Discovery of VU0467485/AZ13713945: An M4 PAM Evaluated as a Preclinical Candidate for the Treatment of Schizophrenia

Article abstract of DOI:10.1021/acsmedchemlett.6b00461

Herein, we report the structure-activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M₄) positive allosteric modulators (PAMs). Compound 6c (VU0467485) possesses robust in vitro M₄ PAM potency across species and in vivo efficacy in preclinical models of schizophrenia. Coupled with an attractive DMPK profile and suitable predicted human PK, 6c (VU0467485) was evaluated as a preclinical development candidate.

Full text of DOI:10.1021/acsmedchemlett.6b00461

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Letter
Discovery of VU0467485/AZ13713945: An M4 PAM Evaluated
as a Preclinical Candidate for the Treatment of Schizophrenia
Michael R Wood, Meredith J. Noetzel, Bruce J Melancon, Michael S Poslunsey, Kellie D. Nance, Miguel
A Hurtado, Vincent B Luscombe, Rebecca L Weiner, Alice L Rodriguez, Atin Lamsal, Sichen Chang,
Michael Bubser, Annie L Blobaum, Darren W. Engers, Colleen M Niswender, Carrie K. Jones, Nicholas J
Brandon, Michael W Wood, Mark E. Duggan, P. Jeffrey Conn, Thomas M. Bridges, and Craig W Lindsley
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00461 • Publication Date (Web): 16 Dec 2016
Downloaded from http://pubs.acs.org on December 19, 2016
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Discovery of VU0467485/AZ13713945: An M4 PAM Evaluated as
a Preclinical Candidate for the Treatment of Schizophrenia
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Michael R. Wood,†,║,Ϩ Meredith J. Noetzel,†,║,Ϩ Bruce J. Melancon,†,║ Michael S. Poslusney,║ Kellie D. Nance,║
Miguel A. Hurtado,║ Vincent B. Luscombe,†,║ Rebecca L. Weiner,†,║ Alice L. Rodriguez,†,║ Atin Lamsal,†,║
Sichen Chang,†,║ Michael Bubser, †,║ Anna L. Blobaum,†,║ Darren W. Engers,†,║ Colleen M. Niswender,†,║, ϒ Caꢀ
rrie K. Jones,†,║, ϒ Nicholas J. Brandon,ψ Michael W. Wood,ψ Mark E. Duggan,ψ P. Jeffrey Conn,†,║, ϒ Thomas M.
Bridges,†,║* and Craig W. Lindsley†,║*
†Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, United States
║Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University School of Medicine, Nashville, TN 37232, United
States
ψAstraZeneca Neuroscience, Innovative Medicines Biotech Unit,, 141 Portland Street, Cambridge, MA 02139, USA
ϒVanderbilt Kennedy Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
ϨThese three authors contributed equally
KEYWORDS: Positive allosteric modulator (PAM), muscarinic acetylcholine receptor 4 (M4), VU0467485, schizophrenia
ABSTRACT: Herein, we report the structureꢀactivity relationships within a series of potent, selective and orally bioavailable musꢀ
carinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs). 6c (VU0467485) possesses robust in vitro M4 PAM
potency across species and in vivo efficacy in preclinical models of schizophrenia. Coupled with an attractive DMPK profile and
suitable predicted human PK, 6c (VU0467485) was evaluated as a preclinical development candidate.
