Discovery and optimization of Lu AF58801, a novel, selective and brain penetrant positive allosteric modulator of alpha-7 nicotinic acetylcholine receptors: Attenuation of subchronic phencyclidine (PCP)-induced cognitive deficits in rats following oral administration

Article abstract of DOI:10.1016/j.bmcl.2013.11.022

In this Letter, we describe a chemical lead optimization campaign starting from a novel, weak α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) hit from a HTS screen. Exploration of the structure-activity relationships for α7 PAM potency, intrinsic hepatic clearance, the structure-property relationships for lipophilicity, and thermodynamic solubility, led to the identification of Lu AF58801: a potent, orally available, brain penetrant PAM of the α7 nicotinic acetylcholine receptor, showing efficacy in a novel object recognition task in rats treated subchronically with phencyclidine (PCP).

Full text of DOI:10.1016/j.bmcl.2013.11.022

Bioorganic & Medicinal Chemistry Letters 24 (2014) 288–293
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of Lu AF58801, a novel, selective
and brain penetrant positive allosteric modulator of alpha-7
nicotinic acetylcholine receptors: Attenuation of subchronic
phencyclidine (PCP)-induced cognitive deficits in rats following oral
administration
a
a
a
a
Jørgen Eskildsen a, , John P. Redrobe , Anette G. Sams , Kim Dekermendjian , Morten Laursen ,
⇑
Jette B. Boll a, Roger L. Papke b, Christoffer Bundgaard a, Kristen Frederiksen a, Jesper F. Bastlund a
a Neuroscience Research DK, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark
b Department of Pharmacology, University of Florida College of Medicine, Gainesville, FL, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter, we describe a chemical lead optimization campaign starting from a novel, weak a7 nicotinic
Received 6 September 2013
Revised 7 November 2013
Accepted 10 November 2013
Available online 20 November 2013
acetylcholine receptor positive allosteric modulator (PAM) hit from a HTS screen. Exploration of the
structure–activity relationships for 7 PAM potency, intrinsic hepatic clearance, the structure–property
relationships for lipophilicity, and thermodynamic solubility, led to the identification of Lu AF58801: a
potent, orally available, brain penetrant PAM of the 7 nicotinic acetylcholine receptor, showing efficacy
in a novel object recognition task in rats treated subchronically with phencyclidine (PCP).
Ó 2013 Elsevier Ltd. All rights reserved.
a
a
Keywords:
Nicotinic acetylcholine receptors
Positive allosteric modulator
Electrophysiology
Lead optimization
Novel object recognition
There is general consensus, that the release of acetylecholine
(Ach) is a key factor in mnemonic mechanisms in humans1 and
rodents.2 Nicotine is known to enhance attention and cognitive
performance by a nonselective activation of multiple nicotinic ace-
tylcholine receptors (nAChRs). nAChRs belong to the super family
of ligand gated ionic channels, and gate the flow of cations includ-
ing calcium. Seventeen subunits of nAChRs have been reported to
diseases associated with memory impairment, including schizo-
phrenia and Alzheimer’s disease.5,6 Taken together, this evidence
suggests that activation of nAChR 7’s may lead to improvement
of cognitive processing in human diseases. To this end, compounds
like EVP6124 and TC5619, acting as agonists of the nAChR 7’s,
have recently been shown to alleviate cognitive disturbances
associated with schizophrenia and Alzheimer’s disease in small
clinical phaseII studies.7–9 Despite the proposed beneficial effects
a
a
date, which are identified as
tionally distinct nAChR complexes exist, for example five alpha7
subunits can form homomeric 7 receptors (nAChR 7), which
are some of the most abundant nicotinic receptors, along with het-
eromeric
4b2 receptors, in the brain (for review see3).
nAChR 7 are expressed in brain areas involved in cognitive
a2–a10, b1–b4, c, d, and e. Many func-
of nAChR
ment with agonists provide suboptimal benefit due to sustained
activation and subsequent desensitization of nAChR 7’s.
In contrast to agonists, nAChR 7 PAMs can reinforce endoge-
a7 agonism, it remains uncertain whether chronic treat-
a
a
a
a
a
a
nous cholinergic transmission without directly stimulating the
processing such as the hippocampus, thalamic nuclei, and frontal
cortex, and their physiological role in the modulation of neuro-
transmitter release, particularly glutamate and GABA, has focused
attention on this subtype.4 In addition to a physiological role,
target receptor.10 Thus, PAMs can selectively modulate the activity
of ACh at nAChR
kinetics of the receptor, without leading to desensitization after
prolonged binding to the receptor. Accordingly, nAChR 7 selective
PAMs have emerged as potential drug candidates.11 Here we report
on a new series of nAChR
7 selective PAMs.12
a7’s, preserving the activation and deactivation
a
nAChRa7 have been implicated in certain pathological conditions
in that decreased nAChR
a
7 expression have been reported in
a
High throughput screening of the Lundbeck compound collec-
tion identified a series of relatively lipophilic secondary amides
(Fig. 1) compounds 1–3.
