Discovery of cyclic sulfoxide hydroxyethylamines as potent and selective β-site APP-cleaving enzyme 1 (BACE1) inhibitors: Structure based design and in vivo reduction of amyloid β-peptides

Article abstract of DOI:10.1016/j.bmcl.2013.07.071

Previous structure based optimization in our laboratories led to the identification of a novel, high-affinity cyclic sulfone hydroxyethylamine- derived inhibitor such as 1 that lowers CNS-derived Aβ following oral administration to transgenic APP51/16 mice. Herein we report SAR development in the S3 and S2′ subsites of BACE1 for cyclic sulfoxide hydroxyethyl amine inhibitors, the synthetic approaches employed in this effort, and in vivo data for optimized compound such as 11d.

Full text of DOI:10.1016/j.bmcl.2013.07.071

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5300–5306
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of cyclic sulfoxide hydroxyethylamines as potent
and selective b-site APP-cleaving enzyme 1 (BACE1) inhibitors:
Structure based design and in vivo reduction of amyloid b-peptides
a
a
a
b,
Heinrich Rueeger a, , Rainer Lueoend , Rainer Machauer , Siem Jacob Veenstra , Laura Helen Jacobson
,
⇑
Matthias Staufenbiel b, Sandrine Desrayaud c, Jean-Michel Rondeau d, Henrik Möbitz a, Ulf Neumann b
a Department of Global Discovery Chemistry, Institutes for BioMedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
b Department of Neuroscience, Institutes for BioMedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
c Metabolism and Pharmacokinetics, Institutes for BioMedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
d Structural Biology Platform, Institutes for BioMedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous structure based optimization in our laboratories led to the identification of a novel, high-affinity
cyclic sulfone hydroxyethylamine-derived inhibitor such as 1 that lowers CNS-derived Ab following oral
administration to transgenic APP51/16 mice. Herein we report SAR development in the S3 and S20 sub-
sites of BACE1 for cyclic sulfoxide hydroxyethyl amine inhibitors, the synthetic approaches employed
in this effort, and in vivo data for optimized compound such as 11d.
Received 14 June 2013
Revised 27 July 2013
Accepted 30 July 2013
Available online 9 August 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
BACE-1
Inhibitor
Alzheimer’s disease
Structure based design
Cyclic sulfoxide hydroxyethylamine
inhibitors
In vivo reduction of amyloid b-peptides
Alzheimer’s disease (AD) is one of the most prevalent neurode-
generative disorders among the elderly worldwide. Considerable
affinity inhibitor which demonstrated Ab reduction in APP51/16
transgenic mice (Fig. 1).18 In this Letter, we report our efforts to
further optimize the cellular activity and thereby reduce CNS-de-
evidence has accumulated indicating
a central role of the
amyloid-b (Ab) peptide and specifically its aggregation in the path-
ogenesis of AD.1 The pathway generating Ab is initiated by BACE1,
a membrane-bound aspartic protease (EC 3.4.23.46), cleaving the
transmembrane protein b-amyloid precursor protein (APP).2–6
The main products generated are the secreted amino-terminal part
of APP (sAPPb) and the carboxy-terminal fragment C99, which is
rived Ab at a lower oral dose (<100 lmol/kg). We hypothesized
that the loss in potency observed between the enzymatic and
S3
CF3
O-
S+
CF3
H2N
O
O
further cleaved by c
-secretase leading to Ab.7,8 Knocking out the
O
S
BACE1 gene not only blocks the generation of Ab but also of C99
for which toxicity has been shown as well.9–14 Knock out of BACE1
leads to hypomyelinization during development but not to general
toxicity.15–17 The inhibition of BACE1, therefore, is an attractive
approach for the development of causal AD therapies.
P1 - P3
N
H
P2'
F
N
H
OH
OH
S1
S2'
18
1
We have previously described the design and synthesis of cyclic
sulfone HEA (cHEA) BACE1 inhibitors exemplified by 1, a high
pKa
BACE1 IC50
CHO APPwt cell IC 50
Papp in MDCK cells
MDCK efflux ratio (ER)
5.4
2 nM
55 nM
40 nm/s
4
⇑
Corresponding author. Tel.: +41 61 6963255; fax: +41 61 6962455.
E-mail address: heinrich.rueeger@novartis.com (H. Rueeger).
Current affiliation: The Florey Institute of Neuroscience and Mental Health,
University of Melbourne, Parkville 3010, Australia.
