Discovery of a Tetrahydrobenzisoxazole Series of γ-Secretase Modulators

Article abstract of DOI:10.1021/acsmedchemlett.7b00178

The design and synthesis of a new series of tetrahydrobenzisoxazoles as modulators of γ-secretase activity and their structure-activity relationship (SAR) will be detailed. Several compounds are active γ-secretase modulators (GSMs) with good to excellent selectivity for the reduction of Aβ₄₂ in the cellular assay. Compound 14a was tested in vivo in a nontransgenic rat model and was found to significantly reduce Aβ₄₂ in the CNS compartment compared to vehicle-treated animals (up to 58% reduction of cerebrospinal fluid Aβ₄₂ as measured 3 h after an acute oral dosing at 30 mg/kg).

Full text of DOI:10.1021/acsmedchemlett.7b00178

Subscriber access provided by University of Victoria Libraries
Letter
Discovery of a Tetrahydrobenzisoxazole Series of #-Secretase Modulators
Zhiqiang Zhao, Dmitri A Pissarnitski, Xianhai Huang, Anandan Palani, Zhaoning Zhu, William
J Greenlee, Lynn A. Hyde, Lixin Song, Giuseppe Terracina, Lili Zhang, and Eric M. Parker
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00178 • Publication Date (Web): 19 Sep 2017
Downloaded from http://pubs.acs.org on September 21, 2017
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
Discovery of a Tetrahydrobenzisoxazole Series of
lators
Zhiqiang Zhao,a,* Dmitri A. Pissarnitski,a Xianhai Huang,a Anandan Palani,a Zhaoning Zhu,a William J.
Greenlee,a Lynn A. Hyde,b Lixin Song,b Giuseppe Terracina,b Lili Zhang,b Eric M. Parker b
a Department of Medicinal Chemistry, MRL, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Department of Neurobiology, MRL, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA
γ
-Secretase Modu-
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
KEYWORDS: Alzheimer's disease; γ-Secretase modulators; tetrahydrobenzisoxazole.
ABSTRACT: The design and synthesis of a new series of tetrahydrobenzisoxazoles and their structure-activity relationship (SAR)
as modulators of γ-secretase activity will be detailed. Several compounds are active γ-secretase modulators (GSMs) with good to
excellent selectivity for the reduction of Aβ42 in the cellular assay. Compound 14a was tested in vivo in a non-transgenic rat model
and was found to significantly reduce Aβ42 in the CNS compartment compared to vehicle-treated animals (up to 58% reduction of
cerebrospinal fluid Aβ42 as measured 3h after an acute oral dosing at 30 mg/kg).
Alzheimer’s disease (AD) is the sixth leading cause of death
in the United States. An estimated 5.5 million Americans have
AD, costing the nation more than $230 billion in 2016.1 As the
elderly population continues to grow, the prevalence of AD
will be increasing greatly. Therefore, there is an urgent need to
develop an effective therapy to prevent Alzheimer's disease.
Aβ proteins, which are metabolites of the amyloid pre-
cursor protein (APP), are considered to be involved in the
degeneration and loss of neurons as well as the onset of
AD.2,3 It is known that Aβ40 is the most abundant species
within the soluble pool of Aβ peptides; however, deposited Aβ
plaques primarily consist of Aβ42,4,5 which is the species that is
most prone to aggregation, forming aggregates of insoluble
fibrils in the brain.6 The higher aggregation potential of Aβ42
may due to the distinct initial folding properties of Aβ40 and
Aβ42.7 These Aβ fragments are produced when APP is first
cleaved by BACE (β-amyloid cleaving enzyme) and subse-
quently by γ-secretase.8 Based on these facts, γ-secretase in-
hibitors (GSIs) and BACE inhibitors have been proposed as
treatments for the purpose of reducing the production of Aβ
fragments.9 However, notch-related adverse effects are
known as a major obstacle for the development of GSIs for the
treatment of AD.10 Therefore, γ-secretase modulators (GSMs)
have attracted much attention in recent years. 11,12
inflammatory drugs (NSAIDs)15,16 and the non-NSAIDs class,
such as 1 from Eisai,17 2 and 3 from Merck.18,19 In addition,
natural product-based GSMs have also been developed.20,21
Following our efforts22-27 to develop in the non-NSAID GSMs,
we developed a new series, represented by structure 4 (Figure
1),28 where the replacement of the double bond present in
structures 1– 3 to mitigiate a potential metabolic. Therefore,
the double bond and the lactam carbonyl in 1 was replaced by
a small heterocyclic fused ring such as tetrahydrobenzisoxa-
zole in 4.