⇑
Corresponding author. Tel.: +45 36301311; fax: +45 36438293.
E-mail address: JORE@lundbeck.com (J. Eskildsen).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.022

J. Eskildsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 288–293
289
O
O
O
O
(S)
(S)
N
H
N
H
N
H
N
H
(S)
O
1
2
3
4
Figure 1. HTS hits 1–3 and pure enantiomer 4.
Table 1
Profile of HTS hit 2
Table 2
SAR exploration of east side phenyl
Flux EC50
Flux MaxE
MW
3.3
37%
265.35 amu
lM
O
R
(S)
N
clogP14
tPSA
3.4
(S)
H
29 Å2
CLint,
CLint,
2.9 L/min
49 mL/min
human
Compd
R
EC50
(lM)
Emax (%)
Clint,human (L/min)
rat
CYP inhibition IC50
(
l
M)
2C9 (28) 2C19 (>40) 2D6 (19) 3A4 (12) 1A2
(>40)
lg/mL
5
6
7
8
H
4.2
3.3
1.9
3.6
>20
3.6
4.5
3.7
2.4
2.7
11
10
4
10
1
4.8
6.4
8.1
5.7
6.9
5.8
2.8
6.6
6.2
5.4
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
2-F
Thermodynamic solubility pH 12
7.4
9
10
11
12
13
14
9
13
15
12
8
A common feature was the 2-phenyl-cyclopropyl moeity on the
carbonyl side and a phenyl group on the amino-side of the amide
connected either via a single bond or via 1 or 2 carbon atoms.
The most active compound, 2, was profiled as shown in Table 1.
The absolute configuration of 2 was not available during the initial
HTS triage of primary hits, but analysis of the NMR spectra of 2
established the configuration of the cyclopropane moeity to be
trans. Compound 2 showed an EC50 of 3.3
tive to an Emax for PNU120596. Incubation of 2 in human liver
microsomes suggested relatively high intrinsic clearance
3-F
4-F
showed lower Emax values and high intrinsic microsomal clearance.
The clogP neutral introduction of a 4-MeO substituent to give 11
demonstrated the best stabilization in human liver microsomes
(Clint,human = 2.8 L/min) combined with a relatively high Emax for
this series of compounds. This general drop in Emax relative to 4
prompted us to investigate compounds containing the S methyl
in the benzylic position. It was clear that a critical issue for the ser-
ies would be optimization with respect to potency, microsomal
stability and aqueous solubility.
Based on experience from earlier projects, involving structurally
related compounds, we knew there was a propensity for potassium
channel modulation. In particular for lipophilic compounds bear-
ing meta substituents. To monitor and mitigate this undesired
cross-reactivity we therefore decided to include a human KCNQ2
screen in our SAR-driving screening cascade.16 Representative
lM and Emax = 37% rela-
a
(Clint,human = 2.9 L/min, liver blood flow 1.3 L/min). With respect
to physicochemical properties, 2 showed a thermodynamic aque-
ous solubility at pH 7.4 of 12
suggested to us that an 7 PAM tool compound may be derived
from these initial hits and we decided to invest in a hit-to-lead
campaign with the aim of indentifying a brain penetrant 7 PAM
tool compound. Key objectives were improvement in physico-
chemical and pharmacokinetic parameters, and to establish which
degree of in-vitro modulation was necessary to achieve in-vivo ef-
fects at relevant plasma and brain exposures.
l
g/mL.13 The combined observations
a
a
Separation of commercially available racemic trans 2-phenyl-
cyclopropane carboxylic acid by Supercritical Fluid Chromatogra-
examples illustrating the
shown in Table 3.
a7 SAR along with KCNQ2 data are
phy (SFC) gave R,R and S,Strans compounds.15 All
a7 PAM activity
As shown in Table 3, high intrinsic human clearance values
were observed for all these compounds, which may in part be
the result of the relatively high lipophilicity of compounds 4–21.
The clogP for the compounds in Table 3 were between 3.3 and
3.9. Relatively high KCNQ2 potency was observed for 15, 16 and
21. Of particular interest to us was the 4-MeO substituted com-
was displayed by compounds derived from S,Strans 2-phenylcyclo-
propyl carboxylic acid. As both enantiomers of 1-phenylethanam-
ines were commercially available, we were able to establish that
compounds derived from S-1-phenylethanamine were dramati-
cally more active than compounds derived from the R isomer. Thus
we could conclude that practically all
a7 PAM activity resided in
pound 18, which offered an attractive balance between the a7
compound 4 (Fig. 1).
