Initial optimization of a new series of γ-secretase modulators derived from a triterpene glycoside

Article abstract of DOI:10.1021/ml300256p

The discovery of a new series of γ-secretase modulators is disclosed. Starting from a triterpene glycoside γ-secretase modulator that gave a very low brain-to-plasma ratio, initial SAR and optimization involved replacement of a pendant sugar with a series of morpholines. This modification led to two compounds with significantly improved central nervous system (CNS) exposure.

Full text of DOI:10.1021/ml300256p

Subscriber access provided by University of Sussex Library
Letter
The Initial Optimization of a New Series of Gamma-
Secretase Modulators Derived from a Triterpene Glycoside
Nathan O Fuller, Jed L Hubbs, Wesley F Austin, Steffen P Creaser, Timothy D. McKee, Robyn
M.B. Loureiro, Barbara Tate, Weiming Xia, Jeffrey L Ives, Mark A Findeis, and Brian S Bronk
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml300256p • Publication Date (Web): 29 Aug 2012
Downloaded from http://pubs.acs.org on September 2, 2012
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Medicinal Chemistry Letters is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
The initial optimization of a new series of gamma-secretase
modulators derived from a triterpene glycoside.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nathan O. Fuller*, Jed L. Hubbs, Wesley F. Austin, Steffen P. Creaser,† Timothy D. McKee, Robyn
M.B. Loureiro, Barbara Tate, Weiming Xia,† Jeffrey L. Ives, Mark A. Findeis,† and Brian S. Bronk
Satori Pharmaceuticals, Inc., 281 Albany Street, Cambridge, MA 02139, USA.
KEYWORDS: Alzheimer’s disease, amyloid beta, gamma-secretase, modulator, black cohosh.
Supporting Information Placeholder
ABSTRACT: The discovery of a new series of gamma-secretase modulators is disclosed. Starting from a triterpene glycoside
gamma-secretase modulator that gave a very low brain-to-plasma ratio, initial SAR and optimization involved replacement of a
pendant sugar with a series of morpholines. This modification led to two compounds with significantly improved central nervous
system (CNS) exposure.
Alzheimer’s disease (AD) is a progressive neurodegenera-
tive disorder that is the leading form of dementia and affects
an estimated 5.4 million Americans1 and 35.6 million people
worldwide.2 AD is the sixth leading cause of death in the
United States, and with the aging baby boomer generation and
increasing life expectancy, the prevalence and mortality rates
associated with this disease are projected to rise dramatically.
Healthcare costs associated with AD are already costing the
United States government close to $200 billion annually, and
recent estimates project that by 2050 the numbers of individu-
als afflicted with AD will increase to between 11 and 16 mil-
lion in the United States1 and 115.4 million people world-
wide.2 These cumulative social and economic factors make
this one of the major healthcare crises facing the world today.
Despite significant research efforts, there are currently no
treatments which effectively slow or stop the progression of
AD. Several symptomatic treatments have been approved, but
these only temporarily improve symptoms of AD patients.3
Thus, there is an urgent unmet medical need for a disease-
modifying therapy for AD that can slow, halt, or reverse dis-
ease progression.
particularly neurotoxic.10,11,12 As a result, there has been much
focus on both β-secretase and γ-secretase as therapeutic targets
to interrupt the amyloid cascade by decreasing the amount of
Aβ42 and thereby preventing the buildup of the amyloid
plaques that initiate the disease.
Significant drug discovery research efforts have focused
on inhibiting γ-secretase with small molecules to reduce the
overall amount of amyloid production. Several different clas-
ses of these γ-secretase inhibitors (GSIs) have been reported in
the literature and been shown to be potent inhibitors of γ-
secretase activity, lowering Aβ levels in vitro and in vivo.13,14,15
However, in addition to its role in the cleavage of APP, γ-
secretase also cleaves multiple essential proteins, including
Notch.16 Thus, by inhibiting γ-secretase, GSIs also interfere
with Notch processing, which leads to toxicities that include
severe gastrointestinal abnormalities and skin cancer.17,18 Sev-
eral GSIs have been advanced to the clinic, but most clinical
GSI studies have failed or been halted early due to observed
toxicity, likely associated with the inhibition of Notch.19,20
In order to develop drugs with a safer profile, recent efforts
have shifted from γ-secretase inhibition to γ-secretase modula-
tion.21,22,23 With the role of Aβ42 in the initiation of the dis-
ease process,9 there is growing evidence that suggests it may
be more important to lower the ratio of Aβ42/Aβ40 (Aβ40
being the most prevalent Aβ peptide residue) or Aβ42/total Aβ
rather than reduce the total amount of Aβ.24 γ-Secretase mod-
ulators (GSMs) achieve this profile by shifting the sites of
APP cleavage away from the more neurotoxic Aβ42 to shorter,
non-toxic peptides such as Aβ37, Aβ38, and Aβ39. Im-
portantly, GSMs have an added advantage in that they do not
