Discovery of Investigational Drug CT1812, an Antagonist of the Sigma-2 Receptor Complex for Alzheimer's Disease

Article abstract of DOI:10.1021/acsmedchemlett.1c00048

An unbiased phenotypic neuronal assay was developed to measure the synaptotoxic effects of soluble Aβ oligomers. A collection of CNS druglike small molecules prepared by conditioned extraction was screened. Compounds that prevented and reversed synaptotox

Full text of DOI:10.1021/acsmedchemlett.1c00048

pubs.acs.org/acsmedchemlett
Letter
Discovery of Investigational Drug CT1812, an Antagonist of the
Sigma‑2 Receptor Complex for Alzheimer’s Disease
Gilbert M. Rishton, Gary C. Look, Zhi-Jie Ni, Jason Zhang, Yingcai Wang, Yaodong Huang,
Xiaodong Wu, Nicholas J. Izzo, Kelsie M LaBarbera, Colleen S. Limegrover, Courtney Rehak,
Raymond Yurko, and Susan M. Catalano*
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ABSTRACT: An unbiased phenotypic neuronal assay was developed to measure
the synaptotoxic effects of soluble Aβ oligomers. A collection of CNS druglike small
molecules prepared by conditioned extraction was screened. Compounds that
prevented and reversed synaptotoxic effects of Aβ oligomers in neurons were
discovered to bind to the sigma-2 receptor complex. Select development
compounds displaced receptor-bound Aβ oligomers, rescued synapses, and restored
cognitive function in transgenic hAPP Swe/Ldn mice. Our first-in-class orally
administered small molecule investigational drug 7 (CT1812) has been advanced
to Phase II clinical studies for Alzheimer’s disease.
KEYWORDS: CT1812, amyloid oligomer, amyloid oligomer-displacing, AβO, AβO-displacing, Alzheimer’s disease drug, sigma-2,
transmembrane protein 97, TMEM97, progesterone receptor membrane component 1, PGRMC1, supercritical fluid extraction, SFE,
conditioned extraction, CE
he role of amyloid 1−42 (Aβ) in neurodegeneration and
Alzheimer’s disease (AD) has been widely studied.1
membrane trafficking, reduce the binding of AβOs to neurons,
and prevent dendritic spine loss.
T
Disease-modifying drug development focus has shifted from
Aβ fibril and plaque onto soluble Aβ oligomers (AβOs) that
are known to be one of the most potently synaptotoxic species
of Aβ.2−12 Particularly synaptotoxic soluble AβOs are freely
diffusible within the brain, versus localized fibril- and plaque-
associated Aβ.13 Soluble AβOs exert reversible effects in the
human cortex,7 in neuronal assay systems,14,15 and in animal
models of cognition.16−18 The pharmacologic blockade of
reversible AβO binding to neuronal receptors and its
synaptotoxic effects may slow the AβO-induced events
associated with synaptic dysfunction and gognitive deficits in
MCI and AD. The MTT assay19 is commonly used as a
measure of toxicity in cultured cells and was adapted for use
measuring membrane trafficking changes in neurons (“Traffick-
ing Assay”, Table 1).20,21 We used this assay to discover
compounds that blocked the synaptotoxic effects of preformed
soluble AβOs at sublethal concentrations.22 Administration of
soluble AβOs to this neuronal system demonstrated that both
synthetic and human brain-derived soluble AβOs behaved as
pharmacologic ligands, bound to and saturated neuronal
receptors, and exerted functional synaptotoxic effects related
to binding.22,23 We have utilized this unbiased phenotypic
neuronal trafficking assay to screen for CNS druglike small
molecules. These molecules also displace bound AβO from
neuronal synaptic receptors to prevent and reverse synaptotox-
icities.22−24 The active compounds identified in this assay were
able to treat and prevent oligomer-induced deficits in
The AβO-displacing compounds identified in our unbiased
phenotypic neuronal trafficking assay were subsequently
screened biochemically against a broad panel of CNS relevant
receptors (EuroFins/Cerep panel of 117 trans-membrane
proteins and soluble receptors, ion channels, and monoamine
transporters). Compound 7 was found to be >100-fold
