Discovery of amino-1,4-oxazines as potent BACE-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.05.003

New amino-1,4-oxazine derived BACE-1 inhibitors were explored and various synthetic routes developed. The binding mode of the inhibitors was elucidated by co-crystallization of 4 with BACE-1 and X-ray analysis. Subsequent optimization led to inhibitors with low double digit nanomolar activity in a biochemical and single digit nanomolar potency in a cellular assays. To assess the inhibitors for their permeation properties and potential to cross the blood-brain-barrier a MDR1-MDCK cell model was successfully applied. Compound 8a confirmed the in vitro results by dose-dependently reducing Aβ levels in mice in an acute treatment regimen.

Full text of DOI:10.1016/j.bmcl.2018.05.003

Accepted Manuscript
Discovery of amino-1,4-oxazines as potent BACE-1 inhibitors
Siem Jakob Veenstra, Heinrich Rueeger, Markus Voegtle, Rainer Lueoend,
Philipp Holzer, Konstanze Hurth, Marina Tintelnot-Blomley, Mathias
Frederiksen, Jean-Michel Rondeau, Laura Jacobson, Matthias Staufenbiel, Ulf
Neumann, Rainer Machauer
PII:
S0960-894X(18)30393-7
https://doi.org/10.1016/j.bmcl.2018.05.003
BMCL 25819
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 February 2018
27 April 2018
2 May 2018
Please cite this article as: Veenstra, S.J., Rueeger, H., Voegtle, M., Lueoend, R., Holzer, P., Hurth, K., Tintelnot-
Blomley, M., Frederiksen, M., Rondeau, J-M., Jacobson, L., Staufenbiel, M., Neumann, U., Machauer, R., Discovery
of amino-1,4-oxazines as potent BACE-1 inhibitors, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.05.003
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Discovery of amino-1,4-oxazines as potent
BACE inhibitors
Leave this area blank for abstract info.
Siem Jakob Veenstra a, Heinrich Rueeger a, Markus Voegtle a, Rainer Lueoend a, Philipp Holzer a, Konstanze
Hurth a, Marina Tintelnot-Blomley a, Mathias Frederiksen a, Jean-Michel Rondeau b, Laura Jacobson c, Matthias
Staufenbiel c, Ulf Neumann c and Rainer Machauer a,*