The rapid onset of clinical efficacy of the M1/M4 preferring
Me
Me
NH2
NH2
Me
N
NH2
Me
agonist xanomeline in both schizophrenic and Alzheimer’s
patients led to a revolution in muscarinic receptor drug disꢀ
covery efforts targeting allosteric mechanisms to afford highly
subtype selective M1 and M4 positive allosteric modulators
(PAMs).1ꢀ6 Of these, M4 has emerged as the favored mAChR
subtype responsible for antipsychoticꢀlike efficacy as well as
modest cognitive enhancement in multiple preclinical rodent
models via a fundamentally new molecular mechanism.7ꢀ15
While M1 PAMs with diverse chemotypes (and without major
species differences in pharmacology) have rapidly advanced to
potentially address cognitive dysfunction and negative sympꢀ
toms,5,6,16,17 M4 PAMs have progressed more slowly for many
reasons, including species differences in M4 PAM potency
(i.e., affinity and cooperativity), challenges with respect to M2
selectivity, and Pꢀgp efflux as well as limited chemical diversiꢀ
ty (Figure 1).9ꢀ14,18ꢀ20 Clearly, these are significant roadblocks
en route to an M4 PAM preclinical candidate. In this Letter,
we detail our navigation of these issues within the VU0467154
series of M4 PAMs, leading to the discovery of a potent, selecꢀ
tive and orally bioavailable M4 PAM 6c (VU0467485) with
robust efficacy in behavioral models that was evaluated as a
preclinical candidate. This is the first disclosure of the strucꢀ
ture activity relationships (SAR) and preclinical profile of 6c.
O
O
Cl
O
N
S
HN
S
HN
Me
N
N
S
HN
MeO
1
2
, VU0152100
, LY2033298
OMe
3
, VU0467154
SO2CF3
Figure 1. Structures of reported M4 PAMs. LY2033298 (1) was the first
reported M4 PAM, but was humanꢀpreferring in potency. VU0152100 (2)
was the first centrally active M4 PAM in rodents, and VU0467154 (3) has
proven to be the bestꢀinꢀclass M4 PAM rodent in vivo tool compound.
Our discovery of 6c originated from an optimization
campaign centered on 2, and from which 3 was identified, but
discontinued due to a human and rat M4 PAM potency disconꢀ
nect that precluded development.10,12ꢀ14 A major thrust of the
initial lead optimization effort was to survey alternatives for
the pyridine ring in 2, and while other pyridine regioisomers
and pyrimidines afforded modest M4 potentiation, the pyriꢀ
dazine core, found in 3, proved optimal for imparting both
potency, metabolic stability, and favorable physiochemical
properties to the series21. SAR was steep and divergent across
rat and human M4, and CNS penetration was low in this series;
however, exceptional DMPK properties could be achieved21.
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Table 1. Structures and Activities of Analogues 6
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The synthesis of analog 6 was straightforward and reꢀ
quired only two steps from known materials (Scheme 1).12ꢀ14
1
2
3
Condensation of 3ꢀchloroꢀ5,6ꢀdimethylpyridazinꢀ4ꢀ
4
5
Scheme 1. Synthesis of Analogues 6a
hM4 EC50 (nM)a
hM4 pEC50
6
Entry
Ar (Het)
7
[% ACh Max ±SEM]
(±SEM)
8
9
6a
130
[83.7+3.5]
6.89+0.06
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6b
6c
96.7
[69.8+2.2]
7.01+0.08
7.10+0.01
aReagents and conditions: (a) Methyl thioglycolate, MeOH, 1M aq.
NaOH, 150 C, microwave, 30 min, 78%; (b) NH2CH2Ar(Het), HATU,
o
DMF, DIEPA, 2 h, 45ꢀ92%.