selective for all measured targets save for the sigma-1 receptor
for which compound 7 was approximately 10-fold more
selective. The effective AβO-displacing compounds reported
here were found to be high affinity binders to the sigma-2
receptor.22,23 We have characterized the binding of our own
AβO-displacing compounds in the presence of other known
sigma-2 ligands23 and have found our AβO-displacing
compounds to bind potently and selectively to the canonical
sigma-2 site.22−24 A structure−activity relationship emerged
between the AβO-displacing activity in our neuronal assay and
sigma-2 receptor binding affinity. The sigma-2 receptor has
been the subject of ongoing characterization studies and, via
affinity purification and micro protein sequencing, identified as
Received: January 22, 2021
Accepted: August 3, 2021
https://doi.org/10.1021/acsmedchemlett.1c00048
© XXXX American Chemical Society
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Table 1. Structure−Activity Relationships of the Anti-AβO
Compounds 1−7
Scheme 2. Preparation of Isoindoline Compounds 4−745
Table 2. Physiochemical Properties and Brain-to-Plasma
Ratios (AUCbrain/AUCplasma) of Anti-AβO Compounds
Scheme 1. Synthesis of Anti-AβO Compounds 2 and
337−39,45
Brain/plasma
AUC
Brain/plasma 24 h
postdose
cmpd
MW
cLogP tPSA
(R)-2 376.2
6.48
5.89
4.65
3.26
12.3
12.0
32.7
66.8
NT
NT
5.64
2.51
8.2
2.0
5.4
5.7
3
6
7
341.8
357.5
431.6
Figure 1. Aβ oligomers (3 μM, red bars) decrease membrane vesicle
trafficking rate compared to vehicle (blue bars). Compound 7
(CT1812) significantly reduces these deficits when added before Aβ
oligomers (Prevention, EC50 = 6.8 uM and 71% recovery) or after Aβ
oligomers (Treatment, EC50 = 358 nM and 88% recovery).24 There
is no effect of CT1812 on membrane vesicle trafficking on its own at
30X EC50 for either prevention or treatment bound AβO when
added 1 h after addition of AβO.
transmembrane protein 97 (TMEM97) in tumor cell lines.25
The TMEM97 protein is likely to form a tight complex with
other molecules such as PGRMC1.26 Sigma-2-selective small
molecules have been developed as imaging agents and as
potential therapeutic agents for oncologic27,28 and neuro-
logic29−32 end points.
AβOs bind saturably and reversibly to a single receptor site
at neuronal synapses that mediates synaptotoxic effects and
neurodegenerative processes resulting from AβO expo-
sure.3,33,34 Previous reports indicate this receptor is a
multiprotein complex most likely consisting of cellular prion
protein (PrPC),14 NgR1,35 and LilRB2.36
als.37−39 SFE effectively selects for low molecular weight
compounds and simultaneously excludes high molecular
weight compounds, polyphenolic compounds, polyionic
compounds, and cellulosic materials that would likely interfere
with the screening process. The light oil SFE fractions are
commonly comprised of relatively volatile low molecular
weight components including reactive aldehydes, ketones,
lactones, Michael acceptors, epoxides, etc. Extract mixtures
containing these reactive natural products are readily
“conditioned” by application of one-, two-, or three-step CE
procedures including, for example, hydride reduction, organo-
metallic addition, oxidation/reduction, and reductive amina-
A CNS druglike chemical library was prepared using our
proprietary “conditioned extraction” (CE) platform beginning
from supercritical fluid extraction (SFE) of natural materi-
B
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Table 3. Compound 7 (CT1812) Reduced Mean Morris Water Maze Swim Length in Transgenic Mice24
nTg + vehicle
Tg + vehicle
SEM
Tg + CT1812
nTg + CT1812
Day
Mean, cm
SEM
N
Mean, cm
3518
N
Mean, cm
SEM
N
Mean, cm
SEM
N
1
2
3
4
3791
2666
2173
1797
150
294
213
219
12
12
12
12
138
329
267
287
10
10
10
10
3230
2332
1987
1611
115
218
265
231
10
10
10
10
3317
2739
2262
2008
192
265
259
160
12
12
12
12
a
3252
2854
a
2426
a
p < 0.5 vs nontransgenic + vehicle.