affect the release of the Notch intracellular domain (NICD)
following the processing of Notch by γ-secretase, a vital factor
in developing safe and tolerable therapeutics for AD.25 The
first reported GSMs were NSAIDs that were found to decrease
the amounts of Aβ42 while increasing Aβ38.26,27 A repre-
sentative example is (R)-flurbiprofen, which advanced to clin-
ical trials but failed due to lack of potency and poor brain ex-
posure, a common problem amongst the NSAID GSMs.28,29
There have been more recent disclosures of carboxylic acid
The pathology of the Alzheimer’s brain is characterized
by amyloid plaques and neurofibrillary tangles.4,5,6 A prevail-
ing viewpoint for the underlying cause of the disease is the
amyloid hypothesis, which contends that amyloid beta peptide
(Aβ) dysregulation initiates a cascade of neuropathological
changes – formation of amyloid plaques, neurofibrillary tan-
gles, synaptic loss and neurodegeneration - that ultimately
results in the precipitous decline in cognition and ability to
function in daily life that define AD dementia.7
Amyloid
plaques consist of Aβ peptides which are formed by the pro-
cessing of amyloid precursor protein (APP). Aβ peptides are
produced through a series of sequential cuts by two mem-
brane-bound enzymes; first β-secretase cleaves APP into the
β-C-terminal fragment, then γ-secretase makes further cuts to
generate Aβ peptides ranging from 37-49 amino acids in
length.8,9 The main component of the amyloid plaques is the
aggregation-prone 42 amino acid form of amyloid beta
(Aβ42), a relatively minor component in the total Aβ pool but
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
GSMs and aryl imidazole GSMs which more potently reduce Recognizing the need to resolve the issues of intrinsic liabil-
levels of Aβ42 while raising levels of Aβ38.21,22 A recent
report of three distinct pyrimidine-based GSMs showed reduc-
tion of both Aβ40 and Aβ42 in vitro but different effects on
Aβ(37-39), with some examples raising Aβ38 and some hav-
ing no effect on Aβ38 production.30 These “second genera-
tion” GSMs have achieved much higher activities toward
Aβ42 reduction than NSAIDs, and several examples have
shown efficacy and selectivity both in vitro and in vivo, sup-
porting modulation of γ-secretase as an effective approach to
decreasing toxic species of Aβ without preventing the pro-
cessing of other proteins, including Notch. Thus, modulation
of γ-secretase has great potential as a therapeutic approach for
the treatment of Alzheimer’s disease.
ity presented by the functionality native to this triterpene gly-
coside, the primary focus of initial medicinal chemistry efforts
was placed on exploring the SAR around the core structure of
the lead compound. Thus, we investigated the effects of re-
moval or modification of selected structural features of 1 on
the overall profile of modulation of Aβ processing by γ-
secretase. In our initial exploratory forays into the chemistry
of 1, we exposed the compound to aqueous HCl, which result-
ed in acyl migration and subsequent formation of bicyclic
ketal 2 and a 30-fold loss in potency towards Aβ42 reduction
(Scheme 1). The bicyclic ketal was found to be quite stable
and was resistant to a variety of reaction conditions, including
many attempts to re-open the ketal. Thus, we turned our atten-
tion to exploring transformations that could be carried out on
the enol ether and would not result in this irreversible cycliza-
tion. While many attempts at direct reduction of the C16-C17
olefin were unfruitful, we discovered that ZrCl4 catalyzed the
isomerization of the enol ether of 1 in CH2Cl2 to provide the
C15 ketone 3 with cis-configuration between the C16 and C23
of the resulting trans-fused tetrahydropyran ring. While ke-
tone 3 saw a 6-fold diminution of potency relative to 1, subse-
quent treatment with NaBH4 to reduce the C15 ketone and
provide the corresponding C15 hydroxyl 4 moderately im-
proved upon the potency of the initial lead (Aβ42 IC50 = 60
nM). The ketone reduction proceeded with excellent stereose-
lectivity (>95:5, only one product observed by 1H-NMR), with
the angular methyl group at C14 presumably creating the steric
environment that accounts for the high degree of selectivity in
the reaction. The stereochemistry resulting from the two-step
reduction process was confirmed by obtaining an X-ray crystal
structure (see Supporting Information). Thus, in the process
of removing one of the more troubling structural features from
a metabolic and chemical perspective, the primary pharmacol-
ogy of the lead compound was improved upon.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As part of a program aimed at developing a series of novel
γ-secretase modulators (GSMs) as therapeutics for AD, Satori
identified an initial lead compound via screening of natural
product extracts for selective reduction of Aβ42.31 This initial
lead (1, Scheme 1) was isolated from extracts of the black
cohosh plant (Actaea racemosa) and represents the first entry
in a unique class of GSMs that features a complex molecular
architecture characterized by a plant sterol core structure.