78.8
[80.6+0.7]
carbonitrile 4 with methylthioglycolate under microwave irraꢀ
6d
diation
delivered
the
core
sodium
5ꢀaminoꢀ3,4ꢀ
in yields
90.6
[75.5+2.4]
7.04+0.14
7.38+0.05
7.07+0.03
dimethylthieno[2,3ꢀc]pyridazineꢀ6ꢀcarboxylate
5
averaging 78%. Next, a HATUꢀmediated amide coupling
reaction with various amines provides analogues 6 in yields
ranging from 45ꢀ92% after HPLC purification.21 The direct
congener of 2, VU0464090 (6a), demonstrated an ~3ꢀfold
increase in M4 PAM potency (human M4 PAM EC50 = 130
nM, pEC50 = 6.89±0.06, 83.7±3.5 ACh Max and rat M4 PAM
EC50 = 59.7 nM, pEC50 = 7.22±0.06, 78.1±1.7 ACh Max),
increased fraction unbound in plasma (fuplasma (r, h) = 0.022,
0.035) and reduced hepatic microsomal intrinsic clearance
(CLint (r, h) = 81 and 36 mL/min/kg; predicted CLhep (r, h) =
37 and 13 mL/min/kg) by virtue of the pyridazine ring system;
however, metabolic instability of the PMB group precluded
further advancement of 6a.14 As shown in Table 1, application
of the fluorine walk strategy22,23,24 for allosteric modulator
optimization proved fruitful, affording a number of potent M4
PAMs (6cꢀh). Heteroatom incorporation into the benzyl ring
was met with limited success, with only 3ꢀpyridyl congeners
(6i and 6j) retaining PAM activity (other regioisomeric pyriꢀ
dines, pyrimidines, pyrazines and pyridazines were inactive,
6e
6f
41.4
[68.5+1.4]
84.1
[75.3+1.1]
6g
6h
43.4
[71.2+3.6]
7.36+0.02
6.99+0.06
100
[87.1+1.9]
6i
6j
6k
6l
239
[76.9+2.8]
6.62+0.11
6.85+0.08
7.22+0.07
142
[80.8+1.5]
M4 EC50s >10 ꢀM). Moreover, we suspected CYP450
mediated oxidative dealkylation of 6a led to 6b, an active puꢀ
tative metabolite (human M4 PAM EC50 = 96.7 nM, pEC50
ꢀ
59.1
[83.1+2.9]
=
7.01±0.01, 69.8±2.2 ACh Max), but with high clearance in
vivo (rat) and undesired activity at hM2. As a battery of in
vitro and in vivo DMPK assays (vide infra) quickly identified
6c as the most attractive PAM in the series, we prepared its
putative dealklyated metabolite 6k, which also proved to be
active (human M4 EC50 = 59 nM), but with no activity at hM2,
suggesting the orthoꢀfluoro moiety enhances selectivity versus
hM2. Incorporation of chiral methyl groups at the benzylic
position of 6c led to (R)ꢀ6l (human M4 EC50 >10 ꢀM) and (S)ꢀ
6m (human M4 EC50 = 508 nM), where enantiospecific activiꢀ
ty was noted, but with unacceptable loss in human M4 PAM
>10
>5.0
6m
508
[62.4+2.5]
6.29+0.09
6n
6o
55.7
[87.1+0.8]
7.25+0.04
6.72b
213b
[60.9]
2
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aCalcium mobilization assays with hM4/Gqi5ꢀCHO cells performed in the
rM4 over rM1ꢀ3,5. Data represent means from at least three independent
presence of an EC20 fixed concentration of acetylcholine; values represent
determinations performed in triplicate using CHO cells stably transfected
with the indicated mAChR.
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2
3
4
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6
7
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means from three (n=3) independent experiments performed in triplicate
b
unless otherwise noted. values from one experiment performed in tripliꢀ
cate.
the foldꢀshift data to better understand the allosteric effects of
this promising candidate.5,22 PAM 6c displayed robust potenꢀ
tiation of ACh and cooperativity (log αβ = 2.1, αβ = 134),
significant intrinsic efficacy (log τB = 5.1), and an approxiꢀ
mately 1 µM estimated affinity (pKB 6.025, KB = 944 nM) at
human M4.
potency. Finally, based on previous beneficial disposition by
virtue of the kinetic isotope effect, we evaluated a deuterated
congener, but in this instance, there was no benefit to stability.
9
Thus, efforts focused on 6c as a potential M4 PAM preclinical
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candidate with minimal species differences in PAM potency.
