Optimization of M4 positive allosteric modulators (PAMs): The discovery of VU0476406, a non-human primate in vivo tool compound for translational pharmacology

Article abstract of DOI:10.1016/j.bmcl.2017.04.043

This letter describes the further chemical optimization of the 5-amino-thieno[2,3-c]pyridazine series (VU0467154/VU0467485) of M₄ positive allosteric modulators (PAMs), developed via iterative parallel synthesis, culminating in the discovery of the non-human primate (NHP) in vivo tool compound, VU0476406 (8p). VU0476406 is an important in vivo tool compound to enable translation of pharmacodynamics from rodent to NHP, and while data related to a Parkinson's disease model has been reported with 8p, this is the first disclosure of the optimization and discovery of VU0476406, as well as detailed pharmacology and DMPK properties.

Full text of DOI:10.1016/j.bmcl.2017.04.043

Accepted Manuscript
Optimization of M4 positive allosteric modulators (PAMs): The discovery of
VU0476406, a non-human primate in vivo tool compound for translational
pharmacology
Bruce J. Melancon, Michael R. Wood, Meredith J. Noetzel, Kellie D. Nance,
Eileen M. Engelberg, Changho Han, Atin Lamsal, Sichen Chang, Hyekyung P.
Cho, Frank W. Byers, Michael Bubser, Carrie K. Jones, Colleen M. Niswender,
Michael W. Wood, Darren W. Engers, Dedong Wu, Nicholas J. Brandon, Mark
E. Duggan, P. Jeffrey Conn, Thomas M. Bridges, Craig W. Lindsley
PII:
S0960-894X(17)30421-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.043
BMCL 24896
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 March 2017
11 April 2017
12 April 2017
Please cite this article as: Melancon, B.J., Wood, M.R., Noetzel, M.J., Nance, K.D., Engelberg, E.M., Han, C.,
Lamsal, A., Chang, S., Cho, H.P., Byers, F.W., Bubser, M., Jones, C.K., Niswender, C.M., Wood, M.W., Engers,
D.W., Wu, D., Brandon, N.J., Duggan, M.E., Jeffrey Conn, P., Bridges, T.M., Lindsley, C.W., Optimization of
M4 positive allosteric modulators (PAMs): The discovery of VU0476406, a non-human primate in vivo tool
compound for translational pharmacology, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/
10.1016/j.bmcl.2017.04.043
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Optimization of M4 positive allosteric modulators
(PAMs): The discovery of VU0476406, a non-human
primate in vivo tool compound for translational
pharmacology
Leave this area blank for abstract info.
Bruce J. Melancon, Michael R. Wood, Meredith J. Noetzel, Kellie D. Nance, Eileen M. Engelberg, Changho Han, Atin
Lamsal, Sichen Chang, Hyekyung P. Cho, Frank W. Byers, Michael Bubser, Carrie K. Jones, Colleen M. Niswender,
Michael W. Wood, Darren W. Engers, Dedong Wu, Nicholas J. Brandon, Mark E. Duggan, P. Jeffrey Conn, Thomas M.
Bridges and Craig W. Lindsley

Bioorganic & Medicinal Chemistry Letters
Optimization of M4 positive allosteric modulators (PAMs): The discovery of
VU0476406, a non-human primate in vivo tool compound for translational
pharmacology
Bruce J. Melancon,a,b Michael R. Wood,a,c Meredith J. Noetzel,a,b Kellie D. Nance,a Eileen M. Engelberg,a
Changho Han,a Atin Lamsal,a Sichen Chang,a Hyekyung P. Cho,a,b Frank W. Byers,a Michael Bubser,a
Carrie K. Jones a,b,e Colleen M. Niswender,a,b,e Michael W. Wood,d Darren W. Engers,a,b Dedong Wu,f
Nicholas J. Brandon,d Mark E. Duggan,d P. Jeffrey Conn,a,b,e, Thomas M. Bridges a,b* and Craig W.
Lindsleya,b,c*
aVanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
bDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
cDepartment of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
d AstraZeneca, Neuroscience, Innovative Medicines & Early Development, Waltham, Massachusetts 02451 USA
eVanderbilt Kennedy Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
fAstraZeneca, Pharmaceutical Science, 35 Gatehouse Drive, Waltham, MA 02451, USA.