Figure 1. In vitro profile of sulfone cHEA inhibitor 1.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.071

H. Rueeger et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5300–5306
5301
cellular activity (up to 25-fold) in the sulfone containing cHEA
inhibitor series was due to the low pKa.18 Therefore, our strategy
for increasing the cellular activity was to raise the pKa from 5.4
to >6.5 by replacing the sulfone with a sulfoxide moiety in the
cHEA transition state mimetic (Fig. 1).19 Retaining the key H-bond
to Thr72 and Gln73 of the flap with an axially substituted sulfoxide
(Fig. 2) was expected to provide an equipotent cHEA mimetic with
potentially better pharmacokinetic properties due to the reduced
number of heteroatoms.
The synthesis of the sulfoxide cHEA inhibitors as shown in
Tables 1 and 2 is summarized in Scheme 1. The enantiopure inter-
mediate 2, previously synthesized in larger amounts for the prep-
aration of sulfone containing cHEA inhibitors,18 was initially
extended with the P20 fragment to allow the introduction of differ-
Figure 2. Cocrystal structure of 11a (green) bound to BACE1.
Table 1
Comparison of enzymatic and cellular activity of sulfone and sulfoxide cHEA inhibitors
CF3 CF3
CF3
O-
S+
O-
S+
CF3
H2N
CF3
H2N
CF3
H2N
O
O
O
O
O
S
F
N
H
F
N
H
F
N
H
OH
OH
OH
1
11a
12a
ERd
LogP
pKa
a
c
e
Compd
IC50
(l
M)
Potency
Ratiob
Papp (nm/s)
hBACE1
CHO-APPwt cells
hCatD
MDCK
1
11a
12a
0.002
0.002
0.088
0.055
0.014
0.330
0.45
0.87
>10
22ꢀ
7ꢀ
40
180
110
4
11
13
5.1
5.2
4.9
5.7
6.7
n.d.
4ꢀ
a
b
c
Values are means of at least three experiments.
The potency ratio measures the drop in potency observed in the cell-based assay relative to the enzyme-based assay.
Papp is the permeability through a MDR1-MDCK cell monolayer transfected with human MDR1.
ER is the efflux ratio (PBL-AP/PAP-BL) in MDCK cells transfected with human MDR1.
d
e
pKa measured with a Profiler SGA instrument using multi-wavelength UV spectroscopy and a linear 2–12 pH unit gradient.
Table 2
P3 SAR of sulfoxide cHEA inhibitors
O-
S+
R3
H2N
F
N
H
OH
a
c
Compd
R3
IC50
(
l
M)
Potency
Ratiob
Papp (nm/s)
ERd
LogP
IC50 (lM)
hBACE1
CHO-APPwt cells
hCatD
MDCK
CYP3A4
11b
11c
OCH3
OCH2CF3
0.033
0.008
0.026
0.062
1.24
1.20
1
8
220
190
12
16
3.5
4.3
0.3
0.3
CF3
11d
0.004
0.010
0.82
3
180
9
3.9
0.2
O
O
O
CF3
11f
20
0.007
0.530
0.021
1.34
2.45
1.78
3
3
160
90
86
27
3.7
0.9
OH
OH
n.d.
>10
a
b
c
Values are means of at least three experiments.
The potency ratio measures the drop in potency observed in the cell-based assay relative to the enzyme-based assay.
Papp is the permeability through a MDR1-MDCK cell monolayer transfected with human MDR1.
ER is the efflux ratio (PBL-AP/PAP-BL) in MDCK cells transfected with human MDR1.