O
N
O
OH
N
N
F
N
N
N
N
F
O
O
1 from Eisai
2
Aβ42 IC50 = 80 nM
AβTotal/Aβ42= 224
Aβ42 IC50 = 29 nM
AβTotal/Aβ42=488
R1
O
N
O
N
R2
N
N
F
N
N
N
O
O
3
4
Aβ42 IC50 = 37 nM
AβTotal/Aβ42= 385
tetrahydrobenzoisoxazoles
(This work)
GSMs selectively inhibit the production of the most amy-
loidogenic and neurotoxic Aβ42 by shifting the position of γ-
secretase cleavage toward the formation of shorter, more solu-
ble peptides, without interfering with the overall γ-secretase
function, as measured by the formation of total amyloid (Aβto-
tal).13 A larger ratio of Aβtotal/Aβ42 represents a more selective
GSM. More selective GSMs do not shut down the processing
of Notch by γ-secretase, and thus offer a potentially improved
side effect profile compare to GSIs. Due to this advantage,
GSMs have attracted great interest from researchers.14 There
are two major classes of GSMs, the nonsteroidal anti-
Figure 1. Design of GSMs from known structures
The tetrahydrobenzisoxazole was selected as a linker to lock
the side chain in the same conformation as the double bond
did in the lead structures 1– 3. We synthesized a series of
compounds with a core structure of 4. Our chemical synthesis
involves key intermediates 12 and 13 which is summarized in
Scheme 1. The O-methylation of 5 afforded 6, which was
converted to the oxime compound 7. The key step is a [3 + 2]
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
O
O
N
N
cycloaddition to afford isoxazole 8. After optimization, we
OH
O
O
N
O
N
1
2
3
4
5
6
R
were able to develop a one pot reaction to produce intermedi-
ate 8 with a 67% yield (two steps). The nitro group in 8 was
reduced to the corresponding amine to afford 9. The amine
group in 9 was converted to formamide 10. The N-alkylation
of 10 with 1-chloropropan-2-one, followed by treatment with
ammonium acetate afforded 12, which was reduced by sodium
borohydride to deliver 13.
R-MgBr
THF, 0 °C
N
N
12
15a-b
TMSCl, NaI
2. NaBH4
CH3CN, 60 °C
F
EtOH, 0 °C
1. R-NH2, THF
3. Chiral OD column
14a-d
Ti(OPr-i)4, 60 °C
separation for
7
8
9
H
N
O
O
N
N
F
F
R
O
O
N
Scheme 1. Synthesis of intermediates 12 and 13
N
N
F
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
N
14a-j
16
O
O
HO
O
CH3I, K2CO3
NH2OH.HCl
H
H
O
N
OH
DMF, RT, 99%
EtOH, RT, 90%
O
N
O2N
O2N
5
6
O
N
Ar
Br
O
O
NaH/DMF
0 °C
N
OH
H
13
O
N
1. NCS, DMF
0 °C, 2.5 h
N
N
O
18
N
O
O
R-OH, DEAD
Ph3P, THF
50 °C
O
Br
O2N
7
2.
NaHCO3,67%
8
O2N
O
N
OR
O
O
N
O
O
N
O
O
HCOOH
SnCl2/EtOH
55 °C, 98%
O
N
N
17a-b
HN
Ac2O, 96%
9
10
H2N
H
O
The biological test results are summarized in Table 1. Hu-
man embryonic kidney (HEK) 293 cells were treated with a
variable concentration of GSM compounds for 5 h. Aβ42 and
Aβtotal in conditioned media were measured using MesoScale
Discovery (MSD) sandwich immunoassays.30 Aβ42 was meas-
ured using a pair of labeled antibodies, TAG-G2-11 and bio-
tin-4G8; total Aβ was measured using antibodies TAG-W02
and biotin-4G8. The electrochemiluminescence (ECL) signal
was measured using an ECLM8 instrument (IGEN Interna-
tional, Inc.) according to the manufacturer’s instructions.