*To whom correspondence should be addressed: thomas.m.bridges@vanderbilt.edu and craig.lindsley@vanderbilt.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
This letter describes the further chemical optimization of the 5-amino-thieno[2,3-c]pyridazine
series (VU0467154/VU0467485) of M4 positive allosteric modulators (PAMs), developed via
iterative parallel synthesis, culminating in the discovery of the non-human primate (NHP) in
vivo tool compound, VU0476406 (8p). VU0476406 is an important in vivo tool compound to
enable translation of pharmacodynamics from rodent to NHP, and while data related to a
Parkinson’s disease model has been reported with 8p, this is the first disclosure of the
optimization and discovery of VU0476406, as well as detailed pharmacology and DMPK
properties.
Keywords:
M4
Muscarinic acetylcholine receptor
Positive allosteric modulator (PAM)
Non-human primate (NHP)
Structure-Activity Relationship (SAR)
2017 Elsevier Ltd. All rights reserved.
Muscarinic acetylcholine receptor subtype 4 (M4) positive
allosteric modulators (PAMs) represent a fundamentally new
approach to treat multiple symptom domains of schizophrenia,
Huntington’s disease and Parkinson’s disease (PD).1-12 M4 PAMs
(Figure 1) represent the extreme of allosteric modulator caveats
in terms of steep SAR, species differences in pharmacology (rat
versus human versus cynomolgus (cyno) monkey versus dog M4
PAM potency, affinity/cooperativity and subtype selectivity),
poor solubility, and/or low CNS penetration.3,10-16 Work in this
area has been challenging since the first report by Eli Lilly of 1, a
human-preferring M4 PAM.1 Subsequent optimization efforts
afforded 2, which provided the first in vivo proof-of-concept
(POC) for selective M4 potentiation of endogenous acetylcholine
in reversing amphetamine-induced hyperlocomotion (AHL) in
rats.3 Further efforts improved physiochemical/DMPK properties
and delivered the in vivo rodent POC tool compound, 3.11,15
Finally, 4 afforded a balance in M4 PAM activity across species,
and was evaluated in depth as our first potential M4 PAM clinical
candidate.16 Despite these advances, we required an in vivo non-
Figure 1. Structures of representative M4 PAMs 1-4, exemplifying an
optimized rodent in vivo tool M4 PAM, VU0467154 (3) and the clinical
candidate VU0467485/AZ13713945 (4).

human primate (NHP) POC tool compound for use in
translational efficacy studies, primate PD models and biomarker
studies that could be freely shared with collaborators. In this
Letter, we detail the discovery effort that led to VU0467406, an
in vivo M4 PAM non-human primate (NHP) tool compound.
finally to the 4-position (8f, hM4 EC50 = 36 nM). Other
heterocycles such as pyridazine (8g), pyrazine (8h), pyrimidine
(8i and 8j) were also well tolerated. Attempts to modulate the
pyridine basicity in 8d-f by the incorporation of fluorine atoms,
as in 8k-p, afforded a broad spectrum of activity, with 8k, 8m
and 8p displaying good hM4 PAM potency and efficacy. From
these initial analogs, PAMs 8f, 8k, 8m and 8p were selected for
more in-depth DMPK profiling.
As 4 offered balanced M4 PAM activity across species, we
focused our efforts on this chemotype.16 A multitude of
substituted benzyl amides had previously been explored, and
SAR suggested a large lipophilic binding pocket accessible to the
4-position substituent, e.g., the 4-SO2CF3 moiety of 3 and related
analogs. Interestingly, and despite their prevalence in GPCR
ligands, we never synthesized and evaluated any biaryl or
heterobiaryl methyl amide congeners; therefore, we focused our
search for an NHP in vivo tool within this unexplored chemical
space.
Table 1. Structures and activities for human M4 PAM analogs 7 and
8.
The synthesis of analogs 8 proved to be straightforward.