d

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H. Rueeger et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5300–5306
(O)n
S
O
S
F
F
NO2
NO2
F
b, c
Boc
F
N
H
N
H
OH
OH
3
4
5
n=1, axial sulfoxide
n=1, equatorial sulfoxide
n=2
6, axial sulfoxide
7, equatorial sulfoxide
a
R1
R1
R1
O
O-
S+
O-
S+
F
O
O
NO2
F
NO2
F
NO2
F
S
NO2
F
S
e
d
+
Boc
N
Boc
Boc
Boc
N
H
N
H
N
H
H
OH
OH
OH
OH
2
8
axial sulfoxide
equatorial sulfoxide
9a-f
10a-f
q, e
i, c, k
h, i, c, e, g, f (11e)
b, c, f (11a-d)
b, c, f
O
R1
R1
O-
O-
S+
R3
O
O
S
CF3
O
O
S+
NO2
S
H2N
F
g
NO2
NO2
Boc
N
F
N
N
H
F
N
F
H
H
OH
OH
O
R
OH
O
13, axial sulfoxide
14, equatorial sulfoxide
3 = F
= OH
11a R1 = CH(CF3)2
12a-d
19a
19b
l
m
3
R1 = CH3
R
11b
11c
11d
11e
19c R3 = OMOM
R1 = CH2CF3
R
R
1 = CH(CF3)CH2OCH3
1 = CH(CF3)CH2OBn
n-p, g
g
j
O
O
HO
O
O-
S+
O-
S+
O-
CF3
H2N
CF3
R2
CF3
H2N
O
O
OH
O
S+
S
r, f
s, f (17e)
H2N
F
R4
R3
R5
F
NH2
OH
F
N
N
H
N
H
H
OH
OH
OH
15 R2 = NO2, axial sulfoxide
16
20
11f
17a-n
(Table 3)
R2 = NO2, equatorial sulfoxide
(metabolite)
f
R2 = NH2, axial sulfoxide (metabolite)
18
Scheme 1. Reagents and conditions: (a) potassium peroxymonosulfate, THF/H2O (2:1), 25 °C; (b) TFA, CH2Cl2,1 h, 25 °C, 99%; (c) (1) 3-tert-butyl-benzaldehyde, NaOAc,
CH2Cl2/MeOH (2:1), 2 h, 25 °C, (2) NaBH3CN, MeOH, 1 h, 25 °C, 95%; (d) R-OH, KHMDS, THF, 24 h, 25 °C, 90–95%; (e) H2O2, AcOH/THF (1:3), 6 h, 25 °C, 96% or H2O2, 0.02 equiv
HAuCl4, MeOH, 5 h, 25 °C, 97%; (f) H2, 10% Pd–C, MeOH, 4–6 h, 45 °C, 85–90%; (g) 3 N HCl in MeOH, 24 h, 25 °C, 95%; (h) (R)-3-benzyloxy-1,1,1-trifluoro-propan-2-ol, KOtBu,
THF, 0 °C, 82%; (i) 4 N HCl in dioxane, 25 °C, 99%; (j) BCl3, CH2Cl2, ꢁ78 °C, 83%; (k) carbonyldiimidazole, NEt3, cat. DMAP, acetonitrile, 2 h, 25 °C, 95%; (l) NaH, CH3SO2(CH2)2OH,
DMF, 1.5 h, 25 °C, 95%; (m) MOM-Cl, i-Pr2NEt, CH2Cl2, 0.5 h, 96%; (n) NaIO4, THF/H2O (2:1), 6 days, 25 °C, 74%; (o) H2, 10% Pd–C, MeOH, 2.5 h, 45 °C, 92%; (p) KOTMS, THF, 1 h,
70 °C, 84%; (q) (R)-1,1,1-trifluoro-3-methoxypropan-2-ol, KOH, THF, 24 h, 40 °C, 92%; (r) (1) R-CHO, NaOAc, CH2Cl2/MeOH (2:1), 2 h, 25 °C, (2) NaBH3CN, MeOH, 1 h, 25 °C,
80–95%; (s) (1) 1-(3-(tert-butyl)phenyl)ethanone, 3-methyl-1-butanol, 1 day reflux, (2) NaBH3CN, MeOH, 1 h, 25 °C, 34%.
ent P3 fragments late in the synthesis. A nearly chemo selective
oxidation of 2 to a mixture of sulfoxides 3 and 4 was achieved by
the slow addition of 1 equiv of potassium peroxomonosulfate in
THF–H2O at 0 °C to avoid over-oxidation to the sulfone 5 (<5%).
N-Boc cleavage with trifluoroacetic acid (TFA) and subsequent
reductive amination with 3-tert-butyl-benzaldehyde provided
sulfoxides 6 and 7. The 1:1 mixture of axial and equatorial sulfox-
ide isomers could not be separated by column chromatography at
any stage. On the other hand, preceding P3 extension of 2 followed
by the chemoselective oxidation of thioether 8 with H2O2 in AcOH–
THF provided a 1:1 mixture of sulfoxide isomers 9 and 10 which
could be separated by column chromatography. Various attempts
to increase the amount of the axial sulfoxide isomer by changing
the oxidant or using metal catalysis20 were not very successful.
Only the HAuCl4 catalyzed oxidation with H2O221 in MeOH slightly
favored the formation of the axial isomer (2:1 ratio). Removal of
the N-Boc protecting group with TFA and prime side extension
via reductive amination followed by catalytic reduction of the nitro
group provided the axial and equatorial sulfoxide inhibitors 11a–d
and 12a–d, respectively. The configurational assignment was
confirmed by co-crystallization of 11a (Fig. 2) and 12a with BACE1.
