Quinolizidinone carboxylic acids as CNS penetrant, selective M1 allosteric muscarinic receptor modulators

Article abstract of DOI:10.1021/ml100095k

Positive allosteric modulation of the M₁ muscarinic receptor represents an approach to treat the cognitive decline in patients with Alzheimer's disease. Replacement of a quinolone ring system in a quinolone carboxylic acid series of M₁ modulators with a quinolizidinone bearing a basic amine linkage led to a series of compounds with higher free fraction, enhanced CNS exposure, and improved efficacy in rodent in vivo models of cognition.

Full text of DOI:10.1021/ml100095k

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Quinolizidinone Carboxylic Acids as CNS Penetrant,
Selective M1 Allosteric Muscarinic Receptor Modulators
Scott D. Kuduk,*,† Ronald K. Chang,† Christina N. Di Marco,† William J. Ray,‡ Lei Ma,‡
Marion Wittmann,‡ Matthew A. Seager,‡ Kenneth A. Koeplinger,§ Charles D. Thompson,§
George D. Hartman,† and Mark T. Bilodeau†
†Departments of Medicinal Chemistry, ‡Alzheimer's Research, and §Drug Metabolism, Merck Research Laboratories,
Sumneytown Pike, PO Box 4, West Point, Pennsylvania 19486
ABSTRACT Positive allosteric modulation of the M1 muscarinic receptor repre-
sents an approach to treat the cognitive decline in patients with Alzheimer's
disease. Replacement of a quinolone ring system in a quinolone carboxylic acid
series of M1 modulators with a quinolizidinone bearing a basic amine linkage led to
a series of compounds with higher free fraction, enhanced CNS exposure, and
improved efficacy in rodent in vivo models of cognition.
KEYWORDS Quinolizidinone, M1 muscarinic receptor, allosteric modulator, cogni-
tion, Alzheimer's
agonists8 or positive allosteric modulators9 could confer the
ne of the pathologies of Alzheimer's disease (AD)
high selectivity over their orthosteric counterparts.
In this regard, the quinolone carboxylic acid HTS
lead 110,11 and related analogues12-14 have been reported
as highly M1 selective positive allosteric modulators (Figure
1). Compound 1 has modest M1 receptor activity and brain
penetration, high plasma protein binding, and low solubility
in a neutral form. As a result, high plasma levels are required
for in vivo efficacy. Attempts to address these issues via
incorporation of basic amines extending off a biphenyl
construct augmented some of these issues (Figure 1). How-
ever, this afforded compounds that were substrates for the
human efflux pump P-glycoprotein (P-gp) leading to reduced
CNS exposure.
concerns the progressive degeneration of the neu-
O
rons of the basal forebrain cholinergic system, con-
tributing to the profound cognitive deficits manifest in AD.1
Acetylcholinesterase inhibitors are currently the front line
of treatment for AD symptoms and act by limiting the
degradation of synaptic acetylcholine (ACh) levels to activate
cholinergic receptors. Although these agents do offer cogni-
tive benefit, the magnitude and duration of the effects are
limited.
ACh activates both nicotinic (ligand-gated ion channels)
and muscarinic (metabotropic) receptors, which belong to
the class A family of G protein coupled receptors (GPCRs).
There are five receptor subtypes in the muscarinic family
(classed M1 to M5),2,3 of which the M1 receptor has been
found to be highly expressed in the cortex and hippocampus
areas that are critically involved in memory formation and
are affected early in AD. As such, M1 has been widely viewed
as the subtype most likely modulating cognitive and mem-
ory effects in this region of the brain.4 As a result, a number
of nonselective or partially selective M1 agonists such as
xanomeline have been evaluated clinically, exhibiting effi-
cacy in improving cognitive performance in AD patients.5,6
However, adverse events (gastrointestinal distress, saliva-
tion, sweating), thought to be mediated by activation of
other peripheral muscarinic receptors, have prevented
their utility.
Efforts to identify compounds highly selective for the M1
receptor subtype have failed due to the highly conserved
nature of the orthosteric acetylcholine binding site. One
avenue to address this is to target allosteric binding sites
on the M1 receptor that may be less highly conserved and
provide ample specificity for M1 over the M2-M5 subtypes.7
As such, M1 allosteric activators in the form of allosteric
Figure 1
Accordingly, efforts were focused on the identification of
an alternate scaffold for the quinolone carboxylic acid that
would allow for improvement of these aforementioned
Received Date: May 4, 2010
Accepted Date: May 31, 2010
Published on Web Date: June 04, 2010
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issues.15,16 Specifically, it was of interest to evaluate if a
quinolizidinone ring system could replace the quinolone
(Figure 2) and confer dissimilar properties. Ideally, this
strategy would also allow for the incorporation of a basic
amine at the benzylic position in the form of a piperidine or
piperazine that could not be achieved in the quinolone
context. Herein we report a series of novel brain penetrant
quinolizidinone carboxylic acid M1 positive allosteric mod-
ulators that address many of the concerns found in quino-
lone 1, with improved in vivo efficacy and pharmacokinetic
properties suitable for further development.