PAM potency, especially the high Emax of 65%, and medium to
The initial chemistry focus was the synthesis of amides derived
from achiral benzylamines and S,S-transcyclopropyl carboxylic acid
to explore the SAR without significantly increasing the lipophilic-
ity. Both the measured logP and the clogP of 4 was 3.4. Represen-
tative examples are shown in Table 2. The SAR, as with other
allosteric ligands, was shallow and showed little variability in the
EC50 values. Introduction of methyl-, methoxy- or fluoro in the
meta- and para-portion of the benzylamine had little effect on
the EC50 or Emax values. While the fluoro substituent was allowed
in the ortho-, meta- and para-position (12–14) there was a
tendency for intolerance of bigger substituents like methoxy (9)
in the ortho position. Disappointingly, compounds 5 thru 14 also
low activity in the KCNQ2 flux assay. However, the high intrinsic
human clearance and low aqueous solubility of 1.8 lg/mL was
unacceptable. Next, our exploration focused on the linker region
with the specific aim to optimize the physicochemical and meta-
bolic properties.
Representative examples of these efforts are shown in Table 4.
The first analog 22 demonstrated, as also suggested from the initial
HTS hits, that a two carbon linker was also allowed.
However, this resulted in a dramatic increase in intrinsic human
clearance (Clint,human = 7.5 L/min). A breakthrough was achieved
with the identification of the R hydroxymethyl group as the
optimal replacement of the S methyl linker substituent. The R

290
J. Eskildsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 288–293
Table 3
Exploration of (S)-Me analogs
O
R
(S)
(S)
N
(S)
H
Compd
R
a7
clogP
Clint,
(L/min)
hKCNQ2
human
EC50
(
lM)
Emax (%)
EC50
(lM)
Emax (%)
4
H
3.2
2.6
>20
3.1
1.5
2.0
3.1
2.2
37
31
0
11
65
50
41
60
3.4
3.9
3.3
3.3
3.3
3.5
3.5
3.5
4.1
3.4
7.2
5.7
4.6
3.6
5.4
3.7
8.4
0.5
1.2
4.5
4.9
4.1
3.2
1.8
67
85
84
72
57
58
80
67
15
16
17
18
19
20
21
4-Me
2-OMe
3-OMe
4-OMe
2-F
3-F
4-F
alcohol 23 had a measured logP of 2.8, it displayed good
a
7 PAM
tool compound. Introduction of a 4-CF3O substituent, to give 27,
led to a substantial drop in aqueous solubility (<1 g/mL), and an
potency, and had an aqueous solubility of 12.5 g/mL. Most impor-
l
l
tantly, 23 showed a substantial decrease in intrinsic clearance to
values below human liver blood flow. Compound 23 did not inhibit
cytochrome P450 enzymes and exhibited high membrane perme-
ability with no efflux liability. Compound 23 retained the KCNQ2
increase in intrinsic clearance. Extending the para substituent to
4-OEt, to give 28, led to decreased metabolic stability. Fine tuning
around compound Lu AF58801, by introduction of fluorines in
compounds 29 thru 31, did not result in compounds with an
improved profile.
On the basis of the combined data, Lu AF58801 showed the best
overall profile and was selected to be progressed for further
studies.
The compounds were synthesized following the routes outlined
in Schemes 1–3. Racemic trans-2-phenylcyclopropane carboxylic
acids were separated into pure enantiomers using chiral SFC.17
The synthesis of racemic trans-2-phenylcyclopropane carbox-
ylic acids is well described in the literature, for example, as
outlined in Scheme 1.18 Substituted cinnamic acids were reacted
with N,O-dimethylhydroxylamine to give Weinreb type amides.
Reaction with trimethylsulfonium iodide gave racemic trans
2-phenylcyclopropane carboxamides that could be hydrolyzed to
racemic trans 2-phenylcyclopropyl carboxylic acids. The racemic
compounds were separated into their R,R and S,S isomers by chiral
SFC.19 Chiral methylbenzylamines were either commercially
available or were prepared by methods described by Ellman.20
activity that was observed in 4 (EC50 = 3.8
absolute configuration of the hydroxymethyl group was an impor-
tant SAR element for 7 PAM activity, as was the case for the
methyl group in 4. The S hydroxymethyl epimer 24 was devoid
of any 7 PAM activity. Extending the linker substituent by one
methylene, as in 25, resulted in a drop in 7 PAM activity, and a
significant increase in microsomal clearance.
lM/Emax = 87%). The
a
a
a
In the final stage of the lead optimization campaign, we sought
to combine our phenyl group SAR knowledge, as outlined in
Table 3, with the linker SAR understanding outlined in Table 4.
