MK-7622: A First-in-Class M1 Positive Allosteric Modulator Development Candidate

Article abstract of DOI:10.1021/acsmedchemlett.8b00095

Identification of ligands that selectively activate the M₁ muscarinic signaling pathway has been sought for decades to treat a range of neurological and cognitive disorders. Herein, we describe the optimization efforts focused on addressing key physicochemical and safety properties, ultimately leading to the clinical candidate MK-7622, a highly selective positive allosteric modulator of the M₁ muscarinic receptor that has entered Phase II studies in patients with Alzheimer's disease.

Full text of DOI:10.1021/acsmedchemlett.8b00095

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Letter
MK-7622: a First in Class M1 Positive
Allosteric Modulator Development Candidate
Douglas C. Beshore, Christina Ng DiMarco, Ronald K. Chang, Thomas J. Greshock, Lei
Ma, Marion Wittmann, Mattthew A. Seager, Kenneth A Koeplinger, Charles D. Thompson,
Joy Fuerst, George D. Hartman, Mark T. Bilodeau, William J Ray, and Scott D Kuduk
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00095 • Publication Date (Web): 30 Apr 2018
Downloaded from http://pubs.acs.org on April 30, 2018
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MKꢀ7622: a First in Class M1 Positive Allosteric Modulator Development
Candidate
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Douglas C. Beshore,* Christina N. Di Marco, Ronald K. Chang, Thomas J. Greshock, Lei Ma, Marion
Wittmann, Matthew A. Seager, Kenneth A. Koeplinger, Charles D. Thompson, Joy Fuerst, George D.
Hartman, Mark T. Bilodeau, William J. Ray, Scott D. Kuduk*#
Merck & Co., Inc., MRL, Kenilworth, New Jersey, 07033, USA
Abstract: Identification of ligands that selectively activate the M1 muscarinic signaling pathway has been
sought for decades to treat a range of neurological and cognitive disorders. Herein, we describe the
optimization efforts focused on addressing key physicochemical and safety properties, ultimately leading to
the clinical candidate MKꢀ7622, a highly selective positive allosteric modulator of the M1 muscarinic
receptor that has entered Phase II studies in patients with Alzheimer’s disease.
N
N
OH
O
N
Me
MK-7622
KEYWORDS: M1, muscarinic, positive allosteric modulator, M1 PAM, MKꢀ7622
Alzheimer's disease (AD) involves the progressive and irreversible degeneration of cholinergic neurons in
the basal forebrain, which serve critical functions in the central nervous systems (CNS) with respect to both
memory and cognition, ultimately contributing to cognitive decline in patients. 1 Cholinergic signaling is
mediated by acetylcholine (ACh), the endogenous ligand of both the nicotinic and muscarinic receptors; the
latter are class A Gꢀprotein coupled receptors (GPCR) comprised of five subtypes (M1 through M5).2,3 Of
the subtypes, M1 is most highly expressed in the hippocampal, striatal, and cortical areas crucial in memory
and higher brain function.4 As a result, direct activation of the M1 receptor has represented an attractive
approach to treat the cognitive deficits associated with AD and for more than two decades, the
pharmaceutical industry has pursued this goal. 5
Moderate cognitive improvement has been demonstrated with acetylcholine esterase inhibitors like
donepezil (Aricept®), which represent the current standard of care for AD patients, impacting both
nicotinic and muscarinic signaling pathways. Direct activation of the muscarinic system with orthosteric
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agonists, such as xanomeline, provides evidence that muscarinic activation may improve cognitive
performance in AD patients. 6 However, xanomeline’s clinical utility was ultimately limited by tolerability
believed to arise from nonselective activation of the M2ꢀM5 muscarinic subꢀtypes, a consequence of the
ligand binding to the highly conserved muscarinic orthosteric binding site. Alternate strategies to achieve
preferential activation of the M1 receptor have also been pursued, including bitopic and allosteric agonists
that interact at less conserved binding regions. 7,8,9 Another strategy to pursue is the identification of a
positive allosteric modulator (PAM) of the M1 receptor, an approach that relies on the selective
amplification of endogenous ACh signaling tone with minimal intrinsic activity in the absence of ACh. An
allosteric modulator approach was pursued at Merck Research Laboratories (MRL) and progress in this
area has been reported.