Table 4. Compound 7 (CT1812) Restored Spontaneous Alteration in the Y-Maze Test of Spatial Working Memory
nTg + vehicle
SEM
Tg + vehicle
SEM
Tg + CT1812
SEM
nTg + CT1812
SEM
Mean
62.6%
N
Mean
56.1%
N
Mean
58.5%
N
Mean
65.3%
N
3.7%
11
2.8%
11
2.8%
11
6.0%
12
a
p < 0.5 vs random chance (50%).
Table 5. Compound 7 (CT1812) Restored Freezing Time in the Contextual Fear Conditioning Test
nTg + vehicle
Tg + vehicle
Tg + CT1812
nTg + CT1812
Mean
52%
SEM
5
N
Mean
SEM
6
N
Mean
45%
SEM
6
N
Mean
51%
SEM
5
N
a
11
32%
12
12
11
a
p < 0.5 vs nontransgenic + vehicle.
tion to provide structurally diverse chemically stable CNS
druglike mixtures of alcohols and “alkaloidal” amines and
amino alcohols. A two-step reductive amination CE protocol
was performed on low molecular weight fractions of SFE
ginger oil to produce mixtures of chemically stable low
molecular weight natural and unnatural products.37 The
mixtures were fractionated, and the fraction pools were
screened in our phenotypic neuronal assay. The racemic
secondary amine 1 (Table 1), which was derived from the
reductive amination of natural gingerone present in the original
SFE ginger oil mixture on reaction with 4-(trifluoromethyl)-
benzylamine, protected neurons from AβO-induced synapto-
toxicity and displayed anti-AβO activity. The metabolic
instability of 1 as measured by mouse liver microsomes
(MLM) prompted our search for structurally analogous
metabolically stable anti-AβO compounds for oral admin-
istration in a transgenic AD mouse model. Substitution for or
removal of the HO- and MeO-substituents of compound 1 was
investigated to probe the likely possibilities of oxidative and
conjugative metabolism at these sites. The chlorinated
compounds 2 and 3 exhibited promising anti-AβO activity
and significantly improved metabolic stability and so were
suitable for advancement to animal behavioral studies.
Comparative assay of the enantiomers (R)-2 and (S)-2
demonstrated significant differences in neuronal activity and
hERG activity (EuroFins, IC50 based on hERG-CHO,
automated patch-clamp assay). We proceeded to prepare the
generally superior (R)-isomers for subsequent analogue work
in this series; however, the hERG activity of these relatively
lipophilic amine compounds remained a concern. We
discovered that reintroduction of phenol and alkoxy aryl
substituents resulted in diminished hERG activity likely due to
increased polar surface area (PSA) of the oxygenated
compounds relative to the chlorinated compounds. However,
improvement in hERG activity upon increase in PSA was often
accompanied by diminished neuronal activity and metabolic
instability. A significant breakthrough came with a series of
isoindoline analogues exemplified by compounds 4−7.
Introduction of the isoindoline moiety realized submicromolar
neuronal activity. Furthermore, it allowed for the reintroduc-
tion of oxygenated substituents and seemed to strike a balance
between increased PSA (to address hERG activity40−42) and
improved neuronal activity and metabolic stability. Our
advanced candidate isoindolines 6 and 7 benefited from the
combination of properties including relatively high PSA,
oppositely polarized aromatic rings, a gem-dimethyl sub-
stitution alpha to nitrogen, and the favored isoindoline moiety.