Later, we discovered during N-Boc deprotection of 14 with 3 N HCl
in MeOH, that the equatorial isomer could be equilibrated into the
thermodynamically more stable axial isomer22 providing a 9:1
equilibrium mixture of 15 and 16 after 24 h stirring at ambient
temperature. An even higher ratio of 20:1 to 11a–d was obtained
by equilibration of the final sulfoxide isomer 12a–d. Reductive
amination of 16 with different P3-aldehyde and ketone fragments

H. Rueeger et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5300–5306
5303
followed by catalytic reduction of the nitro group gave access to
17a–n. The deuterated analogs 17b and 17d were prepared from
the corresponding deuterated aldehyde and reduction of the inter-
mediate imine with NaBD3CN. The phenolic metabolite 20 was
synthesized from 2 by intermediate protection of the amino-alco-
hol moiety as the oxazolone 19a after prime side extension to
allow the introduction of the phenolic moiety under strongly basic
conditions with 2-(methyl sulfonyl) ethanol. For the oxidation of
the thioether to the sulfoxide the phenol group in 19b was first
protected by a MOM-ether (19c). Reduction of the nitro group,
cleavage of the oxazolone moiety with KOTMS and subsequent
sulfoxide equilibration with 3 N HCl in MeOH with concomitant
MOM-ether cleavage afforded the metabolite 20.
the CF3 groups provided the less lipophilic compound 11c (LogP
4.3) with much higher plasma and brain levels in APP51/16 trans-
genic mice (Table 4), however, with a fourfold lower potency in the
enzymatic assay (IC50 = 8 nM). A possible explanation for the lower
cellular activity of 11c (IC50 = 62 nM, eightfold potency ratio) com-
pared to the methoxy compound 11b (IC50 = 26 nM) could be the
higher lipophilicity (LogP 4.3 vs 3.5). The substitution of one of
the CF3 group of 11a by a methoxymethyl ether provided the more
polar compound 11d (LogP 3.9) with more potent cellular activity
(IC50 = 10 nM), a lower potency ratio (<threefold), good permeabil-
ity (Papp 180 nm/s) and a slightly lower ER of 9. The more polar
alcohol 11f (LogP 3.7) exhibited a twofold lower potency in both
assays, however, the presence of an additional H-bond donor re-
sulted in a drastic increase in the ER (9 ? 86). Overall, the insertion
of more polar P3 fragments had only a minor effect on the unde-
sired CYP3A4 interaction.
A comparison of the enzymatic potency and the cellular activity
of the initial hexafluoroisopropoxy substituted 4-amino-fluoro-
benzyl sulfone cHEA inhibitor
1 with the sulfoxide analogs
(11a and 12a, Table 1) demonstrated that the axial sulfoxide 11a
was as potent and selective over cathepsin D as the sulfone cHEA
inhibitor. The equatorial analog 12a, on the other hand, which is
not able to form a H-bond interaction with the flap residue
Thr-72 (Fig. 2), showed a 40-fold lower activity. As expected the
sulfoxide cHEA containing inhibitor 11a displayed a higher cellular
activity compared to the sulfone cHEA analog 1 (7-fold vs 22-fold
potency ratio), which can be explained by the higher pKa (6.7 vs
5.7). Equally important was the increase in permeability to a Papp
of 180 nm/s, being in the range commonly observed for central
nervous system drugs (>150 nm/s),23 albeit at the expense of an
increased P-gp-efflux ratio (ER 4 ? 11) in the MDR1-MDCK cell
monolayer assay. The pharmacokinetic evaluation of 11a in mice
indicated significant brain penetration. However, only a modest
bioavailability (14%) was observed due to high clearance
(129 mL/min/kg) and high first-pass metabolism most likely facil-
itated by the high lipophilicity (LogP 5.2).
Previous SAR exploration of the S3 pocket in thesulfone cHEA
series18 revealed the important binding contributions of the CF3
group by H-bonding as well as by nonbonding protein–fluorine
interactions.24 The same interactions could be observed in the
co-crystal structure of the sulfoxide cHEA inhibitor 11d in BACE1,
the H-bonding to Thr232 (3.2 Å) and a deeply buried water
(3.1 Å) and the beneficial short orthogonal C–F contact to the
amide of Gln12 (2.8 Å) (Fig. 3). The two CF3 groups in the P3
fragment of 11a were initially introduced to completely abolish
the formation of the phenolic metabolite 20. Removal of one of
In vitro metabolite identification studies of 11d using liver
microsomes suggested that oxidation at the N-benzylic tethered
prime side fragment was the main site of metabolism as observed
in other HEA inhibitors.25 The different modifications of the prime-
side fragment to improve the metabolic stability are summarized
in Table 3. Since di-alkylation of the benzylic position was prohib-
ited due to steric interference with the cHEA scaffold, the deuter-
ated analogs 17b and 17d were prepared to gain some metabolic
stability from the deuterium isotope effect.26 Subsequent meta-
bolic stability assessment in mouse and human liver microsomes
showed only a minor improvement, indicating a ‘masked’ meta-
bolic reaction in which the formation of the active oxygenating
species had occurred prior to the isotopically sensitive C–H bond
cleavage.27 A few small steric modifications in the vicinity of the
benzylic position were tolerated without considerable loss of po-
tency, like the (R)-phenethylamine analog 17e and the ortho–flu-
oro substituted analog 17g, however, having no positive effect on
the microsomal stability. Subsequently, we focused our attempts
to improve the metabolic stability by reducing the lipophilicity of
the P20 fragment. As shown with several representative examples,
the incorporation of polar functionalities (17h–k) or more polar
heterocyclic ring systems (17l–n) led to a reduction of the CYP3A4
interaction and to an improvement of the metabolic stability in hu-
man (HLM) as well as mouse (MLM) liver microsomes. Unfortu-
nately, this beneficial effect was associated with an at least
threefold lower enzymatic and cellular activity and a higher ER
(17–41). In comparison to the initial 3-tert-butyl-benzyl P20 frag-
ment of 11d, no equipotent prime side fragment with improved
in vitro metabolic stability could be identified.