From previously known SAR, the left side 3-
O
N
O
O
O
Cl
NH4Ac/HOAc
95 °C, 59%
O
N
Cs2CO3, DMF,
55 °C, 54%
11
H
O
O
O
N
N
O
OH
O
N
O
NaBH4, EtOH
0 °C, 97%
N
N
N
12
13
methoxyphenyl-4-methyl-1H-imidazole is required for GSM
activity. Therefore, we focused our efforts on the SAR devel-
opment of the right side of the molecule. The SAR is summa-
From 12 and 13 a variety of final targets were prepared
through traditional chemistry: such as reductive amination (12
to 14a-j), Grignard reaction (12 to 15a-b), Mitsunobu reaction
(13 to 17a-b), dehydroxylation29 (15a to 16) and alkylation
(13 to 18) as illustrated in Scheme 2.
rized in Table 1. When R2 is N-methylamino, the Aβ42 inhibi-
tion activity is only 12 µM with no selectivity (14h, clogD =
1.07, pKa = 8.10). It is known that increasing lipophilicity
often results in improved in vitro activity and improved blood
brain barrier permeability.31 Controlling the pKa was a suc-
cessful approach to improve the CNS penetration.32 We then
introduced fluorine to modulate both lipophilicity and basicity
of the target molecules. Substitution with N-
Scheme 2. Synthesis of final GSM compounds
trifluoroethylamino led to improved Aβ42 inhibition activity
and selectivity (464 nM and 43-fold for 14i, clogD = 2.77,
pKa = 5.42). When the N-trifluoroethylamino (14i) was re-
placed by an N-para-fluorobenzylamino (14e, clogD = 3.71,
pKa = 7.08), the Aβ42 activity of 14e increased more than 3
fold. This result suggests that the phenyl ring can increase
activity. When the N-cyclohexanamino (14j, clogD = 2.86,
pKa = 8.42) was replaced by an N-(4-fluorophenyl) amino, the
Aβ42 inhibition activity and selectivity are greatly improved
(39 nM and 499-fold for 14a, clogD = 4.20, pKa = 5.33). This
result is probably due to the strong electron withdrawing effect
and the lipophilic effect of the fluorine introduced on the phe-
nyl ring. When a tertiary amino (14f) or N-alpha-methyl para-
fluorobenzylamino was introduced (14g), the compounds lost
some activity when compared to 14e. When nitrogen was re-
placed by oxygen (17a, b and 18) the compound also lost
2
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
some activity relative to 14e. Nevertheless, we found that the
1
2
3
4
5
6
7
8
enantiomerically pure compounds 14a and 14c are two com-
pounds in the series with both good activity and high selectivi-
ty. To this point, the double bond and the lactam carbonyl
present in 1 was successfully replaced by a tetrahydroben-
zisoxazole ring. The new GSM compounds, 14a and 14c, are
two-fold more active than GSM 1, and have similar activity to
2 and 3. Additionally, 14a and 14c are more selective than 1
and 3, and have similar selectivity to 2.
15a
OH
0.19
0.15
105
102
15b
OH
9
Table 1. Cellular IC50 of Tetrahydrobenzisoxazole Analogs
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
O
N
O
R1
R2
N
N
16
H
H
H
0.11
0.29
0.20
178
68
4
Comp-
ound
Aβ42 IC50,
(µM)
R1
R2
Aβtot/Aβ42
17a
17b
3.9×10-2
499
(14a)
14a, b
H
98
0.23 (14b)
86
575
28
2.9×10-2
(14c)
14c, d
H
18
H
0.51
39
0.64 (14d)
n = 3
14e
14f
H
H
0.13
32
79
Mass spectrometric analysis of Aβ peptides secreted by
HEK293 cells carrying the London-Swedish double mutation
after treatment with vehicle (DMSO) or 10 ꢀM solutions of
compounds 14a, 14c, 14e is shown in Figure 2. As expected
for γ-secretase modulators, compounds 14a, 14c, and 14e
blocked the production of Aβ42 and Aβ40, while the relative
proportion of Aβ38 and Aβ37 is increased in comparison to
Aβ40 (numbers in spectra are amyloid-β numbering). The Aβ
profile in conditioned medium was analyzed using surface
enhanced laser desorption/ionization (SELDI) mass spectrom-
etry as described previously.33
0.25
14g
14h
14i
H
H
H
0.26
1.2×10
0.46
77
2
43
14j
H
0.31
64
3
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
strate (Pa-b = 68 nm/s, Pb-a = 462 nm/s, ER = 6.8). The efflux
ratio of 14c is 4-fold higher than 14a (Pa-b = 376 nm/s, Pb-a
600 nm/s, and ER= 1.6). The higher efflux ratio of 14c could
1
2
=
3
4
be the cause for its low brain penetration and poor in vivo effi-
cacy.