Most importantly, 1 exhibits a unique and compelling profile
towards γ-secretase modulation, selectively reducing levels of
Aβ42 and Aβ38 in vitro, while raising levels of Aβ37 and
Aβ39. The levels of Aβ40 and total Aβ levels were main-
tained, and 1 also displayed pronounced selectivity over Notch
processing in vitro.
Scheme 1.
More extensive testing was carried out on 4, and it was
found to have excellent selectivity in transporter, off-target,
and safety assays. However, following dosing in CD1 mice,
the compound was found to have an insufficient pharmacoki-
netic profile for advancement, displaying moderate-to-high
clearance and poor brain exposure (data not shown). This
poor in vivo performance was not entirely unexpected, for the
native C3 sugar appendage and the C24 acetate had been
shown to be vulnerable to metabolism when 1 had been dosed
in vivo.31 In addition, little had been done to address other
parameters, such as number of hydrogen bond donors (HBD =
5) and topological polar surface area (tPSA = 155 Å2) that
could preclude a molecule from achieving acceptable levels of
brain exposure.32,33,34 Thus, we sought to improve the overall
CNS disposition by further modification of the structure of 4
that would address these design parameters.
While 1 was an intriguing initial hit, examination of its
structure quickly revealed a number of potential metabolic and
chemical liabilities that limited its potential usefulness as a
therapeutic drug. In particular, the C3 glycoside, C16 enol
ether, and C24 acetate stood out as areas of concern. Indeed,
the concerns over the metabolic stability of 1 were confirmed
upon dosing in CD1 mice. Rapid clearance of 1 precluded its
therapeutic usefulness, as both the C3 sugar and the C24 ace-
tate were readily metabolized in vivo.31 In addition, the high
MW and tPSA of 1 lay well outside the range typical for CNS
drugs, causing concerns that we would not be able to prepare
compounds that gave adequate CNS exposure to be therapeu-
tically useful. Therefore, an important milestone in this pro-
ject would be to prepare compounds that gave sufficient brain-
to-plasma ratios in vivo.
Interrogation of the additional metabolic and chemical soft
spots of 4 revealed some interesting trends in the SAR of this
lead series. Hydrolysis of the C24 acetate to unmask the
C24,C25 diol 5 resulted in a 50-fold reduction in potency in
lowering Aβ42 (Scheme 2). Cleavage of the glycoside to pro-
vide triol 6 resulted in only a 10-fold loss of activity, while
further treatment with K2CO3/MeOH to remove the C24 ace-
tate and provide the tetrol 7 caused the potency to decrease by
more than 17-fold compared to triol 6. These results identi-
fied the C24 acetate as a more critical pharmacophore to the
Aβ42 pharmacology than the C3 glycoside. As a result, the
focus of these investigations shifted to carrying out further
ACS Paragon Plus Environment
2

Page 3 of 7
ACS Medicinal Chemistry Letters
SAR studies at the C3 position of the scaffold through which
Hence, while N-methyl (11) and N-ethyl morpholines (13)
1
2
3
4
5
6
7
8
we hoped to identify a glycoside surrogate that maintained the
overall pharmacological profile while improving on the physi-
cochemical properties of the series. These improvements
would then hopefully translate into an improved pharmacoki-
netic profile.
maintained potency, imparting more lipophilic character by
functionalization of the morpholine nitrogen with propyl, ben-
zyl, or cycloalkyl groups resulted in a loss of activity (14 - 17).
A decrease in potency was also realized for N-trifluoroethyl
morpholine 18. Incorporating a heteroatom into the morpho-
line substituents and thereby increasing the polarity in this
region of the molecule resulted in improved Aβ42 activity
relative to the purely aliphatic analogs. For example, replac-
ing the terminal methyl of the N-propyl derivative 14 with a
hydroxyl (19) or a methyl ether (21) resulted in much im-
proved potency, and the N-oxetane derivative 22 was two-fold
more potent than the corresponding N-cyclobutyl analog 17.
Both hydroxyalkyl and ether appendages on the morpholine
nitrogen showed a strong propensity for reduction of Aβ42 (19
- 23). A similar effect was seen upon incorporation of amino-
alkyls into the morpholine side chain, providing compounds
with strong Aβ42 lowering capabilities (24 - 26). Amide ap-
pendages on the morpholine nitrogen also resulted in extreme-
ly potent compounds (27 – 28), but carboxylic acids were not
as active (29 - 30). Both diastereomers of the γ-lactam moiety
showed good activity (31 – 32), as did the imidazole deriva-
tive 33. Whether or not the improved potency results from the
ability of the heteroatoms to pick up additional interactions or
is simply due to increased polarity is not clear.