Representative examples are shown in Table 5.
The first target molecule was the introduction of a 4-MeO
substituent to give 26, which from now on will be referred to as
Lu AF58801. This modification led to an increase in a7 PAM activ-
ity, an improved selectivity against hKCNQ2, and a slight improve-
ment in metabolic stability. The measured logP of 2.7 and aqueous
solubility of 8 lg/mL was within our target profile for a a7 PAM
Table 4
Exploration of linker analogs
O
(S)
(S)
Compd
R
a
7
Solubility (
lg/mL)
clogP
Clint,
(L/min)
hKCNQ2
M)
human
EC50
2.8
(lM)
Emax (%)
EC50
17
(l
Emax (%)
22
23
21
—
3.2
2.4
7.5
0.9
15
N
H
OH
OH
OH
2.0
65
3
12.5
3.8
87
—
(R)
(S)
N
H
24
25
>20
—
2.4
2.7
0.9
4
>10
N
H
4.3
21
19
25
72
(S)
N
H

J. Eskildsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 288–293
291
Table 5
Connecting the pieces
Compd
a7
Solubility (
l
g/mL)
clogP
Clint,
(L/min)
hKNCQ2
M) Emax (%)
human
EC50
2.0
(lM)
Emax (%)
EC50
3.8
(
l
OH
OH
O
(S)
(S)
23
26
65
12.5
8
2.4
2.3
0.9
0.7
87
(R)
(R)
N
H
(S)
O
0.9
2.5
0.3
1.3
64
26
69
59
6.7
0.8
4.3
2.8
87
60
90
89
N
H
(S)
OMe
OH
O
(S)
(S)
27
28
29
(R)
<1
9
3.5
2.9
2.5
2.6
1.4
1.7
N
H
(S)
OCF3
OH
OH
O
(R)
(R)
N
H
(S)
OEt
F
O
a
(S)
—
N
H
(S)
OMe
F
OH
O
30
0.6
0.6
60
70
3.5
2.5
2.5
1.2
0.4
6.0
6.8
86
90
(S)
(R)
N
H
(S)
OMe
F
OH
O
a
(S)
31
—
(R)
N
H
(S)
OMe
a
Crystalline sample could not be obtained.
N
Chiral 2-amino-2-phenylethanols were either commercially
available or were prepared as outlined for R-2-amino-2-(4-
methoxyphenyl)ethanolin Scheme 2, using methods described by
Ellman.21 Halogen–lithium exchange on 4-bromo-anisol gave a
lithio-intermediate that was reacted with R(S)-2-methylpropane-
2-sulfinic acid [2-(tert-butyldimethylsilanyloxy)ethylidene]amide
to give a diastereomeric addition product with 80–90% de favoring
the R diastereomer. The diastereomers were smoothly separated
by flash chromatography. Global deprotection using HCl in dioxane
gave the desired R aminoalcohol building block.
S
O
OTBS
Li
Br
O
a
b
O
OTBS
OH
O
S
(R)
N
(R)
H2N
(R)
H
c
O
O
Scheme 2. Reagents and conditions: (a) tert-BuLi, MTBE, ꢀ78 °C, 2 h; (b) (RS)-2-
methylpropane-2-sulfinic acid [2-(tert-butyldimethylsilanyloxy)ethylidene]amide,
ꢀ78 °C, 3 h (85% overall yield from steps a and b); (c) 2 M HCl in dioxane, room
temperature, 16 h (quant).
The desired
a7 PAM compounds were prepared by standard
coupling reactions between the trans 2-phenylcyclopropane
carboxylic acids and the amines as outlined in Scheme 3.22
R1
R1
N
R1
OH
a
O
O
R2
O
R3
O
O
H2N
OH
R1
R1
O
S+
O
R1
I-
O
N
OH
R2
O
c
b
R3
N
H
a
racemic, trans
Scheme 1. Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochlo-
ride, EDCI, DMAP, Et3N, DCM, room temperature overnight (70–88%); (b) NaH, DMF,
0–25 °C, 2 h (71–90%); (c) (i) NaOH, MeOH/H2O, reflux 3 h; (ii) SFC separation
(25–30%).
Scheme 3. Reagents and conditions: (a) HATU, DMF, Et3N, 25 °C, 0.5–5 h.