5
6
7
8
Table 2. Drug levels in plasma and brain
Comp- Plasma Concentration
Brain Concentration
ound
14a
14c
(ꢀM)*
10.4
6.01
(ꢀM)*
14.3
2.19
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
measured 3 h after the dosing (n = 3)
In summary, we designed and synthesized a series of GSM
compounds containing a unique tetrahydrobenzisoxazole ring.
The unique tetrahydrobenzisoxazole ring successfully replaced
the double bond in previously published GSMs. This series
contained several compounds which are potent GSMs with
good to excellent selectivity. Specifically, 14a and 14c, are
two-fold more active than 1 and have similar activity to GSM
2 and 3. In addition, 14a and 14c are more selective than GSM
1 and 3, and have similar selectivity to 2. Selected compounds
were tested in vivo. Among them, compound 14a significantly
reduced CSF Aβ42 by 58% after 3 h, dosed orally at 30 mg/kg
in rats compared to vehicle-treated animals.
Figure 2. Mass spectrometric analysis of Aβ profile
Compounds 14a, 14c, 14d and 15a were then tested in vivo
rat model. Compound 14a significantly reduced cerebrospinal
fluid (CSF) Aβ42 by 58% (p = 0.0066, n = 5) 3h after a single
oral dose at 30 mg/kg in rats compared to the vehicle-treated
animals (Figure 3).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
120
100
80
60
40
20
0
Biological assay protocols, general experimental descriptions,
procedures and characterization of final compounds. (File type,
PDF)
AUTHOR INFORMATION
Corresponding Author
* Tel: +1 908-740-2383; E-mail; zhiqiang.zhao@merck.com
Vehicle 14a
14c
14d
15a
Compound
ACKNOWLEDGMENT
We thank Dr. Alexander Pasternak and Dr. Santhosh Neelamkavil
for their encouragement and review of this paper.
Figure 3. CSF Aβ42 reduction in rat
Measured 3 h after an acute oral dosing at 30 mg/kg com-
pared to the vehicle
ABBREVIATIONS
Abbreviations: AD, Alzheimer's disease; Aβ, amyloid β; ; AE,
adverse effects; APP, amyloid precursor protein; BACE, β-
amyloid cleaving enzyme; CSF, cerebrospinal fluid; DEAD, di-
ethyl azodicarboxylate; GSM, γ-secretase modulators; GSI, γ-
secretase inhibitor; NCS, N-chlorosuccinimide; TMS, trimethylsi-
lyl.
Compound 14a was the most efficacious among compounds
tested, albeit it was not as potent as 14c in vitro. To get an
insight into this discrepancy, plasma and brain drug levels
were assessed in the rat tissues collected after dosing. We con-
cluded that the efficacy of 14a was driven by its excellent
penetration into the CNS compartment, which provided a ~6-
fold higher exposure compared to 14c (Table 2). This conclu-
sion was supported by the fact that 14c is a PGP efflux sub-
Notes
The authors are or were employees of Merck.
REFERENCES
4
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
(1) Alzheimer’s Association. 2017 Alzheimer’s Disease Facts and Figures. Alzheimer’s & Dementia 2017; 13, 325-373.