Scheme 2.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
While part of our early work was focused on discovering a
suitable replacement for the C3 glycoside via initial cleavage
of the sugar followed by functionalization or chemical dis-
placement of the exposed C3 alcohol,35 we were also interest-
ed in exploiting rather than replacing the native sugar in our
synthetic endeavors. To this aim, by treating 4 with NaIO4 or
Pb(OAc)4 we were able to carry out a double oxidative cleav-
age event on the sugar to provide dialdehyde 8 (Scheme 3),
which served as a versatile intermediate.36,37 Reduction of
dialdehyde 8 with NaBH4 provided tetrol substrate 9, which
possessed good pharmacology but did not offer any ad-
vantages over 4 from a physicochemical properties standpoint.
Dual reductive amination with dimethylamine provided the
diamine 10, which, although lowering the tPSA, also realized
a diminution in potency. When we carried out the reductive
amination of dialdehyde 8 with methylamine, after undergoing
an initial reductive amination, the intermediate amino-
aldehyde species participated in a second intramolecular re-
ductive amination event to provide C3 morpholine 11. This
two-step chemical transformation of the native sugar into a
morpholine provided a substrate which not only maintained
the primary pharmacology (Aβ42 IC50 = 130 nM) but offered a
significant improvement from a physicochemical properties
perspective, lowering the tPSA to 98 Å2 and the HBD count to
two. Thus, by unlocking the reactive potential of the 1,2,3-
triol of the sugar to synthesize the C3 morpholine as a re-
placement for the C3 glycoside, we had discovered a new lead
series of compounds possessing a modified headpiece which
maintained high potency in lowering Aβ42 in vitro and pro-
vided a versatile handle for further derivatization to allow for
engineering of the overall physicochemical properties.
Scheme 3.
The synthesis of acyl morpholines and related species also
furnished interesting and highly potent analogs. Conversion
of the morpholine nitrogen to sulfonamide 34, urea 35, and
amide derivatives (36 – 40) provided a series of compounds
with excellent activity towards Aβ42 lowering. Although
these acylated derivatives were intriguing, the physicochemi-
cal properties of these compounds were less compelling than
morpholines that maintained a basic center (vide infra). The
morpholine headpiece proved to be a very versatile handle for
incorporating a diverse range of functional groups into the
scaffold, and proved advantageous for tuning the overall mo-
lecular properties and the potential to improve the CNS dispo-
sition. In addition, the selectivity for reduction of Aβ42 ver-
sus Aβ40 was maintained across the series.
To fully exploit the medicinal and synthetic chemistry po-
tential of this C3 morpholine series of compounds, a focused
but diverse range of morpholine compounds was designed and
synthesized, and representative examples from this series of
compounds are shown in Table 1. A scan of the SAR of this
C3 morpholine series reveals that a wide variety of substitu-
tion is tolerated on the morpholine nitrogen. The simple N-H
morpholine 12 was particularly potent with an Aβ42 IC50 of 70
nM. Small aliphatic substituents were also tolerated, and the
general trends suggest that polar substituents on a basic mor-
pholine are preferred over more lipophilic substituents, a trend
that was in concert with one of our key design elements.
The overall profile of two C3 morpholine derivatives, N-
H morpholine 12 and the N-oxetane analog 22, allowed these
to emerge as candidates for further evaluation. While all mol-
ecules had a higher molecular weight than is usually targeted
for a CNS drug, the balance of potency, HBD, tPSA, and lipo-
philicity of analogs 12 and 22 along with the potential benefits
provided by having a basic center in the molecule to assist in
permeating the blood-brain barrier elevated these over
ACS Paragon Plus Environment
3

ACS Medicinal Chemistry Letters
Page 4 of 7
1
2
Table 1. SAR of representative examples from the Satori C3 morpholine series of GSMs.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
icant improvement over the previously disclosed lead com-
pounds from the C3 glycosidic series of GSMs (1 and 4), and
we felt this disposition and overall profile was sufficient to
our initial leads (1 and 4) and other potential candidates in the
series (Table 2).
The C3 morpholines 12 and 22 were dosed in CD1 mice
to evaluate in vivo pharmacokinetics (Figure 1). The results
showed that attenuating amine basicity in N-oxetane morpho-
line species 22 (pKa = 4.6) decreases the volume of distribu-
tion (0.6 L/kg versus 12.1 L/kg for 12) while modestly in-
creasing the clearance (3.3 L/hr/kg for 22 versus 3.1 L/hr/kg)
compared to the more basic N-H morpholine 12 (pKa = 7.7).
Most importantly, we found that these compounds gave en-
couraging brain-to-plasma ratios (1.7 for 12, 0.23 for 22).
While the compounds exhibited acceptable brain-to-plasma
ratios, the bioavailability values were low (%F = 11% for 22
and 37% for 12, respectively) and low levels of brain exposure
were observed. Still, this murine PK data represented a signif-
assess a PK/PD response in vivo.