Bioorganic & Medicinal Chemistry Letters
Discovery of amino-1,4-oxazines as potent BACE-1 inhibitors
Siem Jakob Veenstra a, Heinrich Rueeger a, Markus Voegtle a, Rainer Lueoend a, Philipp Holzer a,
Konstanze Hurth a, Marina Tintelnot-Blomleya, Mathias Frederiksen a, Jean-Michel Rondeau b, Laura
Jacobson c, Matthias Staufenbiel c, Ulf Neumann c and Rainer Machauer a,*
a Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
b Center for Proteomic Chemistry, Structural Biology Platform, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
c Department of Neuroscience, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
New amino-1,4-oxazine derived BACE-1 inhibitors were explored and various synthetic routes
developed. The binding mode of the inhibitors was elucidated by co-crystallization of 4 with
BACE-1 and X-ray analysis. Subsequent optimization led to inhibitors with low double digit
nanomolar activity in a biochemical and single digit nanomolar potency in a cellular assays. To
assess the inhibitors for their permeation properties and potential to cross the blood-brain-barrier
a MDR1-MDCK cell model was successfully applied. Compound 8a confirmed the in vitro
results by dose-dependently reducing Aβ levels in mice in an acute treatment regimen.
Keywords:
Alzheimer’s disease
BACE-1
2016 Elsevier Ltd. All rights reserved.
amino-1,4-oxazines
pKa
P-gp
BACE-1 (Beta-site Amyloid precursor protein Cleaving
Enzyme) is a prime target for the treatment of Alzheimer’s
disease (AD) since it catalyzes the first step of the APP (amyloid
precursor protein) processing.1 The amyloid-β (A) peptides
resulting from the APP processing by BACE-1 and gamma-
secretase are thought to trigger downstream events ultimately
resulting in neurodegeneration and the development of AD
following the widely accepted amyloid hypothesis.2-4 However,
Phase III trials with the BACE-1 inhibitor verubecestat have
recently failed in AD patients at early and at mild-to-moderate
disease states, which are known to have fully established
amyloid-β pathology. This is widely interpreted as a strong hint
that anti-amyloid-β treatment has to start early, possibly at pre-
symptomatic stages of the disease. In line with this, clinical
studies at pre-symptomatic AD stages have been initiated.5
catalytic aspartic acid residues of the protease. Those inhibitors
often only need to fill the S1 and S3 sub-pockets of the protease
(nomenclature according to Schechter and Berger19, the parts of
the inhibitor filling S1 and S3 are designated as P1 and P3,
respectively), leading to potent inhibitors in the drug-like
molecular weight range (<500 Da). In contrast, peptidomimetic
inhibitors usually need to occupy multiple S and/or S’ pockets to
achieve nanomolar potency.
In 2008, when we started our work on this class of inhibitors,
a few early examples had emerged from fragment screening
activities 20 like 6- and 5-membered acyl-guanidines 1 and 2 21, 22
and amino-1,3-thiazines 3 23
.
From our work on macrocyclic peptidomimetic BACE-1
inhibitors carrying hydroxyethylene or ethanolamine transition
6,
7
state mimetics (TSM)
inhibitors
and on cyclic sulfoxide / sulfone
8,
9
we concluded that for drug-like, brain penetrant
Figure 1. Examples of non-peptidic BACE-1 inhibitors.
BACE-1 inhibitors it will be crucial to limit the number of H-
bond donors and acceptors to an absolute minimum and, at the
same time, to adjust the pKa of the moiety interacting with the
catalytic aspartic acids of BACE-1 around the physiological pH,
between 6 and 8.6
Our own efforts led to the discovery of a series of amino-1,4-
oxazine inhibitors exemplified by 4 (Figure 2). When we did a
broad substructure search in the literature on this structural class
we were surprised to see that the core structure A (Figure 2, with
A = any atom, no ring annelated to the cyclic amidine) yielded
10-18
Recently a number of groups
have described BACE-1
only two
5-(dimethylamino)-3,6-dihydropyrazin-2(1H)-one
inhibitors that employ a 2-aminoheterocycle/cyclic amidine,
which engages in hydrogen bonding interactions with the
examples published in 1995.24 Similarly the corresponding