(2) Esparza, T.J.; Zhao. H.; Cirrito, J.R.; Cairns, N. J.; Bateman, R. J.; Holtzman, D. M.; Brody, D. L. Amyloid-beta Oligomerization in
Alzheimer Dementia vs. High Pathology Controls. Ann Neurol. 2013, 73(1), 104–119.
(3) Sadigh-Eteghad, S.; Sabermarouf, B.; Majdi, A.; Talebi, M.; Farhoudi, M.; Mahmoudi, J. Amyloid-Beta: A Crucial Factor in
Alzheimer’s Disease. Med. Princ. Pract. 2015, 24(1), 1–10.
9
(4) Kreft, A. F.; Martone, R.; Porte, A. Recent Advances in the Identification of γ-Secretase Inhibitors to Clinically Test the Aβ Oligomer
Hypothesis of Alzheimer’s Disease. J. Med. Chem. 2009, 52, 6169–6188.
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
(5) Irvine, G. B.; El-Agnaf, O. M.; Shankar, G. M.; Walsh, D. M.; Protein Aggregation in the Brain: The Molecular Basis for Alzheimer’s
and Parkinson’s Diseases, Mol Med. 2008, 14(7-8): 451–464.
(6) Gu, L. and Guo, Z.; Alzheimer's Aβ42 and Aβ40 Peptides form Interlaced Amyloid Fibrils, J. Neurochem. 2013, 126(3), 305–311.
(7) Chen, Y. and Glabe, C. G. Distinct Early Folding and Aggregation Properties of Alzheimer Amyloid-β Peptides Aβ40 and Aβ42. J. of
Biological Chem. 2006, 281(34), 24414-24422.
(8) Jonsson, T.; Atwal, J. K.; Steinberg, S.; Snaedal, J.; Jonsson, P. V.; Bjornsson, S.; Stefansson, H.; Sulem, P.; Gudbjartsson, D.; Malo-
ney, J.; Hoyte, K.; Gustafson, A.; Liu, Y.; Lu, Y.; Bhangale, T.; Graham, R. R.; Huttenlocher, J.; Bjornsdottir, G.; Andreassen, O. A.;
Joensson, E. G.; Palotie, A.; Behrens, T. W.; Magnusson, O. T.; Kong, A.; Thorsteinsdottir, U.; Watts, R. J.; Stefansson, K. A Mutation in
APP Protects Against Alzheimer’s Disease and Age-Related Cognitive Decline. Nature 2012, 488, 96−99.
(9) Yan, R.; Vassar, R. Targeting the β-Secretase BACE1 for Alzheimer's Disease Therapy. Lancet Neurol. 2014, 13, 319-329
(10) Choi, S. H.; Norstrom, E. Moderate Reduction of γ-Secretase: Is There a Therapeutic Sweet Spot? J. Neurosci. 2007, 27, 13579-
13580.
(11) Bursavich, M. G.; Harrison, B. A.; Blain, J-F. Gamma Secretase Modulators: New Alzheimer’s Drugs on the Horizon? J. Med. Chem.
2016, 59, 7389-7409.
(12) Xia, W.; Wong, S. T.; Hanlon, E.; Morin, P. γ-Secretase Modulator in Alzheimer's Disease: Shifting the End. J. Alzheimer's Dis.
2012, 31, 685-696.
(13) Weggen, S., Eriksen, J. L., Das, P., Sagi, S. A., Wang, R., Pietrzik, C. U., Findlay, K. A., Smith, T. E., Murphy, M. P., Bulter, T.,
Kang, D. E., Marquez-Sterling, N., Golde, T. E., and Koo, E. H. A Subset of NSAIDs Lower Amyloidogenic Aβ42 Independently of Cy-
clooxygenase Potency. Nature 2011, 414, 212−216.
(14) Crump, C. J.; Johnson, D. S. Li, Y-M.; Development and Mechanism of γ‑Secretase Modulators for Alzheimer’s Disease, Biochemis-
try 2013, 52, 3197 – 3216.
(15) Joo, Y.; Kim, H.-S.; Woo, R.-S.; Park, C. H.; Shin, K.-Y.; Lee, J.-P.; Chang, K.-A.; Kim, S.; Suh, Y.-H. Mefenamic Acid Shows
Neuroprotective Effects and Improves Cognitive Impairment in in vitro and in vivo Alzheimer's Disease Models. Mol. Pharmacol. 2006,
69, 76−84.