After comparing the in vivo PK and predicted microsomal
stability of 12 versus 22 in mouse (MLM, % remaining after
60 minutes +/-NADPH: 12 = 20%/25%; 22 = 1%/1%), we
decided to focus on 12, which was taken forward for in vivo
efficacy evaluation in CD1 mice.38 Unfortunately, due to the
high clearance of the molecules, we were unable to obtain
sufficient brain exposure to elicit a robust pharmacodynamic
response in vivo (see Supporting Information). Results of
further studies aimed at improving clearance of this series in
order to obtain adequate exposure for a PD response will be
presented in due course.
ACS Paragon Plus Environment
4

Page 5 of 7
ACS Medicinal Chemistry Letters
1
2
3
Table 2. Calculated physicochemical properties of high-interest compounds from the Satori C3 morpholine series of
GSMs.39
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Full Aβ production and pharmacokinetic profiles (CD1 mouse) for compounds 12 and 22.
Aβ From 2B7 Conditioned Media
Cells treated for 5.0 Hours
AβFrom 2B7 Conditioned Media
Cells treated for 5.0 Hours
120
110
100
90
80
70
60
50
40
30
20
10
0
120
110
100
90
80
70
60
50
40
30
20
10
0
Aβ38
Aβ40
Aβ42
AβTotal
Cell Viability
Aβ38
Aβ40
Aβ42
AβTotal
Cell Viability
-10.0 -9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0
-10.0 -9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0
Log (mol/L)
Log (mol/L)
Evaluation of the Aβ profile in our cell-based assay showed
that both N-H morpholine 12 and N-oxetane analog 22 exhib-
ited the ability to lower Aβ42 levels in cells, with similar ac-
tivity versus Aβ38, good selectivity for lowering Aβ42 versus
Aβ40 (ratio of Aβ40 IC50/Aβ42 IC50 = 25 for 12 and 52 for
22), and preserved the levels of total Aβ in cells40 (Figure 1).
The molecules showed excellent selectivity for lowering Aβ42
versus Notch processing (>70-fold selective for both 12 and
22). This level of Notch selectivity is consistent with a GSM
profile and, due to the toxicities associated with inhibition of
Notch processing, offers a distinct advantage over the lack of
Notch selectivity exhibited by GSIs which have advanced to
clinical trials.41 The overall modulation profile of the series,
reducing Aβ42 and Aβ38 while maintaining Aβ40 and total
Aβ represents a different GSM profile when compared to
most other GSMs reported in the literature.
ACS Paragon Plus Environment
5

ACS Medicinal Chemistry Letters
In summary, the Satori GSM program progressed from an
Page 6 of 7
1
2
3
4
5
6
7
8
ABBREVIATIONS
intriguing initial hit, triterpene glycoside 1 identified in a tar-
geted bioassay screening, to a new lead compound (4) with a
modified core. Application of medicinal chemistry design
principles in conjunction with innovative synthetic chemistry
approaches led to the discovery of a new series of C3 morpho-
line compounds. This series represents a new structural class
of GSMs that maintained the compelling pharmacological
profile of the initial leads, lowering Aβ42 and Aβ38 levels in
cells, preserving Aβ40 and total Aβ, and showing no inhibi-
tion of Notch activity. This GSM profile differs from most
other GSMs reported in the literature, which lower Aβ42 but
raise levels of Aβ38. In the process of design, we significant-
ly improved drug-like properties of the lead compounds by
decreasing both the tPSA and the overall count of hydrogen
bond donors (HBD) while keeping the lipophilicity in an ac-
ceptable range (<5). This led to compounds 12 and 22, which
have significantly improved CNS penetration relative to gly-
coside 4, which more closely resembles our natural product
lead 1. In addition, these compounds retained an excellent
profile in standard transporter, off-target, and safety assays in
vitro, and showed improved chemical stability. The morpho-
line nitrogen provides a convenient and versatile handle by
which the overall molecular properties of the compounds can
be readily manipulated. The discovery of this new series of
C3 morpholine GSMs with a plant sterol-derived core struc-
ture represents a novel and exciting entry into the arena of γ-
secretase modulation as a potential therapeutic approach for
Alzheimer’s disease.
Aβ, amyloid beta; AD, Alzheimer’s disease; APP, amyloid pre-
cursor protein; cLogP, calculated lipophilicity; CH2Cl2, di-
chloromethane; CNS, central nervous system; EtOAc, ethyl ace-
tate; GSM, gamma-secretase modulator; GSI, gamma-secretase
inhibitor; HCl, hydrochloric acid; HBD, hydrogen bond donors;
K2CO3, potassium carbonate; Me2NH, dimethylamine; MeNH2,
methylamine; MLM, mouse liver microsomes; MeOH, methanol;
NaBH4, sodium borohydride; NaCNBH3, sodium cyanoborohy-
dride; NaIO4, sodium periodate; NSAID, non-steroidal anti-
inflammatory drug; Pb(OAc)4, lead tetraacetate; PK, pharmacoki-
netic; PO, by mouth; tPSA, topological polar surface area; SAR,
structure-activity relationship; ZrCl4, zirconium tetrachloride.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
1
Alzheimer’s Association, 2012 Alzheimer’s Disease Facts and
Figures, Alzheimer’s & Dementia, Volume 8, Issue 22, 131-168.