292
J. Eskildsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 288–293
Table 6
Electrophysiological effects of Lu AF58801 were evaluated on
human- 7 and rat- 7 receptors expressed in Xenopus oocytes.
receptors were activated by 30 M ACh for 30 s, and the activity
Effect of Lu AF58801 on human and rat
a
7 receptors expressed in Xenopus oocytes
a
a
a7
l
Human alpha7
Rat alpha7
of Lu AF58801 was evaluated by testing Ach compound with a
EC50 (nM)
Max fold mod
EC50 (nM)
Max fold mod
preincubation time of 1 min (Fig. 2).
Lu AF58801 showed concentration-dependent PAM activity on
both human and rat a7 receptors, with corresponding EC50 values
Peak
AUC
1400
1600
43
44
1000
1000
250
2200
on both peak response and on AUC response, while the fold maxi-
mum modulation of control ACh response was much higher on rat
receptors than on human receptors (Table 6). 30 lM Ach was used
as standard. The effect of Lu AF58801 was also investigated on het-
eromeric nicotinic receptors expressed in Xenopus oocytes. No sig-
in-vivo. The compound was not mutagenic in the AMES test either
with or without liver enzyme metabolic activation, and did not in-
duce micronuclei up to 100 lM.
The novel object recognition (NOR) task23 is a test paradigm
thought to reflect visual learning and recognition processing.24
The subchronic administration of phencyclidine (subPCP) induces
a behavioural and neurobiological syndrome in rodents, upon ces-
sation of treatment, which bears a remarkable similarity to some of
the core symptoms in schizophrenic patients, including cognitive
disruption.25
nificant effects were observed at 10 or 30
lM on a3b4, a4b2, or
muscle 1b1 d receptors (data not shown). The selectivity of Lu
a
c
AF58801 was further examined using a panel of more than 108
molecular targets (panel affinity screening at Cerep, France). No
appreciable interaction was found with any of the examined tar-
gets in this panel, other than at the 5-HT2 receptor subtype. Lu
AF58801 was shown to inhibit the 5-HT2a receptor by 51% at 10
NOR testing was performed as previously described.26,27 As
shown in Figure 4, subchronic vehicle-treated rats spent signifi-
cantly more time exploring a novel object during the 3 min test
trial (as evidenced by a high discrimination index score). Vehicle-
treated animals, that had previously received PCP for 1 week
7 days prior, spent approximately an equal amount of time explor-
ing both a familiar and a novel object (reflected in a discrimination
index score close to 0). Acute administration of Lu AF58801
(10–30 mg/kg, po) dose-dependently attenuated PCP-induced
deficits in novel object exploration, an effect that reached statisti-
cal significance for the 30 mg/kg treatment group (P < 0.05). Lu
AF58801 did not affect total object exploration time of objects, or
distance travelled in the test arena at any dose tested (data not
shown). Analysis of brain samples from rats treated with 10 or
30 mg/kg Lu AF58801, harvested at the end of behavioural testing
(i.e., 90 min post-administration), revealed free brain concentra-
tions corresponding to 56 and 145 nM, respectively (n = 6). At this
l
M.
Exposure assessment of Lu AF58801 was performed in Lister
Hooded rats following an oral dose of 10 mg/kg (dose volume:
5 ml/kg, in 100% PEG400). The compound was shown to be rapidly
absorbed systemically with high plasma concentrations, followed
by a first-order elimination profile with a half-life round 1 h
(Fig. 3). Lu AF58801 showed high oral bioavailability of 70% in rats
relative to an intravenous dose and measurement of brain homog-
enate concentrations at the different time-points resulted in a
brain-to-plasma ratio of 0.15, based on AUCs of the respective
matrices. From the total brain exposure measured at 0.5 h
(305 ng/g), an unbound concentration of around 70 nM was esti-
mated based on a free fraction of 7% in rat brain homogenate.
Lu AF58801 was also broadly profiled in our standard battery of
in vitro safety pharmacology and toxicology assays. Briefly, Lu
AF58801 was not cytotoxic and did not lead to formation of reac-
tive oxygen species up to 100
on heterologously expressed hERG, hNav1.5, hKv4.3/KChIP2,
hKCNQ1/mink or hHCN4 when tested at 10 M in automated patch
clamp. However, at 10 M, there was a tendency towards an in-
crease in hKir2.1 (33 22%, mean SE), and a decrease in hCav1.2
currents (35 8%, mean SE), however these effects at high con-
centrations are not judged relevant for the actions of Lu AF58801
lM. The compound had no effect
low drug concentration, AF58801 is only expected to modulate a7
receptors, with no or very little contribution from other targets,
such as KCNQ channels. Taken together with the in-vitro potency
on alpha7 receptors this suggest that only low fold modulation of
the ACh response (2–3 fold) on the alpha7 receptor is needed to
obtain in-vivo efficacy in the NOR assay.