The chemistry employed to access target molecules
examined in this communication is shown in Scheme 1.
Lithiation of 2-picoline with n-butyllithium is followed by
addition of ethoxymethylene diethylmalonate 3 and subse-
quent cyclization in refluxing o-xylene to provide quinolizi-
dinone 4.17 Vilsmeier formylation at the 1-position afforded
the requisite aldehyde 5. Addition of the appropriate
Grignard reagents to 5, which were subsequently hydrolyzed
using NaOH in THF led to acids 9c,d. Oxidation using IBX
followed by hydrolysis yielded ketone 9b. Alternatively,
deoxygentation of 6c,d produces 9c,d after ester hydrolysis.
Intermediate 9d (R = 4-chlorophenyl) underwent Suzuki
cross-coupling with the appropriate boronic acid to result in
9e-g. Lastly, reduction amination of 5 with the appropriate
amines followed by saponification of the ethyl ester afforded
target N-linked quinolizidinone carboxylic acids 10a-i.
Carbon linked homologues of the quinolone carboxy-
lic were evaluated first as shown in Table 1. Modifications
containing a hydroxyl group (9a) or ketone (9b) at the ben-
zylic position were inactive. The benzyl analogue 9c gave
an M1 IP = 0.92 μM, while potency was enhanced with
the p-chloro (9d; M1 IP = 200 nM) and p-phenyl (9e; M1 IP =
110 n M ). However, all of these compounds were fully bound
to plasma proteins. Efforts to enhance free fraction via
incorporation of aryl pyridines12 (9f,g) in lieu of the biphenyl
yielded potent M1 potentiators, but had negligible reduction
on protein binding.
Quinolizidinone carboxylic acids that are nitrogen linked
at the benzylic position were evaluated as shown in Table 2.
Having a simple piperidine (10a) in place gave an M1 IP =
7.6 μM, but addition of a 4-phenyl group (10b) increased
activity ∼6-fold. Ring sizes other than a 6-membered ring
were not tolerated as highlighted by phenyl pyrrolidine 10c.
Incorporation of a piperazine in lieu of the piperidine
afforded 10d, which further improved M1 potentiation and
also gave very high rat and human free fractions (>25%).
Ketopiperazine 10e gave similar M1 potency compared to
10d, and led to further reductions in plasma protein binding.
Attempts to incorporate a pyridine as exemplified by 10f led
to reduction in M1 functional activity. Substitution on the
phenyl ring was tolerated, with the best results observed for
groups at the para position. For example, bromide 10g was
the most potent potentiator in this series with an M1 IP =
132 nM and a reasonable free fraction in rat (∼4%). A
trifluoromethyl group (10h) resulted in a similar free fraction,
with a ∼2-fold loss in M1 potency. A cyano group provided
a reasonably potent potentiator (10i; M1 IP = 520 nM), but
had an improved plasma free fraction relative to 10g,h.
As P-glycoprotein (P-gp) is considered the dominant efflux
transporter at the blood brain barrier (BBB) responsible for
the efflux of a number of xenobiotic substances from the
CNS, select compounds from Table 2 were evaluated for their
passive permeability properties (Papp) and susceptibility
as P-gp substrates in order to further evaluate their CNS
exposure potential. As can be seen from Table 3, most of the
compounds possessed good passive permeability (Papp) and
were not substrates for human (MDR1) or rat (MDR1a) P-gp
Figure 2
Scheme 1a
a Reagents: (a) LDA, THF, -80 to -20 °C; (b) o-xylene, reflux; (c) POCl3, DMF, 0 to 20 °C; (d) RMgBr, THF-CH2Cl2, 0 to 25 °C; (e) NaOH, THF, EtOH, 50 °C;
(f) TFA, Et3SiH, CH2Cl2; (g) ArB(OH)2, Pd(t-Bu3P)2, Cs2CO3, THF, 100 °C; (h) IBX, CH3CN, 75 °C; (i) NaBH(OAc)3, DCE, AcOH, 4 Å molecular sieves.
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Table 1
Table 2
a Values represent the numerical average of at least two experiments.