6c was then evaluated in a panel of in vitro DMPK assays23
where it displayed properties that supported continued proꢀ
gression. Not only did 6c possess an exceptionally clean
CYP450 inhibition (3A4, 2D6, 2C9, 1A2 IC50s >30 ꢀM in huꢀ
man hepatic microsomes) and induction profile (3A4, 1A2,
2B6 EC50s >50 µM, Emaxs ≤ 1.0 in cryopreserved human
hepatocytes) profile, but also moderate plasma protein binding
was noted across species (fuplasma r, h, c = 0.031, 0.054 and
0.091) with moderate fraction unbound in rat brain homogeꢀ
nate (fubr = 0.037). Based on hepatic microsomal CLint data,
moderate predicted hepatic clearance was also observed for 6c
(predicted CLhep r, h, c: 73, 26, and 38 mL/min/kg). Moreoꢀ
ver, 6c was not a Pꢀgp substrate (ER = 1.4 in MDCKꢀMDR1)
and showed good apparent permeability (Cacoꢀ2 Papp = 31 x
10ꢀ6 cm/s). In rat and dog brain distribution studies, 6c disꢀ
played moderate to high CNS penetration with Kps of 0.31 to
1.0 and Kp,uus of 0.37 to 0.84. With regard to toxicity evaluaꢀ
tion in vitro, 6c was clean in GSH trapping experiments (huꢀ
man hepatic microsomes) and a miniꢀAmes test (data not
shown). In vivo PK (Table 2) was assessed in three species
(rat, dog and cynomolgous monkey), which revealed moderate
clearance, low to moderate volume of distribution at steadyꢀ
state, and short to moderate
The molecular pharmacology profile of M4 PAM 6c is shown
in Figure 2. PAM 6c is inactive in the absence of acetylchoꢀ
line (ACh), but in the presence of an EC20 concentration of
ACh (Figure 2A), 6c potentiates M4 in a concentrationꢀ
dependent manner, affording potent activity at both human
and rat M4 (human M4 PAM EC50 = 78.8 nM, pEC50
=
7.10±0.01, 80.6±0.7 ACh Max and rat M4 PAM EC50 = 26.6
nM, pEC50 = 7.57±0.05, 68.7±3.4 ACh Max). For a preclincial
candidate, comparable activity across species is essential for
INDꢀenabling toxicology studies, and this was a major issue
for previously reported M4 PAMs for which the species disꢀ
connect averaged 10ꢀ50ꢀfold.9ꢀ14,18ꢀ20 Thus, we also evaluated
6c against dog and cynomolgous monkey M4 and the key antiꢀ
target, M2. Beyond rat and human, 6c displayed no major
species differences in potency (dog M4 EC50 = 87 nM, 49%
ACh Max, dog M2 >30 ꢀM and cyno M4 EC50 = 102 nM, 74%
ACh Max, cyno M2 >30 ꢀΜ). In a progressive foldꢀshift asꢀ
say with human M4 (Figure 2B), 6c afforded a maximal ~45ꢀ
fold leftward shift of the human M4 ACh concentrationꢀ
response curve (CRC) at 10 ꢀM (~40ꢀfold shift at rat M4).
Moreover, PAM 6c was selective versus human and rat M1ꢀ3,5
The operational model of allosterism was applied to
.
Table 2. Pharmacokinetic Parameters of 6c
Parameter
Rata
(SD)
Doga
(beagle or
mongrel)
1/3
NHPa
(cyno)
Dose (mg/kg) iv/po
CLp (mL/min/kg)
Vss (L/kg)
1/3
29
0.2 (iv only)
21
1.5
25
1.5
4.2
1.2
1.5
3.8
79
0.88
Elimination t1/2 (h)
Cmax (ꢀM) po
1.3
0.48
0.22
0.75
0.59
9.5
ꢀ
ꢀ
ꢀ
ꢀ
Tmax (h) po
AUCoꢀinf (ꢀM*hr) po
F (%) po
Figure 2. Molecular pharmacology profile of M4 PAM 6c. (A) Enhanced
intracellular calcium release induced by a subthreshold concentration of
acetylcholine (EC20), a PAM CRC on both rat and human M4, with EC50s
of 26.6 nM and 78.8 nM, respectively. (B) 6c induces an ~45ꢀfold maxiꢀ
mal leftward shift of the hM4 acetylcholine response curve at 10 ꢀM. (C)
6c is highly selective for hM4 over hM1ꢀ3,5. (D) 6c is highly selective for
3
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Total brain:total plasma (Kp)
0.31
0.37
1.0
ꢀ
ꢀ
1
2
Unbound
0.84
3
4
brain:unboundplasma (Kp,uu
)
aValues represent means from two to three animals.
5
6
7
8
9
Figure 4. P450 phenotyping and CYP mapping for 6c, indicating that
multiple CYP450s (3A4/2C19/1A2 and to a lesser extent, 2C9) catalyze
biotransformation.