l
l
In summary, we have developed a potent, brain penetrant,
positive allosteric modulator of the
receptor, Lu AF58801, based on
campaign of an initial HTS hit. The optimization of hit 2 involved
the identification of a hydroxymethyl group in the linker region
which dramatically improved the microsomal stability of the
a
7 nicotinic acetylcholine
hit-to-lead optimization
a
AF58801(nM)
Control
Wash out
30
100 300
Plasma
Brain
5000
1000
100
= Control (30 µM ACh)
= AF58801 + ACh
0.2 µA
0.0
0.5
1.0
1.5
2.0
2.5
5 min
Time (h)
Figure 2. Lu AF58801 showed potentiation of the control response to ACh (30
l
M)
Figure 3. Plasma (ng/ml) and brain (ng/g) concentration–time courses of AF58801
following oral administration of 10 mg/kg to Lister Hooded rats. Each data point
represents an average of n = 3 SEM.
even at low concentrations (30, 100 and 300 nM) at human
in oocytes.
a7 receptors expressed

J. Eskildsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 288–293
293
13. All aqueous solubilities were determined in phosphate buffer pH 7.4 based on
crystalline material. For 2 the aqueous solubility measurement was based on
crystalline or partly crystalline material.
14. clogP was calculated using Daylight Version 4.9 from Daylight Chemical
Information Systems, Inc.
0.6
0.4
0.2
0.0
*
15. The absolute configuration was performed by comparing optical rotary power
with literature values.
16. A CHO cell line with stable expression of human KCNQ2 receptors was used in
a 86 Rb efflux assay to determine the activity of new compounds on the KCNQ2
receptor. KCNQ2 cells were seeded in 96 well plates and loaded with 86 Rb.
KCNQ2 channels were activated by adding 30 mM KCl to the external buffer,
and the activity of new compounds was measured by calculating the change in
efflux of 86 Rb in control compounds. EC50 values and maximum stimulation
was calculated for the new compounds. Retigabine was used as standard
positive control.
###
17. Commercially available, racemic trans 2-phenyl-cyclopropanecarboxylic acid
(Sigma–Aldrich, catalog no P22354) was subjected to chiral SFC separation. The
separation was performed on a Berger Multigram II machine, operating at
50 mL/min at 35 °C and 100 bar backpressure using stacked injections. The
column was a ChiralpakAD 5u, 250 ꢁ 21 mm. The eluent was CO2 (70%) and
ethanol (30%). (1S,2S)-2-Phenyl-cyclopropanecarboxylic acid was obtained as
an oil that slowly solidified upon standing. ½a D20
ꢂ
¼ þ300:9ꢃ (c 1% EtOH). (Lit:
Figure 4. The effects of Lu AF58801 in the subPCP NOR task in rats.28 ###P < 0.001
versus vehicle + vehicle-treated group; ⁄P < 0.05 versus PCP + vehicle-treated group
(n = 12).
½
a 2D0
ꢂ
¼ þ389ꢃ (c 0.61, CHCl3) Kozikowski et al., J. Med. Chem. 2009, 52, 1885;
Lit: ½a 2D0
ꢂ
¼ þ311:7ꢃ (c 1.776, EtOH) Walborsky et al., Tetrahedron 1964, 20,
1695).
18. Rodriques, K. E. Tetrahedron Lett. 1991, 32, 1275.
19. In all cases, the dextrorotary trans 2-phenyl-cyclopropanecarboxylic acids
were used for further synthesis.
20. (a) Datta, G. K.; Ellman, J. A. J. Org. Chem. 2010, 75, 6283; (b) Tanuwidjaja, J.;
Peltier, H. M.; Ellman, J. A. J. Org. Chem. 2007, 72, 626; (c) Robak, M. T.; Herbage,
M. A.; Ellman, J. A. Chem. Rev. 2010, 110, 3600.
compounds. Lu AF58801 was active in the subchronic PCP novel
object recognition model in rats, a paradigm with some relevance
to cognitive deficits associated with schizophrenia. Lu AF58801 is
considered an attractive tool compound for
a7 nicotinic receptor
21. Tang, T. P.; Volkman, S. K.; Ellman, J. A. J. Org. Chem. 2001, 66, 8772.
22. The a solution of 4-bromo-anisol (7.0 g, 37.4 mmol) in 70 mL of MTBE was
added 1.3 M of tert-butyllithium (60.5 mL) dropwise at ꢀ78 °C. The resulting
mixture was stirred at ꢀ78 °C for 2 h under Ar. To this solution was added
modulation in-vivo. Further evaluation of this probe is underway.