lactam precursor B (Figure 2) was barely known. Two literature
references described them as intermediates for aldosterone
synthase inhibitors.25, 26
fluorines next to the basic amidine functionality without
significantly altering the core structure. Our first activities
therefore focused on the fluorination pattern at the quaternary
methyl group (table 1).
Figure 2. First example of new amino-1,4-oxazine inhibitors,
corresponding Markush structure and potential precursor.
For the first examples of this new compound class we
envisioned synthetic routes via suitable lactams B, whereas for
later examples no lactam intermediates were isolated. Since all
routes required at some point a chiral amino-alcohol intermediate
C, the first challenge was always to establish high yielding,
highly stereoselective approaches to those amino-alcohols.
Classical amino-acid chemistry or more recent Ellman
methodologies 27 proved to be very versatile. The amino-alcohols
were then further transformed to intermediates which were
cyclized under a broad variety of conditions, depending on the
substitution pattern of the final amino-1,4-oxazine core D
(Scheme 1).28, 29
Figure 3. Crystal structure of BACE-1 in complex with 4, showing key
H-bonded interactions.
Scheme 1. General overview/key intermediates of selected approaches to amino-1,4-oxazine cores D.
The profiling of the first examples showed that this series of 1,4-
As shown in previous studies at Novartis, the pKa of the
moiety interacting with the catalytic aspartic acids of BACE-1
(Asp32 and Asp228) is of pivotal importance for cellular potency
and brain penetration.6 We also anticipated a relationship
between pKa and efflux.8, 9, 30 Therefore it was important to have
a flexible synthetic strategy allowing fine-tuning of the pKa of the
basic amidine unit.
oxazines indeed is capable of inhibiting the BACE-1 enzyme in
the low micromolar (9) to double-digit nanomolar (8b) activity
range. The more active enantiomers have the (R) configuration.
The introduction of one or two fluoro substituents at the
quaternary methyl group gave potent inhibitors with
incrementally lowered pKa by approximately one unit for every
fluorine introduced. Three fluoro substituents at the quaternary
methyl (7) led to considerable loss of potency, the CF3 group
presumably being too bulky, in view of the close contacts to
Tyr71 and Asp32 mentioned above. In cellular assays all
compounds showed nanomolar activity for the inhibition of Aβ
release. Introduction of fluorine substituents to the P1 phenyl ring
led to measurable, albeit smaller, reduction of the pKa.
Introducing an additional fluorine in the 4-position of the P1
phenyl (8a/b), pointing toward Tyr71, reduced the pKa from 7.1
(6) to 6.8 and the efflux ratio from 7 to 4-5, at the same time
improving the potency by roughly a factor of 2 (Table 1). For
these reasons, we decided to keep this fluorine in all later
inhibitors. A second fluorine on the central aromatic core resulted
in a more complex picture. The 4,6-difluoro substitution (9) led
to considerable loss of potency. Presumably, a repulsive
interaction between the 6-F substituent and the amide carbonyl
affected the conformation of the latter, thereby weakening the H-
bonded interaction to the main-chain carbonyl of Gly230 (Figure
3 and 4).
The first compound prepared (4) was reasonably potent on
BACE-1 with an IC50 of 70 nM; it had a pKa of 9.5 and exhibited
strong P-glycoprotein (P-gp)-mediated efflux, as measured in
MDR1-transfected MDCK cells.31 The binding mode of the
compound to BACE-1 was elucidated by co-crystallization of
racemic 4 with BACE-1 and X-ray analysis at 1.54 Å resolution
(Figure 3).32 The exocyclic nitrogen interacted with both catalytic
aspartic acids and the endocyclic nitrogen interacted with Asp32.
The P1/P3 amide NH formed a hydrogen bond to the carbonyl
oxygen of Gly230. The oxazine ring oxygen and amide carbonyl
were not involved in any direct H-bonded contacts. The
quaternary methyl group was in close contact to Tyr71 (3.5Å)
and Asp32 (3.5Å) while the P1 phenyl ring made hydrophobic
interactions to Leu30, Tyr71, Phe108, Trp115 and Ile118.
To adjust the pKa to the desired range we investigated the
incremental effects of fluorine substituents in proximity of the
amidine.33 We looked for an opportunity to introduce one to three