In general, all compounds from this series displayed high sys-
temic blood clearance. To assess the effect of BACE1 inhibition on
brain Ab40 levels, compounds with the highest cellular activity
(IC50 <50 nM), good permeation properties and a moderate ER
(<15–20)28 were tested in male or female human wild-type APP
transgenic mice (APP51/16).14,18,29 After oral administration expo-
sure in blood and brain, as well as Ab40 forebrain concentrations
were measured at 4 h post-dose.
The results of these experiments are shown in Table 4. The only
moderate Ab40 reduction of 11a despite high brain exposure can be
explained with the very low unbound brain concentration
(fu <0.01) probably induced by the lipophilic OCH(CF3)2 P3 residue,
which was introduced as a means to completely block the forma-
tion of the phenolic metabolite 20.30 The expected over-propor-
tional increase of blood levels at higher oral doses, caused by
saturation of the first-pass clearance, could be demonstrated with
11c and 11d in male mice. The most efficacious inhibitor 11d low-
ered brain Ab40 levels by 62% at the highest dose of 180
lmol/kg
and 39% at 100 mol/kg (p <0.0001 vs vehicle treated group,
l
2-tailed Student’s t-test). A slightly lower Ab40 reduction (38%)
Figure 3. Cocrystal structure of 11d (green) bound to BACE1.
was achieved with 11c, despite higher blood and brain exposure,

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H. Rueeger et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5300–5306
Table 3
P20 SAR of sulfoxide cHEA inhibitors
CF3
O-
S+
O
O
H2N
F
N R2'
H
OH
R2
H
IC50
(
l
M)
Papp (nm/s)
ERc
LogP
IC50
(
l
M)
HLM/MLMd
190/180
(lL/min/mg)
a
b
0
Compd
hBACE1
150
CHO-APPwt cells
hCatD
MDCK
CYP3A4
>10
18
17a
>10
O
11d
17b
0.004
0.010
0.012
0.82
1.20
180
190
9
3.9
n.d.
3.7
3.7
0.2
0.1
500/480
600/530
D
D
D
0.004
10
17c
17d
17e
0.018
0.016
0.010
0.044
0.031
0.032
4.03
3.29
0.75
200
n.d.
170
17
n.d.
9
0.7
n.d.
0.1
690/550
600/660
600/730
D
(R)
17g
0.010
0.038
0.29
240
9
4.1
3.4
0.1
400/580
F
OH
17h
17i
0.012
0.013
0.134
0.126
3.39
7.90
100
80
17
25
1.0
2.4
80/210
40/150
OH
O
17j
0.080
0.212
0.075
0.382
0.78
6.83
210
310
26
17
3.6
3.7
0.9
4.7
280/190
300/180
O
F
F
17k
F
17l
0.021
0.019
0.226
0.124
0.764e
0.422
9.68
3.57
>10
100
190
190
24
41
26
2.9
3.1
3.5
3.6
1.1
1.6
140/380
580/510
140/400
N
N
O
17m
17n
N
N
a
b
c
Values are means of at least three experiments.
Papp is the permeability through a MDR1-MDCK cell monolayer transfected with human MDR1.