Condensation of commercially available 3-chloro-5,6-
dimethylpyridazine-4-carbonitrile 5 with methyl thioglycolate
under basic conditions smoothly affords the sodium carboxylate
6 in 78% yield. A HATU-mediated amide coupling with 2-, 3- or
4-bromobenzyl amines then delivers analogs 7 in yields ranging
from 45-92%. A subsequent Stille or Suzuki cross-coupling
reaction with a diverse range of aryl and heteroaryl moieties
delivers analogs 8 in yields ranging from 58-90%.17
hM4
EC50 (nM)a
[% ACh Max ±SEM]
>10,000
hM4
pEC50
(±SEM)
Cpd
R
7a
7b
>5
[31+2]
185
[84+2]
6.77+0.13
6.74+0.07
>5
187
7c
8a
[82+2]
>10,000
[30+1]
Scheme 1. Synthesis of M4 PAM analogs 8.a
1,650
8b
5.80+0.12
[41+3]
319
[49+6]
232
8c
6.53+0.12
6.67+0.12
7.20+0.31
7.57+0.19
8d
[75+5]
103
[63+4]
8e
36
[83+8]
56
8f
8g
7.26+0.06
aReagents and conditions: (a) Methyl thioglycolate, MeOH, 1M aq. NaOH,
150 oC, microwave, 30 min, 78%; (b) bromobenzyl amine, HATU, DMF,
[96+5]
159
[63+6]
8h
8i
.
DIPEA, 2 h, 45-92%; (c) 5 mol% Pd(dppf)Cl2 CH2Cl2, Bu3SnAr(Het), THF,
7.00+0.29
7.08+0.04
6.67+0.11
.
65-80% or 5 mol% Pd(dppf)Cl2 CH2Cl2, (HO)2BAr(Het), K3PO4, THF/H2O,
100 oC, 58-90%.
84
[91+9]
230
[65+11]
8j
SAR was driven using activity at the human M4 receptor
(potency in functional assay), but key compounds were assessed
on rat and cyno M4 as well, as the goal was an NHP tool
compound. As shown in Table 1, many potent human M4 PAMs
were discovered and several displayed attractive DMPK profiles
and excellent CNS penetration (brain:plasma Kp and Kp,uu) vide
infra. Initial evaluation of the 2-, 3- and 4-bromobenzyl
derivatives, 7a-c respectively, demonstrated a clear preference
for 3- and 4- substitution. Conversion of these into the
corresponding biaryl analogs, 8a-c, confirmed the finding, with
the 2-phenyl derivative 8a inactive (hM4 EC50 >10 M), the 3-
phenyl congener 8b weak (hM4 EC50 = 1.65 M) while the 4-
phenyl derivative 8c displayed good M4 PAM activity (hM4 EC50
= 319 nM). Thus, future efforts focused solely on 4-substituted
biaryl and heterobiaryl analogs 8d-p. PAM 8c showed low
efficacy and poor physiochemical properties and a high cLogP
(>4); therefore, we elected to evaluate diverse azaheterocycles in
an attempt to improve physiochemical properties, enable salt
formation and lower lipophilicity. Exploration of the pyridyl
biaryl analogs 8d-f showed robust SAR. Potency increased as
the pyridine nitrogen was moved from the 2-position (8d, hM4
EC50 = 232 nM), to the 3-position (8e, hM4 EC50 = 103 nM) and
58
[92+10]
8k
7.31+0.16
1,716
[86+5]
8l
5.81+0.14
7.20+0.06
6.84+0.04
64
[79+2]
144
8m
8n
[78+14]
994
[68+1]
8o
8p
6.11+0.22
7.04+0.11
91
[74+3]
aCalcium mobilization assays with hM4/Gqi5-CHO cells performed in the
presence of an EC20 fixed concentration of acetylcholine; values represent
means from three (n=3) independent experiments performed in triplicate.