Table 1. Basic structure-activity / pKa exploration of P1 phenyl and quaternary methyl of amino-1,4-oxazine
Compounds
R
R’
Fn
BACE-1
IC50 [nM]
70
cellular
measured
pKa
9.5
MDR1-MDCK cells
IC50 [nM]
flux [10-6cm/s] efflux ratio
4
CH3
5-Br
5-Br
5-Br
5-Cl
5-Br
-
17
4
19
19
15
33
24
16
15
25
29
30
28
7
5
CH2F
CHF2
CF3
-
170
8.2
6
-
38
15
324
8
7.1
7*
8a*
8b
9*
10*
11
-
352
6.2
1
CHF2
4-F
56
6.8
5
14
3
6.8
4
CHF2
CHF2
CHF2
5-Br
4,6-di-F
2,4-di-F
4,5-di-F
1119
701
300
69
9
6.5
2
5-Br
7.6
2
3-Me, 5-CN
20
6.6
2
* racemate [For synthetic reasons some of the compounds were prepared as racemates].
The 2,4-substitution (10) also led to a loss of potency, albeit to
a lesser extent, presumably due to a direct repulsive interaction
between the 2-F substituent and Gly230 of BACE-1 (Figure 4).
This substitution pattern led to a highly congested structure with
a pKa of 7.6, approximately 1 unit higher than anticipated.
We also wanted to investigate the effect of steric bulk on the
efflux properties of this series of inhibitors. We hypothesized that
steric shielding of the polar amidine functionality could reduce
susceptibility to P-gp recognition and thereby lower the efflux.
The effect of the introduction of various alkyl substituents next to
the amidine moiety are shown in Table 2. A single methyl group
slightly reduced (12a) or maintained (12b) potency, depending
on the stereochemistry. Removal of the second stereo center by
switching to the more bulky dimethyl substitution (13) led to
weakly reduced potency, but overall the methyl substitutions had
little influence on the pKa or permeation properties. An attempt
to slightly reduce the steric bulk by connecting the alkyl
substituents led to the spiro-substituted cyclopropyl derivative
14, which showed better potency but had surprisingly high
efflux. This effect might be partially explained by the polar
cyano-substituent on the P3 moiety which appears to trigger
additional P-gp efflux in other compounds too (see also 8a/b
versus 8c). When the ring size was increased to a 6-membered
spiro cycle (15), the potency dropped only modestly versus the
non-substituted 8b. Despite the slight decrease in pKa we
observed an increase in the cellular efflux for the tetrahydro-
pyran 15, which might be due to improved recognition by the
efflux pump through a polar interaction with the oxygen atom.
We then prepared acetals to reduce the risk of increased
interaction/recognition by P-gp, also hoping to achieve, at the
same time, a lowering of the pKa by the oxygen atom located in
β-position to the amidine. Comparing the monocyclic acetals 16a
and 16b, the (3RS,6RS)-stereoisomer 16b is 10-fold more active
than the (3RS,6SR)-stereoisomer 16a. The bicyclic acetal 17 is
less active (about 3-fold) than 16b. The major draw-back of these
surprisingly stable acetals is their higher pKa of 7.6 (16b) and 8.2
(17), which translated to efflux ratios >10.
Figure 4. Fluorination patterns and amide conformations/interactions.
In contrast, the 4,5-difluoro derivative (11) was devoid of such
intra- or inter-molecular repulsive interactions and was a potent
BACE-1 inhibitor with its pKa in the expected range (pKa 6.6).
All compounds showed stronger potency in the cellular Aβ
release assay in comparison to the biochemical enzyme inhibition
assay. This ratio was not substantially influenced by the changes
in pKa. More striking was the effect of the pKa on the efflux
observed in the MDR1-MDCK cellular transport assay. All
compounds showed good to very good flux, and a clear decrease
in the P-gp-mediated efflux when going from the most basic
amidines 4 and 5 to the less basic 6 and 7. Based on our previous
experience with other chemical series of BACE-1 inhibitors, the
MDR1-MDCK efflux ratio is a good predictor of the in vivo
brain permeation. Therefore, 1,4-oxazine inhibitors with MDR1-
MDCK efflux ratios ≤ 5 appeared attractive to us.
To double-check whether this correlation would also hold true
in the case of this new series of BACE-1 inhibitors, compound 8a
was tested in the APP51/16 mouse model34, using two oral doses
and the 4 h time point. Indeed, brain/blood ratios of 1.3 and 1.4
and reductions of forebrain A of 20% and 63% were observed at
the lower (30 µmol/kg) and higher oral dose (100 µmol/kg),
respectively. Since the in vivo potency of this early example was
already in the range of the previously reported NB-216,6 it
strongly supported further exploration of this series.
To further explore the correlation between brain permeation,
the pKa of the TSM and the P-gp-driven efflux we investigated
whether steric shielding and the pKa lowering effect of fluorine
could be combined. The transfer of the fluorines from the
quaternary methyl-group of 8a-c to the 6,6-geminal di-methyl
group gave compound 18. To avoid the creation of a second
stereo center one fluorine was introduced at each of the two
methyl groups. By this, both fluorines are placed again β to the

Table 2. In vitro data of transition-state-mimic (TSM) exploration
Compounds
TSM
X
R
BACE-1
cellular
measured
pKa
MDR1-MDCK cells
IC50 [nM]
IC50 [nM]
flux [10-6cm/s] efflux ratio
F
F
5-Br
14
27
3
7
6.8
6.7
16
17
4
8b
8c
3-Me, 5-CN-
11
F
F
5-Br
5-Br
92
38
11
5
7.3
7.1
13
31
3
2
12a*
[rac. trans]
12b*
[rac. cis]
13
14
F
F
5-Br
87
25
26
4
7.0
7.4
16
15
5
3-Me, 5-CN
15
15
F
F
3-Me, 5-CN
3-Me, 5-CN
28
11
27
6.5
7.8
14
17
20
12
16a*
207
16b*
17
F
F
F
3-Me, 5-CN
3-Me, 5-CN
3-Me, 5-CN
16
53
24
4
25
3
7.6
8.2
7.3
14
7
21
18
15
18
15
19
20
F
F
3-Me, 5-CN
3-Me, 5-CN
5-Br
12
76
13
663
1161
32
6.4
n.d.
7.3
7.3
24
34
5
4
3
2
2
21a
21b
H
H
1077
20
5-Br
10
* racemate; n.d. = not determined.