ER is the efflux ratio (PBL-AP/PAP-BL) in MDCK cells transfected with human MDR1.
d
Compounds were incubated with human (HML) or mouse (MLM) microsomes at 37 °C. The in vitro metabolic clearance rate was derived from data collected at four time
points (0, 5, 15 and 30 min) in a reaction including cofactor(s) (NADPH and/or UDPGA).
e
The low cellular activity of 17m can be explained by the low pKa of 4.9.
which can be explained with the threefold lower cellular activity. A
significantly higher exposure and stronger pharmacodynamic re-
sponse could not be observed with the deuterated N-benzyl ana-
logs 17b and 17d in comparison to the non-deuterated analogs
11d and 17c, respectively. However, reduced levels of the amine
metabolite 18 could be observed. For 17b 37% versus 120% of

H. Rueeger et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5300–5306
5305
Table 4
PK-PD of 3-alkoxy substituted benzyl sulfoxide cHEA inhibitors
Compd
Dose (
l
mol/kg)
Gender29
Concd blooda (nmol/mL)
% of parent in blood
Concd braind (nmol/g)
Rat brain fu
Brain Ab40 reductione
(%), 4 h pdf
18b
20c
11a
11c
180
180
100
60
Male
Male
Male
Male
Male
Male
Male
Female
Female
Male
Female
Female
Female
0.9
6.1
2.6
0.8
3.8
1.0
0.3
5.2
2.1
0.4
1.6
1.5
5.4
n.d.
25
40
94
38
77
120
4
<1
4
4
0.8
1.3
0.7
0.4
0.7
0.2
0.1
0.8
0.5
0.1
0.2
0.3
0.7
<0.01
0.02
32g
38g 27g 18g
10
25
18
23
<1
<1
10
n.d.
n.d.
n.d.
11d
180
100
60
0.02
62g 39g 23h 60g 39g
60j
20j
5
17b
17c
17d
17e
60
37
22
12
10
0.02
0.04
22i
31g
28i
34g
100
100
100
0.02
a
Concentrations in blood (nmol/mL =
cremophor.
lM) obtained at 4 h after po application to APP51 transgenic mice. The oral formulation was a suspension in water containing 2%
b
Percent of amine metabolite 18 versus parent inhibitor formed in blood after 4 h.
Percent of phenol metabolite 20 versus parent inhibitor formed in blood after 4 h.
Concentrations in brain (nmol/g) obtained at 4 h after po application to APP51 transgenic mice.
% mean reduction compared to the mean of the vehicle control.
pd: post dose.
p 60.0003.
p = 0.02.
c
d
e
f
g
h
i
p 60.003.
j
Coadministration of Ritonavir (12.5 mg/kg) orally applied 0.5 h before administration of inhibitor.
parent 11d at the dose of 60
of parent 17c at a dose of 100
deuterium effect. The higher brain concentration of 17e (0.7 nmol/
g) achieved with the 100 mol/kg dose compared to 11d
(0.2 nmol/g), both having the same brain fu, did not translate into
a higher Ab40 reduction (34% and 31%, respectively) at the 4 h time
point.31
Having successfully demonstrated that potent sulfoxide cHEA
inhibitors can significantly lower brain Ab40 levels, but fell short
on achieving sufficient systemic drug exposure at lower doses,
we investigated the pharmacokinetic boosting with sub-thera-
peutic doses of Ritonavir, to reduce the high clearance caused
by CYP3A4 metabolism. The co-administration of the strong
CYP3A4 inhibitor Ritonavir, also known as ‘pharmaco enhance-
ment’, has been successfully employed in most marketed HIV
inhibitors to reduce drug load.32 Co-administration of Ritonavir
(12.5 mg/kg po, 0.5 h prior to drug application) with the most
efficacious inhibitor 11d produced the same Ab40 reduction after
l
l
mol/kg and for 17d 12% versus 22%
mol/kg (Table 4), indicated a partial
pharmacodynamic studies in different species will be the subject
of future communication.
l
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
07.071.
References and notes
1. Citron, M. Nat. Rev. Drug Disc. 2010, 9, 387.
2. Hussain, I.; Powell, D.; Howlett, D. R.; Tew, D. G.; Meek, T. D.; Chapman, C.;
Gloger, I. S.; Murphy, K. E.; Southan, C. D.; Ryan, D. M.; Smith, T. S.; Simmons, D.
L.; Walsh, F. S.; Dingwall, C.; Christie, G. I. Mol. Cell. Neurosci. 1999, 14, 419.
3. Lin, X.; Koelsch, G.; Wu, S.; Downs, D.; Dashti, A.; Tang, J. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 1456.
4. Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Caccavello, R.; Davis, D.; Doan,
M.; Dovey, H. F.; Frigon, N.; Hong, J.; Jacobson-Croak, K.; Jewett, N.; Keim, P.;
Knops, J.; Lieberburg, I.; Power, M.; Tan, H.; Tatsuno, G.; Tung, J.; Schenk, D.;
Seubert, P.; Suomensaari, S. M.; Wang, S.; Walker, D.; Zhao, J.; McConlogue, L.;
John, V. Nature 1999, 402, 537.
5. Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E. A.; Denis, P.;
Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.;
Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J.
