Identification of amides as carboxylic acid surrogates for quinolizidinone-based M1 positive allosteric modulators

Article abstract of DOI:10.1021/ml300280g

Selective activation of the M₁ muscarinic receptor via positive allosteric modulation represents an approach to treat the cognitive decline in patients with Alzheimer's disease. A series of amides were examined as a replacement for the carboxylic acid moiety in a class of quinolizidinone carboxylic acid M₁ muscarinic receptor positive allosteric modulators, and leading pyran 4o and cyclohexane 5c were found to possess good potency and in vivo efficacy.

Full text of DOI:10.1021/ml300280g

Letter
pubs.acs.org/acsmedchemlett
Identification of Amides as Carboxylic Acid Surrogates for
Quinolizidinone-Based M1 Positive Allosteric Modulators
Scott D. Kuduk,*,† Ronald K. Chang,† Thomas J. Greshock,† 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, P.O. Box 4, West Point, Pennsylvania 19486, United States
S
* Supporting Information
ABSTRACT: Selective activation of the M1 muscarinic receptor via positive allosteric modulation
represents an approach to treat the cognitive decline in patients with Alzheimer's disease. A series of
amides were examined as a replacement for the carboxylic acid moiety in a class of quinolizidinone
carboxylic acid M1 muscarinic receptor positive allosteric modulators, and leading pyran 4o and
cyclohexane 5c were found to possess good potency and in vivo efficacy.
KEYWORDS: carboxylic acid surrogates, quinolizidinone, positive allosteric modulators
holinergic neurons serve critical functions in both the peripheral
Cand the central nervous systems (CNS). Acetylcholine is the
key neurotransmitter in these neurons targeting nicotinic and
metabotropic (muscarinic) receptors. Muscarinic receptors are
class A G-protein-coupled receptors (GPCR) widely expressed in
the CNS. There are five muscarinic subtypes, designated M1−M5,1,2
of which M1 is most highly expressed in the hippocampus,
striatum, and cortex,3 implying that it may play a central role in
memory and higher brain function.
Figure 1.
a
Scheme 1
One avenue to engender selectively for M1 over the other
muscarinic subtypes is to target allosteric sites on M1 that are
less highly conserved than the orthosteric site.8,9 Quinolone
carboxylic acid 1 (Figure 1) has been previously identified as a
selective positive allosteric modulator of the M1 receptor with
exclusive selectivity for the M1 subtype.10 Recent efforts to
improve the potency and CNS penetration of 1 led to a number
of structural enhancements.11−15 Further advancements were
discovered by incorporation of a quinolizidinone ring system
such as 2 in lieu of the quinolone, particularly in the area of
improved free drug levels and in vivo activity.16−18 However,
a
Reagents and conditions: (a) BOP, amine, triethylamine, DMF.
the presence of the acid in this scaffold limited the extent of
CNS exposure, and dominant clearance was by means of direct
One of the hallmarks of Alzheimer's disease (AD) is the pro-
glucuronidation of the parent acid, complicating human
gressive degeneration of cholinergic neurons in the basal forebrain
leading to cognitive decline.4 Accordingly, direct activation of the
pharmacokinetic predictions. Accordingly, it was of interest to
see if noncarboxylic acid M1 potentiators19 could be identified
from this acid scaffold. This communication describes efforts to
M1 receptor represents an approach to treat the symptoms of AD.5
In this regard, a number of nonselective M1 agonists have shown
potential to improve cognitive performance in AD patients but were
clinically limited by cholinergic side effects thought to be due to
activation of other muscarinic subtypes via binding to the highly
conserved orthosteric acetylcholine binding site.6,7
Received: September 12, 2012
Accepted: October 13, 2012
© XXXX American Chemical Society
A
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identify CNS penetrant quinolizidinone carboxylic amides as
replacements for the acid motif.
Table 2. Protein Binding Data and P-gp Data for Select
Compounds
The preparation of requisite amides is shown in Scheme 1.