(16) Davis, K. L. NSAID and Alzheimer's Disease; Possible Answers and New Questions. Mol. Psychiatry 2002, 7, 925−926.
(17) Kimura, T.; Kawano, K.; Doi, E.; Kitazawa, N.; Shin, K.; Miyagawa, T.; Kaneko, T.; Ito, K.; Takaishi, M.; Sasaki, T.; Hagiwara, H.
Preparation of Cinnamide, 3-Benzylidenepiperidin-2-one, Phenylpropynamide Compounds as Amyloid β Production Inhibitors. PCT Int.
Appl. 2005, WO 2005115990 A1 20051208.
(18) Sun, Z.-Y.; Asberom, T.; Bara, T.; Bennett, C.; Burnett, D.; Chu, I.; Clader, J.; Cohen-Williams, M.; Cole, D.; Czarniecki, M.;
Durkin, J.; Gallo, G.; Greenlee, W.; Josien, H.; Huang, X.; Hyde, L.; Jones, N.; Kazakevich, I.; Li, H.; Liu, X.; Lee, J.; MacCoss, M.;
Mandal, M. B.; McCracken, T.; Nomeir, A.; Mazzola, R.; Palani, A.; Parker, E. M.; Pissarnitski, D. A.; Qin, J.; Song, L.; Terracina, G.;
Vicarel, M.; Voigt, J.; Xu, R.; Zhang, L.; Zhang, Q.; Zhao, Z.; Zhu, X.; Zhu, Z. Cyclic Hydroxyamidines as Amide Isosteres: Discovery of
Oxadiazolines and Oxadiazines as Potent and Highly Efficacious γ-Secretase Modulators in vivo. J. Med. Chem. 2012, 55, 489–502.
(19) Huang, X.; Zhou, W.; Liu, X.; Li, H.; Sun, G.; Mandal, M.; Vicarel, M.; Zhu, X.; Bennett, C.; McCraken, T.; Pissarnitski, D.; Zhao,
Z.; Cole, D.; Gallo, G.; Zhu, Z.; Palani, A.; Aslanian, R.; Clader, J.; Czarniecki, M.; Greenlee, W.; Burnett, D.; Cohen-Williams, M.;
Hyde, L.; Song, L.; Zhang, L.; Chu, I.; and Buevich. A. Synthesis and SAR Studies of Fused Oxadiazines as γ‑Secretase Modulators for
Treatment of Alzheimer's Disease. ACS Med. Chem. Lett. 2012, 3, 931−935.
(20) Haugabook, S. J.; Yager, D. M.; Eckman, E. A.; Golde, T. E.; Younkin, S. G.; Eckman, C. B. High Throughput Screens for the Identi-
fication of Compounds That Alter the Accumulation of The Alzheimer’s Amyloid Beta Peptide (Abeta). J. Neurosci. Methods 2001, 108,
171−179.
(21) Findeis, Mark A.; Schroeder, F. C.; Creaser, S. P.; McKee, T. D. and Xia, W. Natural Product and Natural Product-Derived Gamma
Secretase Modulators from Actaea Racemosa Extracts. Medicines 2015, 2, 127 -140.
(22) Huang, X.; Aslanian, R.; Zhou, W.; Zhu, X.; Qin, J.; Greenlee, W.; Zhu, Z.; Zhang, L.; Hyde, L.; Chu, I.; Cohen-Williams, M.; Palani,
A. The Discovery of Pyridone and Pyridazone Heterocycles as γ-Secretase Modulators. ACS Med. Chem. Lett. 2010, 1, 184−187.
(23) Qin, J.; Zhou, W.; Huang, X.; Dhondi, P.; Palani, A.; Aslanian, R.; Zhu, Z.; Greenlee, W.; Cohen-Williams, M.; Jones, N.; Hyde, L.;
Zhang, L. Discovery of a Potent Pyrazolopyridine Series of γ-Secretase Modulators. ACS Med. Chem. Lett. 2011, 2, 471−476.
