site, some flap residues
have been removed for the sake of clarity (PDB code: 4bek). The protonated amidine
motif in the thiazine head group forms tight hydrogen bonds to the catalytic
aspartates D289 (not shown) and D93 (modeled amide compound in green).

T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4239–4243
4241
Table 1
In vitro data of amides 2–25 described in this study
0
0
d
Compounds
R
R3
R5
Head group(HG)
IC50
hBACE1 enzyme
(l
M)a
Ab40 cell-basedb
P-gp
hERc
pKa
H2N
O
N
2
3
4
5
H
H
H
H
H
H
H
H
Cl
H
2.02
8.98
1.02
0.55
0.2
Nt
Nt
Nt
Nt
8.3
8.3
7.3
9.7
H2N
S
N
1.72
0.34
0.21
H2N
N
N
Cl
Cl
O
H2N
N
H2N
O
S
N
6
7
CH2(to Me group)
CH2(to Me group)
H
H
Cl
Cl
10.78
4.00
1.3
Nt
Nt
8.3
8.3
H2N
N
0.38
H2N
N
O
8
9
H
F
H
H
Cl
Cl
0.015
0.012
0.001
0.012
18.6
Nt
8.4
N
H2N
H2N
H2N
H2N
H2N
N
>11
N
N
N
N
N
N
N
N
N
N
10
11
12
13
14
15
16
H
H
F
H
H
H
Cl
H
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
0.348
0.032
0.137
0.039
0.031
0.045
0.76
1.04
Nt
Nt
O
0.037
0.010
0.001
0.003
0.004
0.14
9.6
10.2
9.8
9.7
9.2
Nt
O
O
10.7
14.5
37.2
Nt
F
H2
N
N
O
O
F
H2
F
H2N
H2N
S
H
1.1
Nt
H
H
17
18
F
F
H
H
Cl
Cl
0.031
0.096
0.015
0.031
Nt
>11
>11
N
H
H2N
14.8
N
(continued on next page)

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T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4239–4243
Table 1 (continued)
0
0
d
Compounds
R
F
R3
R5
Cl
Head group(HG)
IC50
hBACE1 enzyme
(l
M)a
Ab40 cell-basedb
P-gp
hERc
pKa
H2N
N
19
H
0.061
0.025
21.2e
10.8
H2N
20
21
F
F
H
H
Cl
Cl
0.12
0.067
0.011
1.8
Nt
7.3
N
O
O
O
H2
N
N
0.014
10.4
N
H2
22
23
24
F
F
F
H
H
H
Cl
Cl
Cl
0.023
0.046
0.37
0.001
0.011
0.073
13.2
10.9
>10
Nt
N
N
H2N
H2N
O
5.9
O
16.6 (R = H)e
N
N
H
H
S
H2N
25
H
H
Cl
0.027
0.002
5.5e
8.9
0
0
R, R3 and R5 refer to the positions of R’s in Figure 1. Nt, not tested.
a
IC50 values are means of at least two independent experiments.
HEK293 cells transfected with wild-type human APP.
ER = efflux ratio in LLCPK1 cells stably expressing human MDR1.
Measured by capillary electrophoresis (pKa Analyzer ProTM by Advanced Analytical).
b
c
d
e
Racemic compound tested.
exposure 4 h after 30 mg/kg p.o.: plasma: 81 78 ng/ml, brain:
2 ng/g, brain/plasma ratio: 0.1. 18: <20% reduction of Ab40 at
in vitro highly active compound (BACE1 enzyme IC50 = 0.027
lM,
8
cellular assay IC50 = 0.002 M) but it also increased its P-gp sub-
l
30 mg/kg p.o.; exposure 4 h after 30 mg/kg p.o.: plasma:
48 69 ng/ml, brain: 32 9 ng/g, brain/plasma ratio: 0.67).
Modifying the benzoannelated amidine 5 by ring enlargement
with one CH2 group gave compound 1919 which exhibited high ba-
sicity (pKa = 10.8) and due to its P-gp substrate properties (hER
21.2) was not tested in our mice model. Exchanging the newly
introduced CH2 group by oxygen as in compound 2019 lowered
the pKa to the more desirable value of 7.3, almost retained the
in vitro activity and finally reduced the P-gp substrate abilities
(hER 1.8), but compound 20 was despite its good exposure still
inactive in the in vivo model (<20% reduction of Ab40 at 30 mg/
kg p.o.; exposure 4 h after 30 mg/kg p.o.: plasma: 629 70 ng/ml,
brain: 1569 89 ng/g, brain/plasma ratio: 2.49). When trying to
isolate the pure enantiomers of 20 after separation on chiral HPLC
we discovered a quick racemization due to the chemical instability
of the aminal group which unfortunately made this head group
unattractive for deeper exploration.
strate abilities (hER 5.5) (<10% reduction of Ab40 at 30 mg/kg
p.o.; exposure 4 h after 30 mg/kg p.o.: plasma: <9 ng/ml, brain:
<26 ng/g).
In summary: exceedingly basic compounds like 9, 17 and 18 re-
sulted in very low exposure and in vivo inactivity. Together with
the high exposure of the less basic, P-gp free and more lipophilic
20 (logD 2.51) this demonstrates the importance of a lower pKa
to achieve a more CNS-drug like profile. The 2-amino-1,3-oxazines
12 and 1321 show despite their basicity and P-gp substrate abilities
because of compensation through high and long-lasting exposure
some in vivo activity. They appear also from their chemical struc-
ture of the HG to be good starting points for further modifications
and concomitant favorable profile changes (physicochemical prop-
erties, P-gp and especially pKa) and therefore are positioned well
for further drug development. Efforts in this direction were re-
warded with the development of BACE1 inhibitors with high
in vivo efficacy suitable for clinical evaluation in AD.22
Further enlargement of the promising 2-amino-1,3-oxazine 12
to a 7-membered 2-aminooxazepine gave the in vitro potent com-
Acknowledgments
pound 21 (BACE1 enzyme IC50 = 0.140
lM, cellular assay
IC50 = 0.011 M) but it also unfavorably increased the pKa from
l
We cordially thank R. Wermuth, Ch. Schnider, R. Humm, H.-P.
Marty, R. Halm, D. Rombach, F. Ricklin, D. Krummenacher and
B. Wagner for their skillful technical assistance.
9.8 to 10.4. Shifting the oxygen in more remote positions from
the amidine moiety as in 2218b,20 and 2320a also gave in vitro highly
potent compounds, but did not result in a decrease of the basicity.
Again annelation of a cyclopropyl group to oxazepine 22 gave
the derivative 2420a which substantially lost in vitro activity and
still was a good P-gp substrate.
Supplementary data
Supplementary data (supplementary material with experimen-
tal details for the preparation of compounds 12, 13, 16, 17 and 18
Enlarging the ring size of the annelated thiazine 16 by one CH2
group to the corresponding thiazepine 25 (pKa = 8.9) resulted in an

T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4239–4243
4243
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Med. Chem. 2013. http://dx.doi.org/10.1021/jm400225m. Published online.