As shown in Table 2, all four PAMs showed attractive
cLogPs (2.66 to 2.92) and conserved TPSAs with molecular

weights below 500. All were highly bound in plasma, except 8f,
which also showed low rat clearance in vivo (despite high in vitro
7.04±0.11, 74±3% ACh Max), dog (dM4 EC50 = 111 nM, pEC50 =
6.95±0.12, 49±4% ACh Max) and cyno receptors (cM4 EC50
=
predicted rat CLhep
,
a
likely consequence of excluding
87.3 nM, pEC50 = 7.06±0.25, 64±2% ACh Max). ACh
concentration response curve (CRC) fold-shift Ca2+ mobilization
and ACh competition binding (with [3H-N-methylscopolamine)
assays provided operational model parameters for 8p at rM4 (KB
= 0.37 µM, α = 8.4, β = 9.1),hM4 (KB = 2.8 µM, α = 7.0, β = 21),
and cyno M4 (KB= 2.1 µM, α=28.2, β=2.5); all experiments are
n=3-4, performed in duplicate. Moreover, 8p was inactive (EC50
> 30 M) at both rat and human M1-3,5 (and also inactive at dog
and cyno M2, data not shown). These data were highly
noteworthy and indicated that 8p was a potent and highly-
selective NHP M4 PAM. A broad secondary pharmacology panel
(Cerep) revealed only one sub-micromolar off-target activity for
fuplasma/fumic terms from the well-stirred equation used for
prediction) and high rat CNS penetration (brain:plasma Kp =
0.94, Kp,uu = 1.1). However, 8f, as expected due to the naked
pyridine ring, was a potent inhibitor of multiple CYP450s (IC50s <
10 M), including 3A4 (IC50 = 340 nM). Similarly, both 8k and
8m were potent inhibitors of 1A2 (IC50s of 2.5 M and 790 nM,
respectively) with low rat in vivo clearance (CLps of 3.6 and 1.7
mL/min/kg, respectively) and moderate total brain distribution
(Kps <0.21). Here, 8p stood out, with an improved CYP
inhibition profile (all IC50s ≥11 M) andfavorable rat PK (CLp =
1.6 mL/min/kg, elim. t1/2 = 2.7 hr) despite a moderate Kp (0.11),
an attractive Kp,uu (1.2). Moreover, 8p was not a human P-gp
substrate in vitro (ER = 1.7). However, we noted a lack of an in
vitro/in vivo correlation (IVIVC) between the in vitro predicted
CLhep and the in vivo CLp. Employing a revised prediction
method (inclusion of binding terms in the well-stirred model), the
predicted rat CLhep decreased to 0.49 mL/min/kg, and a more
robust IVIVC resulted.
8p – a rat GABAA receptor (benzodiazepine site) binding IC50
0.44 µM, which was subsequently de-risked by a functional assay
determination (no activity at 10 µM). functional
=
A
electrophysiology hERG assay with 8p was likewise clean (IC50
> 33 µM), and a mini-Ames assay (TA98 and TA100 strains ±
S9) was negative for mutagenicity. In addition, 8p was devoid of
evidence for CYP 1A2/2B6/3A4 induction potential in
cryopreserved human hepatocytes (48 hr incubation with enzyme
activity readout; EC50s > 50 M, Emaxs < 2), and in a 4 day sub-
chronic dosing rat study (10 mg/kg, QD PO, n = 2), the day 4
versus day 1 AUC0-∞ ratio was 0.77, indicating little to no auto-
induction in vivo in rat.
Table 2. In vitro and in vivo DMPK properties of 8f, 8k, 8m and 8p.
Property
MW
cLogP
TPSA
8f
8k
8m
407.4
2.92
8p
407.4
2.92
93.8
398.4
2.66
93.4
407.4
2.92
93.8
93.8
In vitro PK parameters
CLINT (mL/min/kg), rat
CLHEP (mL/min/kg), rat
CLINT (mL/min/kg), human
CLHEP (mL/min/kg), human
Rat fuplasma
415
59.9
20.1
15.2
0.037
0.013
0.042
57.1
31.5
19.6
17.8
0.002
0.029
ND
81.6
37.7
20.2
17.1
0.004
0.023
ND
134.0
46.0
19.2
14.8
0.002
0.011
0.020
Human fuplasma
Rat fubrain
Cytochrome P450 (IC50, M)
1A2
2C9
2D6
3A4
9.7
2.3
1.3
2.5
25
>30
24
0.79
26
>30
25
11
>30
19
0.34
>30
Figure 2. M4 PAM concentration response curves (CRCs) for 8p
(VU0476406) at human (hM4 EC50 = 91.0 nM, pEC50 = 7.04±0.11, 74±3%
ACh Max), rat (rM4 EC50 = 13.5 nM, pEC50 = 7.93±0.17, 72±4% ACh Max),
cyno (cM4 EC50 = 87.3 nM, pEC50 = 7.06±0.25, 64±2% ACh Max) and dog
(dM4 EC50 = 111 nM, pEC50 = 6.95±0.12, 49±4% ACh Max).