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Screening of the MRL sample collection provided lead BQCA (Figure 1, IP is defined as the inflection
point of the potentiation curve) 10 and has been wellꢀcharacterized.11 Optimization efforts aimed toward
improving physicochemical properties led to the identification of quinolizidinone PQCA.12,13,14 While both
BQCA and PQCA served as excellent tools to probe the pharmacological effects of allosteric
modulation,15 there were a number of improvements required to render a suitable development compound.
Firstly, these carboxylic acidꢀcontaining modulator classes exhibited generally low central nervous system
exposure in which a relationship was not observed between CNS exposure and Pꢀgp efflux ratios (as
measured by CSF/Uplasma ratios), suggesting the involvement of additional transport mechanisms.
Furthermore, due to the propensity for acyl glucuronide formation and low confidence in predicting human
pharmacokinetics, a suitable candidate could not be identified. While amideꢀbased strategies that addressed
pharmacokinetic and metabolic properties were pursued by MRL and others, 16 , 17 these approaches
enhanced Pꢀgp efflux and hERG channel activity, which were ultimately limiting. This manuscript
summarizes key data and medicinal chemistry efforts], which culminated in the identification of MKꢀ7622,
a CNS penetrant, M1 PAM suitable for clinical development.
Figure 1. BQCA and PQCA M1 positive allosteric modulators.
To address these limitations, replacement of the carboxylic acid became a key focus of our medicinal
chemistry efforts. The favorable CNS properties of BQCA are unique, as most carboxylic acids are
peripherally restricted.18 The measured pKa of BQCAꢀlike carboxylic acids are higher (ranging between
6.8ꢀ7.4) than typical carboxylic acids, suggesting an intramolecular ketoꢀhydrogen bond (Figure 2).
Mimicry of this motif was pursued through the introduction of a third ring fused to the bicyclic core.
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Bioisostereꢀlike replacement of the intramolecular ketoꢀhydrogen bond with removal (isoxazolinone 1) or
retention (pyrazolone 2) of hydrogen bond donating ability proved unfruitful. Further substitution of the
tricyclic ring system was required to establish activity and the addition of an Nꢀphenyl ring provided
modest potency (3, M1 IP = 8.9 µM). Introduction of an ortho substituent on the phenyl ring was critical
for achieving potent M1 activity. In the case of compound 4, an orthoꢀfluorine provided M1 potency
comparable to BQCA. Extensive SAR studies with pyrazoloneꢀcontaining compounds revealed that
exquisite potencies could be achieved (IP <5 nM,).19 However, increases in potency were orthogonal to
desirable physicochemical properties (i.e., solubility), as compounds like 4 possess few rotatable bonds and
high aromatic ring count.
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Figure 2. Optimization of nonꢀcarboxylic acid M1 PAMs
This structureꢀbased observation proved to be critical, as physicochemical properties were modulated by
changes to the tricyclic core cores. Exploration of core replacements with the simplified paraꢀ
methoxybenzyl moiety
revealed that the fused pyridinone 5 (Table 1) possessed weak activity and a
dramatic increase in cLogP. Potency was restored with the introduction of the pyrimidinone 6, but the low
sp3ꢀcharacter of these naphthylpyridinones also impaired physicochemical properties, resulting in only
modest improvements in ligandꢀlipophilic efficiency (LLE).20 Replacement of the fluorophenyl with transꢀ
cyclohexanols, as observed with lactam 7, proved fruitful through the introduction of significant sp3ꢀ
character with gains in potency and LLE. While improvements in physicochemical properties were
21
achieved with lactams, Pꢀgp efflux remained endemic and was not resolvable within this series.
Interestingly, as the transꢀcyclohexanol was introduced into the pyrimidinone scaffold 8, further
improvements in LLE were realized (compared to 6) and Pꢀgp efflux was attenuated. Further
improvements in LLE were realized with introduction of the chloropyridine 9.
Table 1. Properties of PAMs 4ꢀ9
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Compound
4
5
6
7
8
9
M1 Potency (IP, µM)a 0.41 3.3 0.82 0.052 0.22 0.015
cLogPb
LLEc
3.4
6.5
5.5
4.0
3.3
4.3
2.3
3.6
4.2
3.0 ꢀ1.0 0.56
a
Values represent the numerical average of at least two experiments. Interassay variability was ± 30% (IP, nM), unless otherwise
noted. b cLogP calculated with ACD version 12. c LLE was calculated as follows: pIPꢀpLogD where pIP= ꢀlog10(IP[M]).