Significant increases in PSA were of particular benefit to our
advanced candidate isoindolines 6 and 7. The introduction of
the methyl sulfone substituent increased PSA between 2- and
5-fold in our various structural analogue series. However, the
dramatic increase in PSA and the accompanying increase in
molecular weight often resulted in loss of neuronal activity or
metabolic instability. The methyl sulfone substitution was well-
tolerated in the isoindoline series and particularly wins the case
of the isoindoline compound 7. Compound 7 exhibited
excellent neuronal activity, high affinity binding at sigma-2, and
excellent selectivity versus a blockade of hERG ion currents.
Notably, the chiral center present in compounds 1−4 had been
replaced by gem-dimethyl substitution alpha to the isoindoline
nitrogen. The gem-dimethyl substitution in compounds 6 and
7 likely blocked oxidative metabolism at the carbon alpha to
nitrogen and was also likely responsible for inducing
entropically favorable restrictions to bioactive conformations,.
The use of the gem-dimethyl substitution is a strategy that has
been used by medicinal chemists when designing clinical useful
compounds.43
The available37−39 methyl ketones 8 and 9 (Scheme 1) were
converted by applying Ellmann’s method44 to the N-tert-
butanesulfinyl ketimines 10 and 11. Subsequent hydride
reduction provided the chiral alpha-methyl amines 12 and
13. The chiral amines were subsequently converted via
reductive amination to the chiral anti-AβO compounds (R)-
2, (S)-2, and 3. The unoptimized overall yield from ketones 8
or 9 is around 6%.
C
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A general synthesis of isoindoline anti-AβO compounds was
developed (Scheme 2). Ready access to the gem-dimethyl-
substituted isoindolines was realized via palladium-mediated
coupling46 of aryl iodides 14 and 1545 to 2-methyl-3-butyn-2-
amine to provide the gem-dimethyl-substituted propargyl-
amines 16 and 17 (74% yield for 17). These were
subsequently hydrogenated to the intermediate amines 19
and 20 (100% yield for 20). The available chiral alpha-methyl
amines 12 and 1837−39 and the gem-dimethyl-substituted
amines 19 and 20 were condensed with the corresponding bis-
benzylbromides 21−23 to provide the isoindoline anti-AβO
compounds 4−7 (63% yield for 7).45
dependent learning and memory compared to vehicle-treated
animals in the fear conditioning assay (Table 5).
Microelectrodes coated with antibodies for total Aβ or AβOs
were inserted into the brains of live transgenic mice and
measured soluble Aβ concentration by the minute.24,49
Intravenous administration of compound 7 increased release
of AβO oligomers and increased AβO concentration in the
interstitial brain fluid without causing a measurable increase in
Aβ monomer concentration. Remarkably, a concomitant
increase in the concentration of AβO in the cerebrospinal
fluid (CSF) was also observed.24 This strongly suggests that
AβO displacement by compound 7 in the brain facilitates
clearance of interstitial AβO to the CSF.
Our early lead compounds 2 and 3 had exhibited promising
neuronal activity and performed well in animal behavioral
models but were exceedingly lipophilic as reflected in their low
PSA (∼12). These compounds exhibited problematic off-target
activity profiles and hERG activity. Certain trends in
physiochemical properties including a substantial increase in
total polar surface area (PSA) (Table 2) contributed to
progress in the program and enabled the nomination of
suitable development candidates. It became clear that
increased PSA addressed off-target and hERG activities. The
reintroduction of phenol and alkoxy substituents as in
compounds 5 and 6 informed the design of next generation
compounds. Incorporation of the aryl methyl sulfone increased
PSA, minimized off-target activities and hERG activity, and
enabled the nomination of our clinical candidate 7. Compound
7 exhibits good absorption and a particularly high brain-to-
plasma ratio of 5.7 at 24 h postdose (Kp,uu = 6.75). The free
fraction of compound 7 in the brain is 13% and in the plasma
is 5%. Compound 7 is not a Pgp substrate and has a CACO-2
Papp(B-A)/Papp(A-B) = 0.95.