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Evaluation in a rat amphetamineꢀinduced hyperlocomotion
(AHL) study, a traditional preclinical model of antipsychotic
activity,10,12ꢀ14 revealed that 6c (Figure 3) showed robust activꢀ
ity with a minimum effective dose (MED) of 10 mg/kg p.o.
(43.2% reversal) that correlates with terminal (time = 1.5 hr)
plasma and brain concentrations of 1.8 ꢀM (0.06 ꢀM unꢀ
bound) and 0.56 ꢀM (0.02 ꢀM unbound), respectively, and
showing greater efficacy than our previously reported rat tool
M4 PAM, VU0467154.13.,14
Figure 3. Metabolite identification studies for 6c across species (rat, dog,
cyno monkey and human).
elimination halfꢀlife with high oral bioavailability in rat but
low bioavailability in dog (3 mg/kg suspension dose of a
monoꢀHCl salt). In vitro metabolite identification experiments
found good coverage of human metabolites in dog and cynoꢀ
molgous monkey and no evidence for humanꢀunique metaboꢀ
lites (Figure 3). Human CYP450 phenotyping experiments
revealed that multiple CYPs contribute to 6c’s metabolism
(Figure 4) with a generally low potential for drugꢀdrug interꢀ
actions (no metabolismꢀrelated DDI liabilities anticipated in
Alzheimer’s disease clinical population as concomitant mediꢀ
cations (acetylcholinesterase inhibitors possess a 3A4/2D6
phenotype) and, in schizophrenia populations, common antiꢀ
Figure 5. 6c has antipsychoticꢀlike activity in an AHL rat model. 6c doseꢀ
dependently (1ꢀ10 mg/kg, po) reverses AHL (amphetamine, 0.75 mg/kg,
s.c., *p < 0.05 vs. vehicle + amphetamine, ** p < 0.01 vs. vehicle + amꢀ
phetamine, *** p < 0.001 vs. vehicle + amphetamine. N = 6ꢀ8rats /group.
psychotics
(e.g.,
clozapine;olanzapine)
possess
a
1A2/2D6/3A4 phenotype). Furthermore, in a functional
hERG assay, 6c was inactive when tested at 11 µM, as well as
against a larger cardiac ion channel panel (IC50s > 33 µM).
Finally, ancillary pharmacology was assessed in an internal
AZ/Cerep panel against 200 targets, and no significant offꢀ
target activities (IC50s or EC50s > 10 µM) were noted, includꢀ
ing both binding and functional assays for cardiac ion chanꢀ
nels, with the exception of a 1.2 µM IC50 (radioligand binding)
at the rat GABAA receptor.21
Based on the fact that 6c was the first M4 PAM discovered
in our program with similar M4 PAM activity across all preꢀ
clinical species and man, an attractive DMPK and ancillary
pharmacology profile, as well as robust efficacy in a rodent
model of antipsychotic activity, 6c was further profiled as a
putative preclinical candidate, including evaluation in a rat
modified Irwin test for effects on autonomic and somatomotor
functions. In this study, following a single high dose (56.6
mg/kg, p.o., N = 6), no significant effects were observed over
a sixꢀhour observation period. However, based on these aniꢀ
mal model acute concentrationꢀeffect data (rat AHL) and 6c’s
predicted human PK profile (Table 3), efficacious human oral
doses projected to provide 12ꢀhour daily coverage of the tarꢀ
get/mechanism were undesirably high and frequent (e.g., >
450 mg, TID).
Table 3. Predicted Human Pharmacokinetics of 6c
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Parameter
Value
3.7 – 8.9
The authors would also like to thank NIH (U01MH087965, Vanꢀ
CL (mL/min/kg)a
1
2
3
4
5
6
7
8
9
derbilt NCDDG). We also thank William K. Warren, Jr. and the
William K. Warren Foundation who funded the William K.
Warren, Jr. Chair in Medicine (to C.W.L.).