Having identified scaffold 9 as displaying low potential for Pꢀgp efflux (BA/AB ratio = 1.1), further
evaluation of the southern benzyl ring was undertaken, providing compounds 9ꢀ13 (Table 2). The
substituent changes had little effect on compound potency with all compounds exhibiting good M1
potentiation. Compounds 9ꢀ12 were evaluated for and exhibited good CNS penetration as determined by
CSF/Uplasma ratios. Interestingly, as pyridine basicity was modulated in this class through electron
withdrawing groups (9, 12, 13), a marked increase in LogD was observed. This was reflected in decreased
solubility at pH 2, which was even more pronounced at physiologically relevant pH 7 at which nonꢀbasic
compounds exhibited no detectable solubility. More basic pyridines (i.e., 10, 11) had good pH 2 solubility
with moderate solubility at pH 7. Additionally, nonꢀbasic and unsubstituted pyridines inhibited hERG. 2ꢀ
Methylpyridine 11 represented the optimized balance of properties with respect to hERG inhibition, Pꢀgp
efflux (Papp = 33⋅10ꢀ6 cm/s; MDR1 BA/AB ratio = 1.6), solubility profile, and LLE.
Table 2. Properties of PAMs 9ꢀ13
M1 IP
(nM)a CSF/Uplasma
Rat
HPLC
Solubility
hERG
b
Compound
R
LogD LLEc pH 2, 7 (µM)d IC50 (µM)
H
Me
Cl
42
19
13
18
21
0.5
0.5
0.9
1.0
ND
2.4
2.6
3.1
3.0
3.2
5.0
5.1
4.8
4.7
4.5
181, 34
191, 17
< 2, <2
71, <3
12
60
13
6.4
7.5
10
11
9
12
13
OMe
SMe
102, < 2
a
Values represent the numerical average of at least two experiments. Interassay variability was ± 30% (IP, nM), unless otherwise
noted. b Determined in SpragueꢀDawley rats following oral dose of 10 mg/kg in 90% PEG 400. Interanimal variability was less than
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c
30% for all values. Ratios determined by rat CSF / (plasma concentration x fraction unbound in plasma). LLE was calculated as
follows: pIPꢀpHPLCLogD where pIP= ꢀlog10(IP[M]). d Kinetic solubility measured at noted pH.
5
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9
The synthetic approach for preparing pyrimidinones 9ꢀ13 is shown in Scheme 1. Methylnaphthalene 14
was treated with Bredereck’s reagent to form intermediate 15. Oxidative cleavage and ester formation
afforded methyl ester 16. Nitro reduction and subsequent bromination afforded 17. Upon hydrolysis,
HATUꢀmediated coupling of acid 18 with (S,S)ꢀtransꢀ1ꢀhydroxyꢀ2ꢀaminoꢀcyclohexane provided amide 19,
which was cyclized with DMF⋅DMA to form benzoquinazoline 20. Negishiꢀtype cross coupling of 20 with
(2ꢀchloroꢀ5ꢀpyridyl)methylzinc chloride afforded chloropyridine 9, which was used for the synthesis of
compounds 10ꢀ13 via standard transformations.
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Scheme 1a: Synthetic Scheme
aReagents: (a) tertꢀbutoxybis(dimethylamino)methane, toluene 120 °C (96%). (b) KMnO4, K2CO3, tertꢀBuOH, H2O (61%). (c) HCl,
MeOH, 90 °C (88%). (d) i. Pd/C, H2, MeOH, THF. ii. Br2, dioxane, CCl4 (95%). (e) LiOH, THF, H2O (94%). (f) (1Hꢀ1,2,3ꢀ
benzotriazolꢀ1ꢀyloxy)[tris(dimethylamino)]phosphonium hexafluorophosphate, (1S,2S)ꢀ2ꢀaminocyclohexanol, DMF, TEA (73%). (g)
DMFꢀDMA, 80 °C (78%). (h) Pd(tBu3P)2, (2ꢀchloroꢀ5ꢀpyridyl)methylzinc chloride, THF, (80%). (i) N,Nꢀdimethylꢀtransꢀ
diaminocyclohexane, CuI, MeOH, 160 °C (41%). (j) MeZnCl, Pd(tBu3P)2, , THF, 90 °C (95%). (k) MeOH, transꢀN1,N2ꢀ
dimethylcyclohexaneꢀ1,2ꢀdiamine, Cs2CO3, CuI, 160 °C (10%). (l) MeSNa, DMF, 120 °C (84%).