Quinolizidinone carboxylic acids 2 and 3, as well as related
analogues, were prepared using previously described proce-
dures.17 Amide bond formation with the appropriate amine
using benzotriazole-1-yl-oxy-tris-(dimethylamino)-phospho-
nium hexafluorophosphate (BOP) provided amides 4 and 5.
Compound potencies were determined in the presence of an
EC20 concentration of acetylcholine at human M1 expressing
CHO cells using calcium mobilization readout on a FLIPR384
fluorometric imaging plate reader. A large number of amines were
used in library format in Scheme 1 with either 4-cyanopiperidine
(2)- or piperazine (3)-linked quinolizidinone carboxylic acids.
Select examples are shown in Table 1. Replacement of the acid in
a
Table 1. M1 FLIPR Data for Select Compounds
a
b
Passive permeability (10−6 cm/s). MDR1 (human) and MDR1a
(rat) directional transport ratio (B to A)/(A to B). Values represent the
average of three experiments, and the interassay variability was 20%.
Figure 2.
the ring size (4k−m) led to an increase in functional activity, with
cyclohexyl 4l (M1 IP = 253 nM) being the optimal fit. Insertion of
a heteroatom such as oxygen into the cyclohexane ring in the form
of a pyran was tolerated, with preference for the 4-position (4o)
over the 3-position (4n), while the sulfide analogue (4q) was
similarly active. N-Methylation of 4o to tertiary amide 4p lost
>10-fold activity, highlighting the requirement for a secondary
amide for M1 activity.
Substitution on the cyclohexane ring identified the 2-position in
the trans configuration as the most promising. The 2-methyl
derivative 4r gave an M1 IP = 190 nM, and similar results were also
noted with gem-difluoro 4s. The 2-fluoro derivative is noteworthy
as it was similarly equipotent to carboxylic acid 2. Thiomethyl ether
4u was similar to methyl 4r, although methyl ether 4v was less
preferred. Moreover, hydroxyl 4w, with the (1S,2S) configuration,
was the most potent among the group (M1 IP = 31 nM), ∼4-fold
better than acid 2. The associated (1R,2R) enantiomer 4x was
∼7-fold less active. Interestingly, while addition of a methyl at the
2-position of 4w in the form of tertiary alcohol 4y was
tolerated, addition of the methyl at the 1-position (4z) led to a
complete loss of all M1 activity. It is worth pointing out that
select compounds were examined in functional assays at other
muscarinic subtypes and showed no activity at M2, M3, or M4
a
IP, inflection point. Values represent the numerical average of at least
two experiments. The interassay variability was 30% (IP, nM), unless
otherwise noted.
2 with NH2 (4a) led to a >10-fold drop in potency, but at least
some activity was preserved. Methylamine (4b) or pyrrolidine
(4c) further decreased activity. Several aromatic amines were
investigated as represented by 4d−h, with 2-aminopyridine 4h
highlighting the most promising among the group.
While cyclopropyl amide 4i was similarly equipotent to amide
4a, cyclobutane 4j was a submicromolar compound. Increasing
B
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Letter
receptors, highlighting that quinolizidinone amides maintain
selectivity for M1.
Table 4. Bioanalysis of Plasma, Brain, and CSF Levels in Rat
for Selected Compounds
Having identified a number of potent amides, select com-
pounds were examined to see if they were substrates for the
multidrug resistant (MDR) efflux transporter P-glycoprotein
(P-gp). In addition, plasma protein binding was determined
using the equilibrium dialysis method in the presence of rat and
human serum (Table 2).
Cyclohexane 4l was not a P-gp substrate and exhibited good
passive permeability with protein binding in the range of 93−
95%. The corresponding pyran analogue 4o increased efflux
and was a substrate for rat P-gp (7.5) but showed markedly
enhanced free fraction (27 and 49% in rat and human,
respectively). Substitution at the 2-position of the cyclohexane
with fluorine (4s,t) maintained good permeability and P-gp
profiles with improved free fraction relative to cyclohexyl 4l.
The most potent potentiator alcohol 4w unfortunately was a
substrate for P-gp, as was the tertiary alcohol analogue 4z.