C.; Collins, F.; Treanor, J.; Rogers, G.; Citron, M. Science 1999, 286, 735.
6. Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M. C.; Pauley, A. M.;
Brashier, J. R.; Stratman, N. C.; Mathews, W. R.; Buhl, A. E.; Carter, D. B.;
Tomasselli, A. G.; Parodi, L. A.; Heinrikson, R. L.; Gurney, M. E. Nature 1999, 402,
533.
7. Duff, K.; Eckman, C.; Zehr, C.; Yu, X.; Prada, C. M.; Perez-tur, J.; Hutton, M.;
Buee, L.; Harigaya, Y.; Yager, D.; Morgan, D.; Gordon, M. N.; Holcomb, L.; Refolo,
L.; Zenk, B.; Hardy, J.; Younkin, S. Nature 1996, 383, 710.
8. Scheuner, D.; Eckman, C.; Jensen, M.; Song, X.; Citron, M.; Suzuki, N.; Bird, T. D.;
Hardy, J.; Hutton, M.; Kukull, W.; Larson, E.; Levy-Lahad, E.; Viitanen, M.;
Peskind, E.; Poorkaj, P.; Schellenberg, G.; Tanzi, R.; Wasco, W.; Lannfelt, L.;
Selkoe, D.; Younkin, S. Nat. Med. 1996, 2, 864.
9. Oster-Granite, M. L.; McPhie, D. L.; Greenan, J.; Neve, R. L. J. Neurosci. 1996, 16,
6732.
10. Cai, H.; Wang, Y.; McCarthy, D.; Wen, H.; Borchelt, D. R.; Price, D. L.; Wong, P. C.
Nat. Neurosci. 2001, 4, 233.
4 h (39%, p <0.0001) at the fivefold lower oral dose of 20 lmol/
kg in APP51/16 transgenic mice. This considerable boosting ef-
fect was further substantiated by the >twofold higher plasma
(2.1 nmol/mL) and brain levels (0.5 nmol/g) in comparison to
the standard dose of 100 lmol/kg. In addition, the formation of
the main amine metabolite 18 as well as the phenolic metabo-
lite 20 were dramatically reduced (65%, respectively, <1% of
parent).
In conclusions, the replacement of the sulfone cHEA scaffold by
the slightly more basic sulfoxide cHEA scaffold led to highly potent,
selective inhibitors with improved cellular activity (11d, IC50
10 nM). Sufficient CNS exposure could be achieved with a single
oral dose of 60 lmol/kg to effect significant reduction in brain Ab
in APP51/16 transgenic mice. The high CYP3A4 mediated metabolic
clearance inherent to this class of cHEA inhibitors could not be
reduced to an acceptable level by chemical modifications. With
the co-administration of the strong CYP3A4 inhibitor Ritonavir this
liability could be minimized leading to a substantial exposure
enhancement and subsequent brain Ab40 reduction at an accept-
11. Luo, Y.; Bolon, B.; Kahn, S.; Bennett, B. D.; Babu-Khan, S.; Denis, P.; Fan, W.;
Kha, H.; Zhang, J.; Gong, Y.; Martin, L.; Louis, J. C.; Yan, Q.; Richards, W. G.;
Citron, M.; Vassar, R. Nat. Neurosci. 2001, 4, 231.
12. Roberds, S. L.; Anderson, J.; Basi, G.; Bienkowski, M. J.; Branstetter, D. G.; Chen,
K. S.; Freedman, S. B.; Frigon, N. L.; Games, D.; Hu, K.; Johnson-Wood, K.;
able oral dose of 20 lmol/kg. Detailed pharmacokinetic and

5306
H. Rueeger et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5300–5306
Kappenman, K. E.; Kawabe, T. T.; Kola, I.; Kuehn, R.; Lee, M.; Liu, W.; Motter, R.;
21. Yuan, Y.; Bian, Y. Tetrahedron Lett. 2007, 48, 8518.
Nichols, N. F.; Power, M.; Robertson, D. W.; Schenk, D.; Schoor, M.; Shopp, G.
M.; Shuck, M. E.; Sinha, S.; Svensson, K. A.; Tatsuno, G.; Tintrup, H.; Wijsman, J.;
Wright, S.; McConlogue, L. Hum. Mol. Genet. 2001, 10, 1317.
22. (a) Johnson, C. R.; McCants, D. J. Am. Chem. Soc. 1964, 86, 2935; (b) Johnson, C.
R.; McCants, D. J. Am. Chem. Soc. 1965, 87, 1109.
23. Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L.
J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W. J. Pharmacol. Exp. Ther. 2002,
303, 1029.