Acknowledgments
dropwise
a solution of R(S)-2-methylpropane-2-sulfinic acid [2-(tert-butyl-
dimethyl-silanyl-oxy)ethylidene]amide (11.0 g, 41.2 mmol) in dry MTBE
(100 mL). The resulting mixture was stirring at ꢀ78 °C for 3 h and then at
room temperature overnight. The mixture was quenched with saturated
aqueous NH4Cl solution and extracted with EtOAc. The combined organic
layers were washed with brine, dried over MgSO4 and evaporated to dryness.
Purified by HPLC (Gilson GX281, Gemini column, gradient 55–85% acetonitrile
in water over 25 min at 25 mL/min flowrate) gave (R)-N-((R)-2-
((tert-butyldimethylsilyl)oxy)-1-(4-methoxyphenyl)ethyl)-2-methylpropane-
Dr. Jane Wang and Miao Wei from WuXi Apptec Shanghai are
acknowledged for expert organic synthesis assistance. We thank
Sascha Bull for excellent technical assistance with in vivo behav-
ioural experiments.
References and notes
2-sulfinamide as an oil (1.22 g, 85%). HNMR (CDCl3 400 MHz)
d 7.28
(d, J = 1.2 Hz, 1H), 6.90 (s, 1H), 6.88 (s, 1H), 4.48–4.51 (m, 1H), 4.28 (d,
J = 0.8 Hz, 1H), 3.82 (s, 3H), 3.74–3.77 (m, 1H), 3.60 (t, J = 9.6 Hz, 1H), 1.24 (s,
1. Green, A.; Ellis, K. A.; Ellis, J.; Bartholomeusz, C. F.; Ilic, S.; Croft, R. J.; Phan, K. L.;
Nathan, P. J. Pharmacol. Biochem. Behav. 2005, 81, 575.
9H), 0.93 (s, 9H), 0.08 (d, J = 5.2 Hz, 1H). ½a D20
ꢂ
¼ ꢀ90:6ꢃ (c 0.106, MeOH). To a
solution of (R)-N-((R)-2-((tert-butyldimethylsilyl)oxy)-1-(4-methoxyphenyl)
ethyl)-2-methylpropane-2-sulfinamide (2.2 g, mmol) in anhydrous dioxane
(20 mL) was added HCl (2 M in dioxane, 2 mL) at room temperature. The
mixture was stirred at room temperature for 16 h and the mixture was
evaporated to dryness. The material was used without further purification. To a
mixture of (R)-2-amino-2-(4-methoxyphenyl)ethanol hydrochloride (2.15 g,
13.2 mmol) and HATU (5.47 g, 14.4 mmol) in DMF (20 mL) was added Et3N
(2.42 g, 24 mmol). The resulting mixture was stirred at room temperature for
30 min. To this mixture was added (1S,2S)-2-phenyl-cyclopropanecarboxylic
acid (2.0 g, 12 mmol) and the mixture was stirred at room temperature for 5 h.
The mixture was evaporated to dryness and purified by preparative HPLC
(Gilson GX281, Gemini column, gradient 35–60% acetonitrile in water over
25 min at 80 mL/min flowrate) to give compound AF58801 as a colorless solid
(1.5 g, 47%). HNMR (CDCl3 400 MHz) d 7.16–7.28 (m, 5H), 7.06–7.08 (m, 2H),
6.86–6.90 (m, 2H), 6.25 (d, J = 6.8 Hz, 1H), 5.02–5.06 (m, 1H), 3.84–3.92 (m,
2H), 3.79 (s, 3H), 2.89 (d, J = 0.8 Hz, 1H), 2.48–2.53 (m, 1H), 1.63–1.69 (m, 2H),
1.25–1.32 (m, 2H). CNMR (DMSO-d6, 150 MHz) d 170.9, 158.6, 141.7, 133.7,
128.8, 128.5, 126.4, 126.2, 114.0, 65.2, 55.5, 55.0, 26.1, 24.2, 16.0. LC–MS m/
2. Rogers, J. L.; Kesner, R. P. Neurobiol. Learn. Mem. 2003, 80, 332.
3. Gotti, C.; Clementi, F.; Fornari, A.; Gaimarri, A.; Guiducci, S.; Manfredi, I.;
Moretti, M.; Pedrazzi, P.; Pucci, L.; Zoli, M. Biochem. Pharmacol. 2009, 78, 703.
4. Broad, L. M.; Sher, E.; Astles, P. C.; Zwart, R.; O’Neill, M. J. Drugs Future 2007, 32,
161.