It was thought that the P-gp efflux of highly potent alcohol
4w could be modulated by finding a different amine in place of
the 4-cyano-4-(2-pyridyl)piperidine. Replacement of the pyridyl
with a phenyl (4w′) led to a modest reduction in M1 potency
and free fraction but substantially reduced P-gp efflux.
However, this change also led to an increase in human ether-
a
Sprague−Dawley rats. Oral dose 10 mg/kg in 0.5% methocel. The
b
interanimal variability was less than 20% for all values. Determined
using rat plasma protein binding from Tables 2 and 3.
(IC50 = 5.1 μM), a ∼3-fold increase over pyridyl 4w. Previously
in the quinolizidinone carboxylic acid series, aryl piperazine
derivatives were identified as potent and CNS penetrant.16 The
4-cyanophenylpiperazine 5a was reasonably potent (M1 IP =
244 nM), with good free fraction, and was not a substrate for P-gp.
Unfortunately, hERG binding (IC50 = 0.39 μM) was an issue.
Incorporation of a methylpyridine (5b) for the cyanophenyl
decreased the hERG binding but markedly increased P-gp
efflux. Isoquinoline 5c alleviated the P-gp issues observed with
5b but did so at the expense of higher protein binding. Lastly,
benzothioazole 5d represented the most optimized potentiator
among the group in terms M1 potency, free fraction, P-gp
profile, and hERG binding.
̀
a-go-go-related gene (hERG) potassium channel binding
Table 3. M1 FLIPR, Protein Binding, P-gp, and hERG Data
for Select Compounds
Selected compounds were examined for CNS exposure in rat
as shown in Table 4. Brain, plasma, and cerebrospinal fluid
(CSF) levels were measured after 2 h following a 10 mg/kg oral
dose. Data for carboxylic acid 2 are shown for comparison.
Fluorocyclohexane 4t gave a CSF/Uplamsa ratio of 0.33, which
was similar to that observed with acid 2, although absolute CSF
levels were lower. Pyran 4o gave robust plasma (2.6 μM) and
CSF (215 nM) levels, with a comparable CSF/Uplamsa ratio of
0.30. The hydroxycyclohexane 4w′ possessed similar CSF levels
to 4t and had the highest total brain levels of the compounds
tested. The high CSF level of 4o was believed to be driven by
the large free fraction due to the pyran (27%), while all other
amides had greater than 90% protein binding. Interestingly,
benzothiazole 5d gave very low drug levels in all matrices
examined, which was surprising since 5d is not a P-gp substrate,
with excellent permeability (Papp = 37) and free fraction similar
to other compounds.
On the basis of the significant CSF levels of pyran 4o,
additional studies were conducted to further investigate the
properties of amide derived modulators. Fold potentiation with
a fixed concentration of modulator 4o was evaluated on the M1
dose response with acetylcholine as the agonist. As can be seen
from Figure 2, with increasing concentration of potentiator,
a left shift was observed up to 52-fold at 34 μM in the
acetylcholine dose response, showing that 4o is a potent positive
allosteric modulator of the human M1 receptor.
a
Values represent the numerical average of at least two experiments.
The interassay variability was 30% (IP, nM), unless otherwise noted.
b
Passive permeability (10−6 cm/s) in Madin Darby canine kidney
c
(MDCK) cells. MDR1 directional transport ratio (B to A)/(A to B).
Values represent the average of three experiments, and the interassay
variability was 20%. Values represent the numerical average of at
d
least two experiments. The interassay variability was 20% (IC50, μM).