(http://www.alz.org/downloads/Facts_Figures_2012.pdf).
2
Wimo, A. P. Alzheimer’s Disease International, World Alz-
ASSOCIATED CONTENT
heimer’s Report 2010: The Global Economic Impact of Dementia, 21
September, 2010.
Exemplary procedures for the synthesis of compounds of interest,
characterization data of key compounds 12 and 22, X-ray crystal
structure of compound 4, and assay protocols are provided in the
supporting information. This material is available free of charge
via the Internet at http://pubs.acs.org.
3
(a) Alzheimer’s Disease Medications Fact Sheet, NIH Publica-
tion No. 08-3431, Department of Health and Human Services, Na-
tional Institute on Aging, U.S. Government Printing Office, Washing-
ton, D. C., 2010. (b) Cummings, J. L. N. Eng. J. Med. 2004, 351,
56.
4
Serrano-Pozo, A.; Frosch, M. P.; Masliah, E.; Hyman, B. T.
AUTHOR INFORMATION
Corresponding Author
Neuropathological Alterations in Alzheimer Disease. In The Biology
of Alzheimer Disease; Selkoe, D. J., Mandelkow, E., Holtzman, D.
M., Eds.; Cold Spring Harbor Perspectives Laboratory Press: Cold
Spring Harbor, NY, 2012; pp 43-65.
* Phone: 617-547-0022. Fax: 617-547-0661.
Email: nathan.fuller@satoripharma.com.
5
Terry, R. D. Alzheimer’s Disease at mid century (1927–1977),
in: Alzheimer: 100 Years and Beyond. Jucker, M.; Beyreuther, K.;
Haass, C.; Nitsch, R., Eds. Springer-Verlag, Berlin Heidelberg,
2006, pp. 58-61.
Present Addresses
†(M.A.F.) Resilientx Therapeutics, 431 School Street, Belmont,
MA 02478 USA.
(S.P.C.) Genzyme Corporation, 500 Kendall Street, Cambridge,
MA 02142.
(W.X.) V.
6
Holtzman, D. M.; Morris, J. C.; Goate, A. M. Alzheimer’s dis-
ease: The challenge of the second century. Sci. Transl. Med. 2011,
3, 77sr1.
7
Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer's
disease: progress and problems on the road to therapeutics. Science
2002, 297(5580), 353-356.
8
Author Contributions
Haass, C.; Kaether, C.; Thinakaran, G.; Sisodia, S. Trafficking
and Proteolytic Processing of APP. In The Biology of Alzheimer
Disease; Selkoe, D. J., Mandelkow, E., Holtzman, D. M., Eds.; Cold
Spring Harbor Perspectives Laboratory Press: Cold Spring Harbor,
NY, 2012; pp 205-229.
All authors have given approval to the final version of the manu-
script.
Notes
9
Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy.
The authors note the following relevant financial interests: the
authors are named as inventors on one or more patents and patent
applications related to compounds discussed in this paper and
either hold equity and/or options on equity in Satori Pharmaceuti-
cals.
Physiol. Rev. 2001, 81, 741-766.
10 Roher, A. E.; Lowenson, J. D.; Clarke, S.; Woods, A. S.; Cotter,
R. J.; Gowing, E.; Ball, M. J. β-Amyloid-(1-42) is a major compo-
nent of cerebrovascular amyloid deposits: Implications for the pa-
thology of Alzheimer disease. Proc. Nat. Acad. Sci. USA, 1993, 90,
10836-10840.
11 Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.;
Ihara, Y. Visualization of Aβ42(43) and Aβ40 in senile plaques with
end-specific Aβ monoclonals: evidence that an initially deposited
species is Aβ42(43). Neuron, 1994, 13, 45-53.
ACKNOWLEDGMENT
The authors would like to thank Ruichao Shen for his comments
on the paper.
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
12 Findeis, M. A. The role of amyloid β peptide 42 in Alzheimer’s
disease. Pharmacol. Ther. 2007, 116, 266-286.
of R-flurbiprofen in healthy elderly individuals. Alz. Dis. Assoc.
Disord. 2007, 21, 292-299.
29
13
Green, R. C.; Schneider, L. S.; Amato, D. A.; Beelen, A. P.;
Kreft, A. F.; Martone, R.; Porte, A. Recent advances in the
Wilcock, G.; Swabb, E. A.; Zavitz, K. H.; and Tarenflurbil Phase 3
Study Group. Effect of tarenflurbil on cognitive decline and activities
of daily living in patients with mild Alzheimer disease: a randomized
controlled trial. J. Am. Med. Assoc. 2009, 302, 2557-2564.
identification of gamma-secretase inhibitors to clinically test the
Abeta oligomer hypothesis of Alzheimer’s disease. J. Med. Chem.