Further profiling revealed that methylpyridine 11 exhibited the lowest intrinsic clearance in human
microsomes (Clint = 15 mL/min/kg) amongst compounds examined from Table 2, and was selected for
advanced characterization. Pharmacokinetic studies with 11 were conducted in rats, dogs, and monkeys
and the parameters obtained are summarized in Table 3. Plasma clearances were low to moderate with
good oral bioavailability. Terminal halfꢀlives were moderate, ranging from 1.3ꢀ8.7 hours. M1 PAM 11 is
eliminated primarily via oxidative metabolism and in vitroꢀin vivo correlation analysis utilizing the “well
stirred” model of hepatic clearance was used to predict in vivo clearances from microsomal stability data.22
Based on this analysis, 11 was predicted to exhibit low to moderate total clearance in human within the
range of 0.7ꢀ2.3 mL/min/kg, supportive of onceꢀdaily dosing.
Table 3. Rat, Dog, and Rhesus Pharmacokinetics of 11
F (%)
t
1/2 (h)
Cl (ml/min/kg)
Vdss (L/kg)
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Rata
Dogb
Rhesusc
56
76
40
1.3
8.7
1.8
18.9
4.5
0.94
2.4
6
7.6
1.2
7
8
9
a
b
SpragueꢀDawley rats (n = 3). Oral dose 10 mg/kg, IV dose = 2 mg/kg. Mongrel dogs (n = 2). Oral dose 3 mg/kg, IV dose = 1
mg/kg. c Rhesus Monkeys (n = 2). Oral dose 1 mg/kg, IV dose = 0.5 mg/kg. Interanimal variability was less than 20% for all values.
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The equilibrium solubility of 11 was tested in various buffer solutions (pH 2ꢀ10), biorelevant media, and
vehicles used for in vivo toxicology studies. Compound solubility was pHꢀsensitive, as observed in the
kinetic solubility in Table 2; at acidic pH (< pH 4) solubility was observed to be between 6 and 9 mg/mL
and at pH > 6 solubility decreased; solubility was <0.001 mg/mL at pH 8 and 10. A crystalline fumarate
salt was identified for development with a solubility of 2.3 mg/mL in simulated gastric fluid and 0.04
mg/mL in fasted state simulated intestinal fluid.
Advanced pharmacological profiling was conducted with M1 PAM 11. Selectivity over M2ꢀM4 muscarinic
subtypes was evaluated in CHO cells overexpressing the human receptor subtypes and neither potentiation
nor agonism of the M2ꢀM4 receptors was observed. This high selectivity for M1 potentiation is consistent
with the properties of the progenitor (BQCA) of 11 and the totality of the molecules prepared during the
course of the optimization efforts. In addition, M1 PAM 11 possessed similar M1 potentiation across
species: rat (M1 IP = 14 nM), mouse (M1 IP = 6.7 nM), dog (M1 IP = 1.4 nM), and rhesus (M1 IP = 3.8 nM)
receptors. M1 PAM 11 was found to have no significant findings in an ancillary pharmacology profile
screen of 140 targets (CEREP panel) with only two hits having less than 5 µM potency (Lipoxygenase 5ꢀ
LO IC50 = 4.4 µM, Phosphodiesterase PDE4 IC50 = 2.1 µM). Lastly, 11 proved to be a weak competitive
reversible inhibitor of CYP2C9 (IC50 = 13.3 ꢁM). The potential for cytochrome P450 induction was
characterized by treatment of human hepatocytes with 11 (0.1ꢀ10 µM) and no observed induction of
CYP3A4 mRNA or activity was observed.