(24) Qin, J.; Dhondi, P.; Huang, X.; Mandal, M.; Zhao, Z.; Pissarnitski, D.; Zhou, W.; Aslanian, R.; Zhu, Z.; Greenlee, W.; Clader, J.;
Zhang, L.; Cohen-Williams, M.; Jones, N.; Hyde, L.; Palani, A. Discovery of Fused 5,6-Bicyclic Heterocycles as γ-Secretase Modulators.
Bioorg. Med. Chem. Lett. 2011, 21, 664−669.
(25) Caldwell, J. P.; Bennett, C. E.; McCracken, T. M.; Mazzola, R. D.; Bara, T.; Buevich, A. V.; Burnett, D. A.; Chu, I.; Cohen-Williams,
M.; Jones, N. T.; Josien, H.; Hyde, L. A.; Lee, J.; McKittrick, B.; Song, L.; Terracina, G.; Voigt, J. H.; Zhang, L.; Zhu, Z. Iminoheterocy-
cles as Gamma-Secretase Modulators. Bioorg. Med. Chem. Lett. 2010, 20, 5380−5384.
(26) Zhu, Z.; Greenlee, W. J.; Li, H.; Vicarel, M. L.; Qin, J.; Dhondi, P. K.; Huang, X.; Palani, A.; Liu, X.; Sun, Z.-Y.; Josien, H. B.; Xu,
R.; Cole, D. J.; Burnett, D. A.; Bennett, C. E.; Mccracken, C. M.; Maccoss, M. Heterocyclic compounds as gamma-secretase modulators
and their preparation and use in the treatment of CNS diseases. PCT Int. Appl. 2010, WO 2010056722 A1 20100520.
5
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
1
2
3
4
5
6
7
8
9
(27) Zhu, Z.; Greenlee, W. J.; Cole, D. J.; Pissarnitski, D. A.; Gallo, G. V.; Li, H.; Josien, H. B.; Qin, J.; Knutson, C. E.; Mandal, M.;
Vicarel, M. L.; Rajagopalan, M.; Dhondi, P. K.; Xu, R.; Sun, Z.-Y.; Bara, T. A.; Huang, X.; Zhu, X.; Zhao, Z.; Clader, J. W.; Palani, A.;
Asberom, T.; Mccracken, T.; Bennett, C. E. Gamma Secretase Modulators. PCT Int. Appl. 2010, WO 2010054078 A1 20100514.
(28) Greenlee, W. J.; Pissarnitski, D. A.; Zhao, Z.; Zhu, Z. Gamma Secretase Modulators. PCT Int. Appl. 2013, WO 2013066740 A1
20130510.
(29) Sakai, T.; Miyata,K.; Utaka,M.; and Takeda, A. Me3SiCl-NaI-CH3CN as an Efficient and Practical Reducing Agent for Benzylic Al-
cohols. Tetrahedron Lett. 1987, 28(33), 3817-3818.
(30) Please see the supporting information for details.
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
(31) Rankovic, Z. CNS Drug Design: Balancing Physicochemical Properties for Optimal Brain Exposure, J. Med. Chem. 2015, 58, 2584-
2608.
(32) Charifson, S. P. and Walters, W. P. Acidic and Basic Drugs in Medicinal Chemistry. J. Med. Chem. 2014, 57, 9701−9717.
(33) Lee, J.; Song, L.; Terracina, G.; Bara, T.; Josien, H.; Asberom,T.; Sasikumar, T. K.; Burnett, D. A.; Clader, J.; Parker, E. M.; Zhang,
L. Identification of Presenilin 1-Selective γ-Secretase Inhibitors With Reconstituted γ-Secretase Complexes. Biochemistry 2011, 50,
4973−4980.
6
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Discovery of a Tetrahydrobenzisoxazole Series of γ-Secretase
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
Modulators
Zhiqiang Zhao,a,* Dmitri A. Pissarnitski,a Xianhai Huang,a Anandan Palani,a Zhaoning Zhu,a
William J. Greenlee,a Lynn A. Hyde,b Lixin Song,b Giuseppe Terracina,b Lili Zhang,b Eric M.
Parker b
a Department of Medicinal Chemistry, MRL, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Department of Neurobiology, MRL, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA
For Table of Contents Use Only.
ACS Paragon Plus Environment