Interassay variability was (30% (IP, nM), unless otherwise noted.
a Values represent the numerical average of at least two experiments.
Interassay variability was (30% (IP, nM), unless otherwise noted.
with efflux ratios less than 2. A notable exception was
ketopiperazine 10f, which had very poor Papp leading to very
low brain and CSF levels despite having a very high rat free
fraction (∼48%). Bromide 10g provided the highest plasma
level and brain levels when dosed orally at 10 mpk. Trifluoro-
methyl piperazine 10h provided a similar CSF/Uplasma ratio com-
pared to 10g, but with a moderately higher B/P ratio (∼2.7). The
highest CSF levels were observed with nitrile 10i, which pre-
sented a CSF/Uplasma ratio of 0.42, albeit with a lower B/P ratio.
The higher total brain levels of 10g-h could be attributed to the
increased lipophilicity of the bromo and trifluoromethyl groups
relative to the nitrile (log D=1.4) moiety in 10i,whichmaylead
to higher nonspecific brain tissue binding.18 In addition, this
greater degree of lipophilicity may be responsible for off-target
activity at the hERG cardiac channel for 10g (IC50 =2.1μM) and
10h (IC50 = 0.1 μM), compared to 10i (IC50 = 7.4 μM). Based
Table 3. Permeability, P-gp, and Bioanalysis of Plasma, Brain, and
CSF Levels in Rat for Selected Compounds
concn (nM)c
brain/
CSF/
d
compds Papp MDR1b MDR1ab plasma brain csf plasma Uplasma
a
10f
10g
10h
10i
<5
16
32
22
2071
59 16 0.03
0.02
0.32
0.25
0.42
0.1
1.4
0.7
0.6
1.3
1.9
3646 4581 70 1.3
1138 3085 14 2.7
1495
248 85 0.17
a Passive permeability (10-6 cm/s). b MDR1 directional transport ratio
(B to A)/(A to B). Values represent the average of three experiments, and
interassay variability was (20%. c Sprague-Dawley rats. Oral dose 10 mg/
kg in 0.5% methocel. Interanimal variability was less than 20% for all
values. d Determined using rat plasma protein binding from table 2.
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Table 4. Rat and Dog Pharmacokinetic Data for 10ia
rat
dog
F (%)
t1/2 (h)
Cl
F (%)
t1/2 (h)
Cl
23
6.4
10.8
33
2.5
13
a F% oral bioavailability, half-life is represented in hours, Cl in mL/min/kg.
Sprague-Dawley rats (n = 3). Oral dose =10 mg/kg in methocel, iv dose =
2 mg/kg in DMSO. Interanimal variability was less than 20%.
in rat (F = 23%) with a good half-life (6.4 h) and relatively
low clearance (10.8 mL/min/kg). The dog clearance was
higher with a shorter half-life. Overall, 10i possessed suitable
pharmacokinetic properties further highlighting the quino-
lizidinone as a suitable replacement for the quinolone
carboxylic acid.
Figure 3. Compound 10i potentiates the acetylcholine dose
response curve for the M1 receptor.
In summary, a quinolizidinone ring system has been
identified as an advanced replacement for the quinolone
core in a series of carboxylic acid M1 positive allosteric
modulators. The quinolizidinone scaffold allowed for in-
corporation of a basic amine in the form of a piperazine
ring, which markedly improved the plasma free fraction.
Quinolizidinone 10i exemplified this leading to a potentia-
tor with markedly improved performance in the mouse
contextual fear conditioning model. Moreover, 10i retains
suitable pharmacokinetic and physicochemical properties
for additional study to determine potential for clinical
evaluation.
Figure 4. Compound 10i in the mouse model of fear conditioning.
on the lower hERG affinity and high CSF levels, 10i was chosen
for further evaluation.
The effect of 10i was evaluated on the affinity of acetylcho-
line for the M1 receptor in a functional assay utilizing calcium
mobilization as a readout. In CHO cells expressing the
human M1 receptor, increasing concentrations of compound
10i from 0.1 to 10 uM potentiate the effect of acetylcholine
leading to a leftward shift in the acetylcholine M1 dose-
response curve (Figure 3). In the absence of acetylcholine,
there was no direct activation (agonism) of M1 by compound
10i up to the highest dose tested (10 uM). In addition, no
agonist or antagonist activity is observed up to 100 μM
on M2, M3, or M4 receptors with or without acetylcholine
present.
SUPPORTING INFORMATION AVAILABLE Representative
assay and experimental procedures and data for test compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: 215-652-5147. Fax: 215-652-3971. E-mail: scott_d_-
kuduk@merck.com.
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