M1 PAM 11 was further characterized regarding receptor allostery (Figure 3) and its ability to modulate the
response of ACh (12 pM to 1.0 µM) to varying doses of allosteric modulator (22 pM to 22 µM).23 The
responses were normalized to the ACh response at 1000 µM and these results indicate that 11 is a highly
cooperative modulator (α = 338) with a 0.948 µM affinity for the unbound receptor (KB) with a τB of
1.067.
Figure 3. Receptor allostery profile of 11 measured by Ca++ flux in human M1 overexpressing CHO cells
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The in vivo properties of M1 PAM 11 were evaluated in a mouse contextual fear conditioning (CFC) assay,
which is dependent on hippocampal function (Figure 4). 24 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 11 exhibited a significant reversal at 3 mg/kg, compared to mice treated with
scopolamine alone. The corresponding plasma levels were 2.4 ꢀM and the estimated free brain levels at
this dose were ~ 24 nM, within ~3ꢀfold range of the M1 inflection point in mouse.
Figure 4. In vivo activity of 11
Contextual Fear Conditioning
70
#
Vehicle
Scop (0.3 mpk)
60
Scop/11 (1 mpk)
Scop/11 (2 mpk)
50
Scop/11 (3 mpk)
*
40
*
30
20
10
0
Baseline
24 hr
In summary, quinolone carboxylic acid HTS lead BQCA was evolved into nonꢀcarboxylic acid modulators
(i.e, 5), which were optimized for physicochemical and safety properties into benzoisoquinolinone 11. M1
PAM 11 is an M1ꢀselective modulator with favorable preꢀclinical pharmacokinetic and pharmacodynamic
properties. On the basis of these properties and the potential for a good human ADMET profile, 11 (MKꢀ
7622) was selected for clinical development and has entered Phase II human proofꢀofꢀconcept studies for
the treatment of cognitive dysfunction in patients with AD. The results of these studies will be disclosed in
due course.
Supporting Information.
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The Supporting Information is available free of charge on the ACS Publications website at DOI:
Experimental procedures of selected compounds and spectra are provided.
ACKNOWLEDGMENTS:
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The authors thank Dr. James Small for obtaining and interpreting NMR spectra.
# Current address: Janssen Pharmaceuticals, 1400 McKean Road (22-21111), Spring House, PA 19477
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Xanomeline, a selective muscarinic receptor agonist, on cognitive function and behavorial symptoms in
Alzheimer. Disease Arch. Neurol. 1997, 54, 465ꢀ473.
7 Greenlee, W.; Clader, J.; Asbersom, T.; McCombie, S.; Ford, J.; Guzik, H.; Kozlowski, J.; Li, S.; Liu, C.;
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Coffin, V.; Watkins, R.; Ruperto, V.; Strader, C.; Taylor, L.; Cox, K. Muscarinic agonists and antagonists
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treatment of CNS. Disorders. Nat. Rev. Drug Disc. 2009, 8, 41ꢀ54.
9 For an example of an allosteric activator of the M1 receptor, see Jones, C.K.; Brady, A.E.; Davis, A.A.;
Xiang, Z.; Bubser, M.; NoorꢀWantawy, M.; Kane, A.S.; Bridges, T.M.; Kennedy, J.P.; Bradley, S.R.;
Peterson, T.E.; Ansari, M.S.; Baldwin, R.M.; Kessler, RM.; Deutch, A.Y.; Lah, J.J.; Levey, A.I.; Lindsley,
C.W.; Conn, P.J. Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates
amyloid processing and produces antipsychoticꢀlike activity in rats. J. Neurosci. 2008, 41, 10422ꢀ10433.
10 Ma, L.; Seager, M.; Wittmann, M.; Jacobsen, M.; Bickel, D.; Burno, M.; Jones, K.; KuzmickꢀGraufelds,
V.; Xu, G.; Pearson, M.; McCampbell, A.; Gaspar, R.; Shughrue, P.; Danziger, A.; Regan, C.; Flick, R.;
Garson, S.; Doran, S.; Kreatsoulas, C.; Veng, L.; Lindsley, C.; Shipe, W.; Kuduk, S.D.; Sur, C.; Kinney,
G.; Seabrook, G.; Ray, W.J. Selective activation of the M1 muscarinic acetylcholine receptor achieved by
allosteric potentiation. Proc. Natl.Acad. Sci. USA 2009, 106, 15950ꢀ15955.
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