2009, 52, 6169-6188.
14
Panza, F.; Frisardi, V.; Imbimbo, B. P.; Capurso, C.; Logrosci-
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Borgegard, T.; Jeréus, A.; Olsson, F.; Rosqvist, S.; Sabirsh, A.;
no, G.; Sancarlo, D.; Seripa, D.; Vendemiale, G.; Pilotto, A.; Solfriz-
zi, V. γ-Secretase inhibitors for the treatment of Alzheimer’s dis-
ease: the current state. CNS Neuroscience & Therapeutics 2010,
16(5), 272-284.
Rotticci, D.; Paulsen, K.; Klintenberg, R.; Yan, H.; Waldman, M.;
Stromberg, K.; Nord, J.; Johansson, J.; Regner, A.; Parpal, S.; Mali-
nowsky, D.; Radesater, A.-C.; Li, T.; Singh, R.; Eriksson, H.;
Lundkvist, J. First and Second Generation γ-Secretase Modulators
(GSMs) Modulate Amyloid-β (Aβ) Peptide Production through Dif-
ferent Mechanisms. J. Biol. Chem. 2012, 287, 11810-11819.
15
D’Onofrio, G.; Panza, F.; Frisardi, V.; Solfrizzi, V.; Imbimbo,
B. P.; Paroni, G.; Cascavilla, L.; Seripa, D.; Pilotto, A. Advances in
the identification of γ-secretase inhibitors for the treatment of Alz-
heimer’s disease. Exp. Opin. Drug Disc. 2012, 7, 19-37.
31
Findeis, M. A.; Schroeder, F.; McKee, T. D.; Yager, D.;
16
Fraering, P. C.; Creaser, S. P.; Austin, W. F.; Clardy, J.; Wang, R.;
Selkoe, D. J.; Eckman, C. B. Discovery of a novel pharmacological
and structural class of gamma secretase modulators derived from the
extract of Actaea racemosa. Manuscript in press ACS Chem. Neuro-
sci. DOI: 10.1021/cn3000857.
Lleo, A. Activity of γ-secretase on substrates other than APP.
Curr. Top. Med. Chem. 2008, 8, 9-16.
17
Searfoss, G. H.; Jordan, W. H.; Calligaro, D. O.; Galbreath, E.
J.; Schirtzinger, L. M.; Berridge, B. R.; Gao, H.; Higgins, M. A.;
May, P. C.; Ryan, T. P. Adipsin, a Biomarker of Gastrointestinal
Toxicity Mediated by a Functional γ-Secretase Inhibitor. J. Biol.
Chem., 2003, 278, 46107-46116.
32
Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. Moving
beyond Rules: The Development of a Central Nervous System Mul-
tiparameter Optimization (CNS MPO) Approach To Enable Align-
ment of Druglike Properties. ACS Chem. Neurosi. 2010, 1, 435-449.
18
Wong, G. T.; Manfra, D.; Poulet, F. M.; Zhang, Q.; Josien, H.;
Bara, T.; Engstrom, L.; Pinzon-Ortiz, M.; Fine, J. S.; Lee, H. J.;
Zhang, L.; Higgins, G. A.; Parker, E. M. Chronic Treatment with the
γ-Secretase Inhibitor LY-411575 Inhibits β-Amyloid Peptide Produc-
tion and Alters Lymphopoiesis and Intestinal Cell Differentiation. J.
Biol. Chem., 2004, 279, 12876-12882.
33
Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M.
D.; Verhoest, P. R.; Villalobos, A.; Will, Y. Defining Desirable Cen-
tral Nervous System Drug Space through the Alignment of Molecular
Properties, in Vitro ADME, and Safety Attributes. ACS Chem. Neu-
rosci. 2010, 1, 420-434.
19
Kreft, A. F.; Martone, R.; Porte, A. Recent advances in the
34
Lipinski, C. A.; Lombardo, R.; Dominy, B. W.; Feeney, P. J.
identification of γ-secretase inhibitors to clinically test the Aβ oli-
gomer hypothesis of Alzheimer’s disease. J. Med. Chem. 2009, 52,
6169-6188.
Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings. Adv.
Drug Deliv. Rev. 1997, 23, 3-25.
20
Hopkins, C. R. ACS Chemical Neuroscience Molecule Spot-
35
Austin, W. F.; Hubbs, J. L.; Fuller, N. O.; Creaser, S. P.;
light on Semagacestat (LY450139). ACS Chem. Neurosci. 2010, 1,
McKee, T. D.; Loureiro, R. M. B.; Findeis, M. A.; Tate, B.; Ives, J.
L.; Bronk, B. S. SAR investigations on a novel class of gamma-
secretase modulators based on a unique scaffold. (Manuscript submit-
533-534.