C
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To examine the in vivo properties, amide 4o was evaluated in
a mouse contextual fear conditioning (CFC) assay, which
serves as a model of episodic memory (Figure 3). In this study,
mice were treated with scopolamine before introduction to a
novel environment to block a new association. Mice dosed
by intraperitoneal injection with 4o exhibited a significant
reversal at 10 and 30 mg/kg, as compared to mice treated
with scopolamine alone. The corresponding plasma levels at
10 mg/kg were 2.4 μM. By way of comparison, the analogous
carboxylic acid 2 showed significant reversal at ∼1 μM plasma
levels, which is consistent with greater in vitro potency than 4o
in the FLIPR assay. In addition, isoquinoline 5c was also
evaluated in mouse CFC and provided full reversal at a very
similar plasma level (2.5 μM) to pyran 4o. Accordingly, it
appears that amides such as 4o and 5c behave similarly in this
rodent cognition model relative to the parent quinolizidinone
carboxylic acid M1 positive allosteric modulators.20
Previously, amides of lead quinolone carboxylic acid 1 have
been examined with little success. As can be seen in Figure 4,
similar amides made in both the quinolone and the
quinolizidinone gave disparate results. For example, while
cyclohexyl and tolyl were accommodated in the quinolizidinone
context for both 4l and 4d, the quinolone congeners 6a and 6b
lost greater than 20-fold potency in the FLIPR assay by way of
comparison. Although the exact reason why amides are viable in
combination with the quinolizidinone nucleus and not the
quinolone is unclear, it serves as another example in which
apparently seemingly minor core modifications lead to distinct
SAR in allosteric modulator lead optimization.
In summary, amides of quinolizidinone carboxylic acids were
found to be potent and CNS penetrant alternatives as selective
M1 positive allosteric modulators. Amides derived from
(1S,2S)-2-aminocyclohexanol were found to be markedly the
most potent variants but were substrates for the P-gp efflux
transporter. Isoquinoline 5c was an exception, which provided
efficacy in a mouse contextual fear model of episodic memory
but possessed low micromolar hERG channel inhibition. Pyran
4o showed minimal inhibition of hERG (IC50 > 15 μM), was
reasonably potent with high CSF levels in rat, and also provided
good in vivo activity in mouse CFC. Further optimization of
these amides and employment of this strategy in other scaffolds
is ongoing.
ASSOCIATED CONTENT
■
S
* Supporting Information
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.
Figure 3. Evaluation of 4o and 5c in the mouse CFC model.
Figure 4.
D
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carboxylic acid M1 positive allosteric modulators. Bioorg. Med. Chem.
Lett. 2010, 19, 2533−2537.
(15) Kuduk, S. D.; DiPardo, R. M.; Beshore, D. C.; Ray, W. J.; Ma,
L.; Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Hydroxy cycloalkyl fused pyridone
carboxylic acid M1 positive allosteric modulators. Bioorg. Med. Chem.
Lett. 2010, 19, 2538−2541.
AUTHOR INFORMATION
Corresponding Author
*Tel: 215-652-5147. Fax: 215-652-3971. E-mail: scott_d_
kuduk@merck.com.
Notes
■
The authors declare no competing financial interest.
(16) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Ray, W. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Identification of Quinolizidinone
Carboxylic Acids as CNS Penetrant, Selective M1 Allosteric Muscarinic
Receptor Modulators. ACS Med. Chem. Lett. 2010, 1, 263−267.
(17) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Ray, W. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Quinolizidinone Carboxylic Acid
Selective M1 Allosteric Modulators: SAR in the Piperidine Series.
Bioorg. Med. Chem. Lett. 2011, 21, 1710−1713.
(18) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Pitts, D. R.;
Greshock, T. J.; Ma, L.; Wittmann, M.; Seager, M.; Koeplinger, K. A.;
Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T.; Ray, W. J.
Discovery of a Selective Allosteric M1 Receptor Modulator with
Suitable Development Properties Based on a Quinolizidinone
Carboxylic Acid Scaffold. J. Med. Chem. 2011, 13, 4773−4780.
(19) For examples of noncarboxylic positive allosteric M1
modulators, see Reid, P. R.; Bridges, T. M.; Sheffler, D. A.; Cho, H.
P.; Lewis, L. M.; Days, E.; Daniels, J. S.; Jones, C. K.; Niswender, C.
M.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W.; Wood, M. R.
Discovery and optimization of a novel, selective and brain penetrant
M1 positive allosteric modulator (PAM): the development of ML169,
an MLPCN Probe. Bioorg. Med. Chem. Lett. 2011, 21, 2697−2701.
(20) In addition, 4o exhibited good preclinical PK properties (dog Cl
= 2.8 mL/min/kg, t1/2 = 11 h).
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