basic amidine functionality. Compound 18 nicely maintained the
enzymatic and cellular potency but the pKa increased by half a
unit compared to 8a-c. In line with the higher pKa, the efflux in
the cellular MDR1-MDCK assay increased. To counter-balance
this effect, and in order to avoid a second stereo center, we
introduced an additional fluorine back into the quaternary methyl
group resulting in a significantly lower pKa of 6.4 for compound
19. As a result, the efflux ratio was reduced to the desired level.
When a second fluorine was added to the quaternary methyl
group a significant drop in activity in the enzymatic and cellular
BACE assays was observed (20). While there was no compound
available for pKa measurements, the previously observed
incremental pKa reduction (Table 1) predicts a pKa of 20
significantly below 6.0. Possibly, this amidine was not
protonated enough for the interaction with the catalytic aspartates
of the enzyme. In contrast to the disappointing potency, the
permeation properties showed some improvement. Overall, the
introduction of three fluorines seemed optimal, we therefore
decided to explore another arrangement, concentrating all three
fluorines in one methyl of the geminal di-methyl group. Since
this design generates a new stereocenter both epimers were
explored. The (6S)-epimer 21a showed a significant drop in
enzymatic and cellular BACE-1 inhibition compared to 13, with
IC50s greater than 1 M in both assays. Its pKa of 7.3 was closer
to that of 13 rather than 19, and minimal efflux was observed in
the MDR1-MDCK assay. The higher pKa is noteworthy,
considering the missing fluorine in P1 (expected effect: 0.3
units). The same pKa and similarly low efflux was also observed
for the (6R)-epimer 21b. The enzymatic and cellular activities of
21b were in the same range as the other most potent examples of
the amino-1,4-oxazine BACE-1 inhibitors described herein.
Trifluoromethyl analogues of 21a/b lacking the second geminal
methyl group were also prepared but were stereochemically
unstable and were therefore not further pursued.
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Overall the amino-1,4-oxazines proved to be a highly
interesting chemotype for BACE-1 inhibition. This scaffold lends
itself to fine-tuning of the pKa, enabling the optimization of brain
permeation, an essential pre-requisite for a potential CNS drug
that needs to be applied over a long treatment period. The
presented SAR suggests that the desired pKa window for highly
brain penetrant inhibitors is even more narrow between 6.5 and
7.5. Due to their good biochemical and cellular potency, good
permeation in the MDR1-MDCK assay and little P-gp efflux
examples 8b, 12b, 19 and 21b are particularly attractive chemical
starting points for further optimization. The optimization of the
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selectivity over other aspartyl proteases, metabolic stability, etc.
will be subject of future communications.
12.
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We thank Kerstin Barker, Markus Furegati, Constanze
Hartwieg, Michael Hediger, Sebastien Jacquier, Philipp
Koeninger, Julia Kohlstedt, Thomas Ruppen, Heiner Schütz, Urs
Stauss, Martin Vogt, Jiri Zadrobilek, Thomas Zimmermann for
synthesis support, Tanja Dittmar, Vera Trappe, Irena Brzak,
Ludovic Perrot, Ulrich Furrer for in vitro and in vivo studies,
Karin Beltz and Rita Ramos for compound formulation support,
Sylvie Lehmann and Emmanuelle Bourgier for X-ray support,
and Rene Hemmig for the preparation of refolded BACE1. We
are grateful to the machine and beamline groups at the Paul
Scherrer Institute, Villigen, Switzerland, for the X-ray data
collection of the BACE1 complex with compound 4.

16.
17.
Hilpert, H J.; Guba, W.; Woltering, T. J.; Wostl, W.;
Pinard, E.; Mauser, H.; Mayweg, A. V.; Rogers-Evans, M.;
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Highlights:
The structure activity relationship of new amino-1,4-
oxazine derived BACE-1 inhibitors is elucidated
In vivo activity in a mouse model is demonstrated
already with an early example
The pKa range of 6.5 to 7.5 is identified as optimal fo
this class of compounds
Several starting for further optimization are presented