21
Oehlrich, D.; Berthelot, D. J.-C.; Gijsen, H. J. M. γ-Secretase
Modulators as Potential Disease Modifying Anti-Alzheimer’s Drugs
ted for publication).
J. Med. Chem. 2011, 54, 669-698.
36
22
Du, M.; Hindsgaul, O.
Novel Hybrid Morpholino-
Wolfe, M. S. γ-Secretase inhibitors and modulators for Alz-
Glycopeptides with the Amino Acid Nitrogen Replacing C-3 of the
heimer’s disease. J. Neurochem. 2012, 120(1), 89-98.
23 Wolfe, M. S. γ-Secretase: Structure, Function, and Modulation
for Alzheimer’s Disease. Curr. Top. Med. Chem. 2008, 8, 2-8.
24 Karran, E.; Mercken, M.; De Strooper, B. The amyloid cascade
hypothesis for Alzheimer’s disease: an appraisal for the development
of therapeutics. Nature Rev. Drug Disc. 2011, 10, 698-712.
25 (a) Wanngren, J.; Ottervald, J.; Parpal, S.; Portelius, E.; Strömberg,
K.; Borgegård, T.; Klintenberg, R.; Juréus, A.; Blomqvist, J.; Blen-
now, K.; Zetterberg, H.; Lundkvist, J.; Rosqvist, S.; Karlström, H.
Second generation γ-secretase modulators exhibit different modula-
tion of Notch β and Aβ production. J. Biol. Chem. 2012, article in
press. (b) Okochi, M.; Fukumori, A.; Jiang, J.; Itoh, N.; Kimura, R.;
Steiner, H.; Haass, C.; Tagami, S.; Takeda, M. Secretion of the
Notch-1 Aβ-like Peptide during Notch Signaling. J. Biol. Chem.
2006, 281, 7890-7898.
Pyranose Ring. Synlett, 1997, 395-397.
37
Chan, J. Y. C.; Hough, L.; Richardson, A. C. J. The Synthesis
of (R)- and (S)-Spirobi-1,4-dioxane and Related Spirobicycles from
D-Fructose. J. Chem. Soc. Perkin. Trans. I, 1985, 7, 1457-1462.
38 For results and protocol for in vitro profiling for microsomal
stability in human, mouse, and rat see Supporting Information.
39
The pKa, ClogP, and tPSA predictions in this paper were calcu-
lated using algorithms included in MarvinSketch, version 5.4.1.1;
ChemAxon: Budapest, Hungary, 2011.
40 When dosed at higher concentrations (between 1-4 μM) com-
pound 12 begins to show some reduction in cell viability (20% reduc-
tion @ 4 μM, Cell Titre Glo assay), and the Aβ40 and total Aβ levels
are lowered at these higher concentrations as a result of reduced cell
viability. Compound 22 shows only slight cell viability issues at the
higher end of the concentration spectrum tested (20% reduction @ 20
μM).
26
Weggen, S.; Erikson, J. L.; Das, P.; Sagi, S. A.; Wang, R.; But-
ler, T.; Kang, D. E.; Marquez-Sterling, N.; Golde, T. E.; Koo, E. H.
A subset of NSAIDs lower amyloidogenic Aβ42 independently of
cyclooxygenase activity. Nature 2001, 414, 212-216.
41 Known GSIs which have advanced to clinical trials show no
selectivity for Notch inhibition (ratio of Aβ IC50/Notch IC50 for
semagecestat = 0.6, for begacestat = 0.8, and for avagecestat = 0.6.
Known GSMs in the literature show much better selectivity towards
Notch inhibition (Aβ42 IC50/Notch IC50 for Merck GSM-1 = >350,
for JNJ-40418677 = >20, for Eisai E-2012 = >400. See “An im-
proved cell-based method for determining the gamma-secretase en-
zyme activity against both NOTCH and APP substrates,”T. D.
McKee, et al., AAIC 2012 poster #P2-095,
27
Erikson, J. L.; Sagi, S. A.; Smith, T. E.; Weggen, S.; Das, P.;
McLendon, D. C.; Ozols, V. V.; Jessing, K. W.; Zavitz, K. H.; Koo,
E. H.; Golde, T. E. NSAIDs and enantiomers of flurbiprofen target γ-
secretase and lower Aβ42 in vivo. J. Clin. Invest. 2003, 112, 440-
449.
28
Galasko, D. R.; Graff-Radford, N.; May, S.; Hendrix, S.; Cot-
trell, B. A.; Sagi, S. A.; Mather, G.; Laughlin, M.; Zavitz, K. H.;
Swabb, E.; Golde, T. E.; Murphy, M. P.; Koo, E. H. Safety, tolerabil-
ity, pharmacokinetics, and Aβ levels after short-term administration
http://www.satoripharma.com/news-events/publications-and-
presentations.php.
ACS Paragon Plus Environment