5. Leonard, S.; Breese, C.; Adams, C.; Benhammou, K.; Gault, J.; Stevens, K.; Lee,
M.; Adler, L.; Olincy, A.; Ros, S. R.; Freedman, R. Eur. J. Pharmacol. 2000, 393,
237.
6. Perry, E.; Martin-Ruiz, C.; Lee, M.; Griffiths, M.; Johnson, M.; Piggott, M.;
Haroutunian, V.; Buxbaum, J. D.; Nãsland, J.; Davis, K.; Gotti, C.; Clementi, F.;
Tzartos, S.; Cohen, O.; Soreq, H.; Jaros, E.; Perry, R.; Ballard, C.; McKeith, I.;
Court, J. Eur. J. Pharmacol. 2000, 393, 215.
7. Bencherif, M.; Dunbar, G. C.; Hosford, D. A.; Gatto, G. J.; Hauser, T.; Jordan, K. G.;
Segreti, A.C. Treatment of Cognitive Dysfunction in Schizophrenia,
WO2012099836 A1 (PCT), 2012.
8. Hilt, D. EVP-6124, A Selective Alpha-7 Partial Agonist, has Positive Effects on
Cognition and Clinical Function in Mild to Moderate Alzheimers Disease Patients.
Oral Presentation at Alzheimer’s Association International Conference 2012 (AAIC);
Vancouver: Canada, 2012.
9. Hilt, D. EVP-6124 Shows Statistically Significant Improvement in Cognition,
Function and Impact on Negative Symptoms. Oral Presentation at American
College of Neuropsychopharmacology Annual Meeting 2011, USA.
10. Hurst, R. S.; Hajós, M.; Raggenbass, M.; Wall, T. M.; Higdon, N. R.; Lawson, J. A.;
Rutherford-Root, K. L.; Berkenpas, M. B.; Hoffmann, W. E.; Piotrowski, D. W.;
Groppi, V. E.; Allaman, G.; Ogier, R.; Bertrand, S.; Bertrand, D.; Arneric, S. P. J.
Neurosci. 2005, 25, 4396.
z = 312.1 [M+H]+. ½a D20
ꢂ
¼ ꢀ219:6ꢃ (c 0.1175, MeOH).
23. Ennaceur, A.; Delacour, J. Behav. Brain Res. 1988, 31, 47.
24. Young, J. W.; Powell, S. B.; Risbrough, V.; Marston, H. M.; Geyer, M. A.
Pharmacol. Ther. 2009, 122, 150.
25. Jentsch, J. D.; Roth, R. H. Neuropsychopharmacology 1999, 20, 201.
26. Redrobe, J. P.; Bull, S.; Plath, N. Front. Psychiatry 2010, 11, 146.
27. Redrobe, J. P.; Elster, L.; Frederiksen, K.; Bundgaard, C.; de Jong, I. E.; Smith, G.
P.; Bruun, A. T.; Larsen, P. H.; Didriksen, M. Psychopharmacology 2012, 221, 451.
28. Male Lister Hooded rats (Charles River, Crl:LH, Germany; 220–240 g) were
administered subPCP (synthesized at H. Lundbeck A/S, dissolved in 0.9% saline;
5 mg/kg, 1 ml/kg, ip bid for 7 days) or saline (1 ml/kg ip bid for 7 days) at 7 am
and 7 pm, followed by a 7-day washout period prior to behavioural testing. Lu
AF58801 (dissolved in 100% PEG400) was administered 30 min prior to the
acquisition trial of the NOR procedure via the po route. Data is presented as the
discrimination index, calculated as the novel object exploration time (Tn)
minus the familiar object exploration time (Tf) divided by the total exploration
time (Tn + Tf). One-way ANOVA, followed by appropriate All-Pairwise Multiple
Comparison post-hoc analysis (Bonferroni’s t-test) was used to investigate
statistical differences between test groups (P < 0.05).
11. Faghih, R.; Gfesser, G. A.; Gopalakrishnan, M. Recent Patents CNS Drug Disc.
2007, 2, 99.
12. As the nACha7 receptor is a calcium permeable ion channel, the activity of this
receptor can be measured in cell lines expressing the target using calcium
sensitive dyes. In this paper, we used a rat GH4C1 cell line with stable
expression of nACh
loaded with a calcium sensitive dye (calcium-4). The
with 20 M ACh and the activity of new compounds was measured as a change
a
7 receptors. The cells were seeded in 384-well plates and
a
7 receptor was activated
l
in fluorescence between control compounds, measured on a FDSS 700 reader.
PNU-120596 was used as positive control and the max stimulation of new
compounds was calculated relative to PNU-120596 defined as 100%.