Vss (L/kg)b
t1/2 (hr)
1.5 – 2.1
1.9 – 6.6
71
ASSOCIATED CONTENT
F (%)c
Supporting Information. General methods for the synthesis and
characterization of all compounds, and methods for the in vitro
and in vivo DMPK protocols and supplemental tables. This mateꢀ
rial is available free of charge via the Internet at
http://pubs.acs.org.
ka (1/hr)d
0.55
aPredicted by multiꢀspecies IVIVE; predicted by scaling of unbound Vss
from rat and dog; cpredicted by assumption of an optimized
form/formulation providing an fabs of 1.0 with an fgut of 1.0 and fhep of 0.71
b
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ABBREVIATIONS
d
(i.e., ERhep = 0.29 based on mean predicted human CL/Qhep; predicted by
DCM, dichloromethane; AHL, amphetamineꢀinduced hyperlocomotion;;
MED, minimum effective dose; muscarinic acetylcholine receptor,
(mAChR); PAM, positive allosteric modulator; HATU, Oꢀ(7ꢀ
azabenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium hexafluorophosphate;
DIEPA, N,Nꢀdiisopropylamine; MED, minimum effective dose; AHL,
amphetamineꢀinduced hyperlocomotion.
the MAT method using rat oral PK data from a 3 mg/kg dose of the monoꢀ
HCl salt formulated as a suspension in 0.1% tween80 0.5% methylcelluꢀ
lose in water.
Additionally, 6c displayed low aqueous solubility (2.4 µM
at pH 7.4) and evidence for solubilityꢀlimited absorption in
dog was observed, even at low doses and when administered
as an HCl salt.
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Still, 6c represents a major advance in the field, as the first
potent M4 PAM to overcome major species differences in poꢀ
tency while maintaining high selectivity versus M2 (rat, dog,
cyno and human EC50s >30 ꢀM), CNS penetration, and in
vivo efficacy. However, given the projected human efficaꢀ
cious dosing and solubility issues, together with an anticipated
challenge in achieving sufficiently high oral exposure in INDꢀ
enabling safety studies to establish acceptable margins, further
advancement of 6c was halted (pending pharmaceutical sciꢀ
ences work), and optimization efforts shifted toward imꢀ
provement of aqueous solubility and longer predicted human
t1/2 while retaining all the desirable properties of 6c. Results
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acetylcholine receptor agonists and allosteric modulators for the
treatment of schizophrenia. Neuropsychopharmacology 2012, 37,
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9. Chan, W. Y., McKinzie, D. L., Bose, S., Mitchell, S. N., Witkin, J.M.,
Thompson, R. C., Christopoulos, A., Lazareno, S., Birdsall, N. J.,
Bymaster, F. P., and Felder, C. C. Allosteric modulation of the muscaꢀ
rinic M4 receptor as an approach to treating schizophrenia. Proc. Natl.
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10. Brady, A. E., Jones, C. K., Bridges, T. M., Kennedy, J. P.; Thompson,
A. D., Heiman, J. U., Breininger, M. L., Gentry, P. R., Yin, H.,
Jadhav, S. B., Shirey, J. K., Conn, P. J., and Lindsley, C. W. Centrally
active allosteric potentiators of the M4 muscarinic acetylcholine reꢀ
AUTHOR INFORMATION
Corresponding Authors
*(CWL). Phone: 1 615ꢀ322ꢀ8700. Fax: 1 615ꢀ936ꢀ4381. Email:
craig.lindsley@vanderbilt.edu. *(TMB). Phone: 1 615ꢀ875ꢀ9771. Fax:
1 615ꢀ936ꢀ4381. Eꢀmail: thomas.m.bridges@vanderbilt.edu
Author Contributions
CWL, TMB, MRW, PJC, CMN, NJB, MED and CJK draftꢀ
ed/corrected the manuscript. BJM, KDN, MSP, MAH and DWE perꢀ
formed the chemical synthesis. CWL, MWR, TMB, PJC, CMN, CJK,
MJN, NJB, MWW, MED and ALR oversaw the target selection and
interpreted the biological data. MJN, ALR, AL, VBL and RLW perꢀ
formed the in vitro molecular pharmacology studies. TMB, ALB and
SC performed the in vitro and in vivo DMPK studies. CKJ and MB
performed the in vivo experiments. All authors have given approval
to the final version of the manuscript.
Acknowledgement
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