This limited structure−activity relationship represents a
medicinal chemistry effort that produced hundreds of
structural analogues. Our effort to optimize anti-AβO activity
in our neuronal assay was complicated by issues of off-target
activities and pharmacokinetics that are beyond the scope of
this Letter. However, the structural features required of the
anti-AβO pharmacophore are well represented here. In
summary, the simple lipophilic benzylic amines 2 and 3
exhibited promising anti-AβO activity and acceptable meta-
bolic stability. They were efficacious in animal behavioral
testing22,24 but suffered from hERG and other off-target
activities due, apparently, to their relatively low PSA. The
isoindolines 4−7 exhibited promising submicromolar neuronal
activity. The isoindoline scaffold tolerated polar substitution
and allowed dramatic increases in PSA leading to highly active
compounds with minimal hERG and off-target activities. Our
advanced candidates 6 and 7 benefitted as well from gem-
dimethyl substitution alpha to nitrogen that imparts metabolic
stability and optimal conformational biases for anti-AβO
activity.
In primary hippocampal and cortical neuronal cultures AβOs
were found to bind specifically and to saturate a single receptor
site on some but not all neurons.22,23 The anti-AβO
compounds reported here prevented and restored trafficking
deficits caused by AβOs in neurons. The offset between sigma-
2 binding affinity and potency in the in vitro membrane vesicle
trafficking assay likely results from several factors such as (1)
lipophilic compound adsorption to microtiter plate plastic that
lowers the effective concentration of the compounds, (2)
greater than 80% of sigma-2 receptors need to be occupied by
compound to achieve an effect on binding and function, or (3)
the high concentration of the low potency synthetic Aβ
preparations needed to achieve adequate testing windows in
the in vitro assays. Clinical candidate 7 prevented and reversed
trafficking deficits caused by AβOs but had no effect in the
absence of AβOs (Figure 1). Compound 7 also prevented
binding AβO to neuronal receptors, displaced prebound AβO,
and was determined by a one-site ELISA assay to have no
effect on AβO assembly or AβO dissociation.24,47
The anti-AβO compounds 2, 3, 6, and 7 restored cognitive
function in transgenic hAPP Swe/Ldn mice (Tables
3−5).22,24,48 The drug-treated transgenic mice performed in
the Morris water maze task significantly better than did the
transgenic vehicle-treated mice (Table 3). Treatment with
compound 7 does not affect nontransgenic animal perform-
ance. Transgenic mice treated with compound 7 remembered
previous arms entered in the Y-maze task significantly better
than chance but vehicle-treated transgenic animals did not
(Table 4). Transgenic mice treated with compound 7
demonstrated significant improvements in spatial and cue-
The CNS druglike small molecules reported here are
potentially first-in-class therapeutics for the treatment and
prevention of early cognitive decline and neurodegeneration in
MCI and AD patients. Our first-in-class clinical candidate
compound 7 (CT1812)45 is an AβO-displacing compound
and a potent and highly selective antagonist of the sigma-2
receptor. Compound 7 has been demonstrated to prevent AβO
binding to neurons and also to displace bound AβO from
neuronal receptors.22−24 It has been determined to have no
effect on AβO assembly or AβO dissociation in a biochemical
ELISA assay.24,47 It is metabolically stable and exhibits good
pharmacokinetics and robust brain exposure and restores
synapse number and cognitive function in transgenic mouse
models.24,48 Remarkably, compound 7, upon AβO-displace-
ment in the rodent brain, facilitates the clearance of AβO from
brain interstitial fluid to CSF as demonstrated in a mouse
microimmunoelectrode study.24 Unlike other Aβ-targeted
therapeutics, by displacing Aβ oligomer binding, CT1812
lowers Aβ oligomer affinity for its receptor, the same
phenotype observed with the protective Icelandic mutation
that confers 4-fold lower incidence of Alzheimer’s disease on
carriers.50
In phase I clinical studies, compound 7 was determined to
be safe and well tolerated at single doses up to 1120 mg and at
multiple doses up to 560 mg in healthy elderly volunteers.51,52
Adverse events, most commonly headache and gastrointestinal
symptoms, were mild to moderate in severity. Plasma
concentrations of drug were found to be dose proportional,
and CSF concentrations upon multiple doses exceeded the
expected minimum target concentrations required to improve
D
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Funding
memory in AD patients. Compound 7 (CT1812) is an orally
administered first-in-class small molecule drug candidate for
Alzheimer’s disease. When administered to mild to moderate
Alzheimer’s patients once daily for 28 days, CT1812
significantly increased CSF concentrations of Aβ oligomers
in AD patient CSF, reduced concentrations of synaptic
proteins and phosphorylated tau fragments, and reversed
expression of many AD-related proteins dysregulated in CSF
compared to placebo.24 Four randomized, double-blind,
placebo-controlled phase II clinical studies are currently
underway in patients with mild to moderate AD: SNAP
(NCT03522129), SPARC (NCT03493282), SHINE
(NCT03507790), and SEQUEL (NCT04735536).