IV Pharmacokinetics
(SD Rat; 0.1-1.0 mg/kg)
CLp (mL/min/kg)
Elimination t½ (hr)
Vss (L/kg)
9.0
1.5
1.1
3.6
1.7
0.50
1.7
0.48
0.60
1.6
2.7
0.34
Compound 8p was found to possess a largely attractive PK
profile across species. In rat, 8p as a 10 mg/kg solution dose
achieved good oral bioavailability (61% F), and the HCl salt of
8p, when dosed as a suspension, achieved 54 %F. PAM 8p
likewise exhibited favorable IV and PO PK in dog (CLp = 5.5
mL/min/kg, elim. t1/2 = 1.0 hour, Vss = 0.70 L/kg, and 47 %F
from 3 mg/kg suspension dose of the HCl salt) and favorable IV
PK in cynomolgus monkey (CLp = 9.3 mL/min/kg, elim. t1/2 = 1.3
hours, Vss = 0.87 L/kg), but with low oral bioavailability (4.7 %F
from 10 mg/kg solution dose of HCl salt). CNS penetration of 8p
was also assessed in dog (brain:plasma Kp = 0.74, Kp,uu = 2.6,
Ccsf:Cplasma,u = 4.8 at 2.0 hr; via a terminal study) and NHP
(brain:plasma Kp = 0.75, Kp,uu = 1.1, Ccsf:Cplasma,u = 1.0 at 1 hr; via
a PET study measuring brain distribution of [18F]-VU0476406
after IV administration),18 providing strong support for the utility
of 8p in NHP behavioral models and biomarker studies.
Brain Distribution (0.25 hr)
(SD Rat; 0.1-0.2 mg/kg IV)
Kp, brain:plasma
Kp,uu, brain:plasm
MDCK-MDR1 (Pgp) ER
0.94
1.1
1.1
0.21
ND
ND
0.13
ND
ND
0.11
1.2
1.7
ND = not determined.
While 8p emerged as the most attractive of the analogs of 8
that were surveyed, we wanted to explore modifications to the
benzylic phenyl ring before initiating a more exhaustive profiling
effort around 8p. While many analogs in which fluorine atoms
and/or nitrogen atoms were added retained excellent M4 PAM
potency (EC50s < 200 nM), all suffered from increased cLogP,
poor in vitro/in vivo clearance and/or unacceptable CYP
inhibition profiles. Thus, 8p was advanced as a potential NHP
tool compound.
Encouraged, we performed additional studies to evaluate 8p
(VU0476406) as a potential candidate for clinical development
before releasing it as a public M4 PAM NHP tool compound. In
vitro metabolite identification experiments employing
Next, 8p was evaluated as an M4 PAM across multiple species
(Figure 2). Unlike all predecessors (except 4), 8p was a potent
M4 PAM at the rat (rM4 EC50 = 13.5 nM, pEC50 = 7.93±0.17,
72±4% ACh Max), human (hM4 EC50 = 91.0 nM, pEC50
=

o
cryopreserved hepatocytes found low turnover and no evidence
for human unique metabolites, with rat and cyno anticipated to
provide adequate coverage of human metabolites (Figure 3).
Human CYP450 phenotyping experiments revealed that multiple
CYPs (3A4 (predominant), 2D6, 2C19, 2C9 and 1A2) contribute
to 8p’s metabolism. Moreover, no significant levels of GSH
conjugates were observed in reactive metabolism/bioactivation
experiments with human hepatic microsomes, which further
bolstered the potential to advance 8p.
decomposes at 243-246 C), suggesting potential challenges to
achieving requisite margins in nonclinical safety and toxicology
studies. An X-ray crystal structure proved telling (Figure 4),
highlighting a network of intra- and inter-molecular hydrogen
bonds forming a tight, helix-like packing with highly ordered -
stacking. Efforts to disrupt this network to enable acceptable
solubility/dissolution with vehicles suitable for IND-enabling
safety and toxicology studies were not successful. Thus, based on
the suboptimal physiochemical properties of 8p and the high
projected human doses, the program team decided to release 8p
as a publicly available NHP M4 PAM tool compound.
Upon release, Surmeier and co-workers evaluated 8p
(VU0476406) in both mouse and NHP models of L-DOPA-
induced dyskinesia (LID), and found that administration of the
M4 PAM ameliorates deficits in synaptic plasticity and behavior
in PD-LID mice and NHPs.20 Specifically, 8p dosed at 10 mg/kg
IV (route chosen due to low cyno oral F, vide supra) significantly
reduced dyskinesia scores and involuntary movements in NHPs,
thus providing early proof-of-concept for M4 PAMs in the
management of Parkinson’s disease and highlighting the utility of
the compound 8p (VU0476406) in NHP studies.