13. Dominguez, D.; Tournoy, J.; Hartmann, D.; Huth, T.; Cryns, K.; Deforce, S.;
Serneels, L.; Camacho, I. E.; Marjaux, E.; Craessaerts, K.; Roebroek, A. J.;
Schwake, M.; D’Hooge, R.; Bach, P.; Kalinke, U.; Moechars, D.; Alzheimer, C.;
Reiss, K.; de Saftig, P.; Strooper, B. J. Biol. Chem. 2005, 280, 30797.
14. Rabe, S.; Reichwald, J.; de Ammaturo, D.; Strooper, B.; Saftig, P.; Neumann, U.;
Staufenbiel, M. J. Neurochem. 2011, 119, 231.
24. Dalvit, C.; Vulpetti, A. ChemMedChem 2011, 6, 104.
25. Charrier, N.; Clarke, B.; Cutler, L.; Demont, E.; Dingwall, C.; Dunsdon, R.;
Hawkins, J.; Howes, C.; Hubbard, J.; Hussain, I.; Maile, G.; Matico, R.; Mosley, J.;
Naylor, A.; O’Brien, A.; Redshaw, S.; Rowland, P.; Soleil, V.; Smith, K. J.;
Sweitzer, S.; Theobald, P.; Vesey, D.; Walter, D. S.; Wayne, G. Bioorg. Med. Chem.
Lett. 2009, 19, 3664.
15. Hu, X.; Hicks, C. W.; He, W.; Wong, P.; Macklin, W. B.; Trapp, B. D.; Yan, R. Nat.
Neurosci. 2006, 9, 1520.
16. Sankaranarayanan, S.; Price, E. A.; Wu, G.; Crouthamel, M. C.; Shi, X. P.;
Tugusheva, K.; Tyler, K. X.; Kahana, J.; Ellis, J.; Jin, L.; Steele, T.; Stachel, S.;
Coburn, C.; Simon, A. J. J. Pharmacol. Exp. Ther. 2008, 324, 957.
17. Willem, M.; Garratt, A. N.; Novak, B.; Citron, M.; Kaufmann, S.; Rittger, A.;
DeStrooper, B.; Saftig, P.; Birchmeier, C.; Haass, C. Science 2006, 314, 664.
18. Rueeger, H.; Lueoend, R.; Rogel, R.; Rondeau, J.-M.; Möbitz, H.; Machauer, R.;
Jacobson, L.; Staufenbiel, M.; Desrayaud, S.; Neumann, U. J. Med. Chem. 2012,
55, 3364.
26. Tung, R. Innov. Pharm. Technol. 2010, 32, 24.
27. (a) Fisher, M. B.; Henne, K. R.; Boer, J. Curr. Opin. Drug Discovery Dev. 2006, 9,
101; (b) Nelson, S. D.; Trager, W. F. 2003, Drug Metab. Dispos. 31, 1481.
28. In our MDR1-MDCK cell monolayer assay, an ER of 10–20 was considered
moderate and acceptable, since substantial brain exposure was observed with
good permeating compounds (Papp >180 nm/s) and an ER of 15–20.
29. (a) Gandhi, M.; Aweeka, F.; Greenblatt, R. M.; Blaschke, T. F. Annu. Rev.
Pharmacol. Toxicol. 2004, 44, 499; (b) Martignoni, M.; Groothuis, G. MM.; de
Kanter, R. Expert Opin. Drug Discovery 2006, 2, 875.
19. An estimate of the pKa shift from a b-substituted sulfonyl amine to a sulfoxyl
amines cannot be given since the data of corresponding analogs have not been
documented. Calculation with Advanced Chemistry Development (ACD/Labs)
30. The AMES test of the phenol 20 did not show any evidence of a mutagenic
potential.
Software V11.02 (Ó 1994–2012 ACD/Labs) predict
b-substituted sulfoxyl amine analogs.
a
higher pKa for the
31. The observed exposure differences could also be explained by the gender
difference of the individual animal groups.
20. (a) Zhao, H.; Samuel, O.; Kagan, H. B. Tetrahedron 1987, 43, 5135; (b) Legros, J.;
Bolm, C. Chem. Eur. J. 2005, 11, 1086; (c) Yamaguchi, T.; Matsumoto, K.; Saito,
B.; Katsuki, T. Angew. Chem., Int. Ed. 2007, 46, 4729; (d) O’Mahony, G. E.; Ford,
A.; Maguire, A. R. J. Org. Chem. 2012, 77, 3288.
32. (a) Moyle, G. L.; Back, D. HIV Med. 2001, 2, 105; (b) Boffito, M.; Maitland, D.;
Samarasinghe, Y.; Pozniak, A. Combinations Curr. Opin. Infect. Dis. 2005, 18, 1.