Portions of this research were supported by the National
Institutes of Health’s National Institute on Aging, grants
R43AG037337 and R01AG051593, and by the National
Institute of Neurological Disorders and Stroke, grant
R43NS083175. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institutes of Health.
Notes
The authors declare the following competing financial
interest(s): Gilbert M. Rishton, Gary C. Look, Nicholas J.
Izzo, Kelsie M. LaBarbera, Colleen S. Limegrover, Courtney
Rehak, Raymond Yurko, and Susan M. Catalano are current or
former employees of Cognition Therapeutics, Inc.
ASSOCIATED CONTENT
■
ACKNOWLEDGMENTS
sı
* Supporting Information
■
The authors gratefully acknowledge the expert conduct of
animal behavioral studies by Prof. Mehrdad Shamloo and
Marie Monbureau at Stanford University and the pivotal
microimmunoelectrode (MIE) studies developed and per-
formed by Prof. John R. Cirrito and Carla M. Yuede at
Washington University School of Medicine, St. Louis.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00048.
Details of materials and methods used in this work
including NMR, MS, and chemical synthesis (PDF)
AUTHOR INFORMATION
■
ABBREVIATIONS
■
Corresponding Author
AD, Alzheimer’s disease; AβO, soluble Aβ oligomer; MCI,
mild cognitive impairment; CNS, central nervous system;
TMEM97, transmembrane protein 97; SFE, supercritical fluid
extraction; CE, conditioned extraction; PSA, polar surface area;
CSF, cerebrospinal fluid; MIE, microimmunoelectrode
Susan M. Catalano − Cognition Therapeutics, Pittsburgh,
Pennsylvania 15203, United States; Email: scatalano@
cogrx.com
Authors
Gilbert M. Rishton − Cognition Therapeutics, Pittsburgh,
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Gary C. Look − Cognition Therapeutics, Pittsburgh,
Pennsylvania 15203, United States
Zhi-Jie Ni − Acme Bioscience, Inc., Palo Alto, California
94303, United States
Jason Zhang − Acme Bioscience, Inc., Palo Alto, California
94303, United States
Yingcai Wang − Acme Bioscience, Inc., Palo Alto, California
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Yaodong Huang − Acme Bioscience, Inc., Palo Alto, California
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Xiaodong Wu − Acme Bioscience, Inc., Palo Alto, California
94303, United States
Nicholas J. Izzo − Cognition Therapeutics, Pittsburgh,
Pennsylvania 15203, United States; orcid.org/0000-
0002-0196-2055
Kelsie M LaBarbera − Cognition Therapeutics, Pittsburgh,
Pennsylvania 15203, United States
Colleen S. Limegrover − Cognition Therapeutics, Pittsburgh,
Pennsylvania 15203, United States
Courtney Rehak − Cognition Therapeutics, Pittsburgh,
Pennsylvania 15203, United States
Raymond Yurko − Cognition Therapeutics, Pittsburgh,
Pennsylvania 15203, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00048
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
themanuscript.
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