In summary, we have detailed the discovery and
characterization of the first reported M4 PAM non-human
primate in vivo tool compound 8p (VU0476406), with similar M4
PAM activity across species. The studies presented here
produced a required in vivo NHP POC tool compound for use in
translational efficacy studies, primate PD models and biomarker
studies that could be freely shared with collaborators. Initial
results demonstrated that 8p significantly reduces dyskinesia
scores and involuntary movements in NHPs, and provided early
POC for M4 PAMs in the management of PD-LID. Further
optimization efforts en route to M4 PAM clinical candidates for
the treatment of schizophrenia and other disorders will be
reported in due course.
Figure 3. In vitro biotransformation of 8p (VU0476406) in cryopreserved
hepatocytes from multiple species (rat, dog, cynomolgus monkey, and
human).
Human PK prediction, utilizing multiple approaches,
suggested that 8p would exhibit low clearance in man (CLp
between 1.3 to 3.6 mL/min/kg) with a 6-17 hour half-life.19
However, for projected 12-hour daily coverage (targeting an
efficacious Cmin scaled from rat in vivo pharmacodynamic
studies; data not shown), 8p was projected to require moderate to
high BID oral doses (370 - 850 mg) due in large part to moderate
predicted human oral F. In parallel, pharmaceutical science work
on both the free base and HCl salt was performed, which found
8p to be highly crystalline with low aqueous solubility (< 0.5 µM
for free base at pH 7.4) and without a clean melt (free base
X-ray crystallographic data
The X-ray crystal structure data for 8p was submitted to the
Cambridge
Crystallographic
Data
Centre
(http://www.ccdc.cam.ac.uk) and assigned CCDC 1538487.
Acknowledgments
We thank the NIH for funding via the NIH Roadmap Initiative
1X01 MH077607 (C.M.N.), the Molecular Libraries Probe
Center Network (U54MH084659 to C.W.L.), R01MH073676
(PJC) and U01MH087965 (Vanderbilt 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.). We also thank key CROs that performed key higher
species PK (Frontage), pharmaceutical science/X-ray work
(Crystal Pharmatech) and Pgp assays (Absorption Systems).
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Hz, 1H), 8.1-8.0 (m, 1H), 7.6 (d, J=8.0 Hz, 2H), 7.5-7.4 (m, 3H),
6.9 (bs, 2H), 4.5 (d, J=4.0 Hz, 2H), 2.71 (s, 3H), 2.70 (s, 3H).
HRMS calc’d for C21H19FN5OS (M+H), 408.1294; found
408.1298.
18. Whole and regional brain distribution of total and calculated
unbound VU0476406 was determined in NHP (cynomolgus
monkey, n = 1) via a PET study employing a single IV
administration of [18F]-VU0476406 (145 MBq) and an
approximate 2 hour data acquisition time post-administration;
manuscript in preparation.
19. Prediction of human CLp was performed using two approaches: 1)
the mean hepatic extraction ratio (ERhep) observed in rat, dog, and
NHP (0.17) applied to human Qhep; 2) in vitro to in vivo
extrapolation of CLint (human hepatic microsomes) using the well-
stirred model of organ clearance with inclusion of human fuplasma
and predicted fumic terms. Predicted human t1/2 was obtained from
predicted CLp and a predicted Vss (1.9 L/kg) scaled allometrically
from the observed rat and dog Vss with correction for species
7.
8.
9.
differences in fuplasma.
20. Shen, W.; Plotkin, J.L.; Francardo, V.; Ko, W.K.D.; Xie, Z.; Li,
Q.; Fieblinger, T.; Wess, J.; Neubig, R.R.; Lindsley, C.W.; Conn,
P.J.; Greengrad, P.; Bezard, E.; Cenci, M.A.; Surmeier, D.J.
Neuron 2015, 88, 762-773.
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Vinson, P.N.; Sheffler, D.J.; Jones, C.K.; Morrison, R.; Wood,
M.R.; Daniels, J.S.; Conn, P.J.; Niswender, C.M.; Lindsley, C.W.
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A.E.; Shirey, J.K.; Jones, C.K.; Conn, P.J.; Lindsley, C.W.
ChemMedChem. 2009, 4, 1600-1607.
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T.J.; Blobaum, A.L.; Bridges, T.M.; Le, U.; Jones, C.K.; Wood,
M.R.; Daniels, J.S.; Conn, P.J.; Niswender, C.M.; Lindsley,
C.W.; Hopkins, C.R. Bioorg. Med. Chem. Lett. 2012, 22, 5084-
5088.
11. Bubser, M.; Bridges, T.M.; Thorbeck, D.D.; Gould. R.W.;
Grannan, M.; Noetzel, M.J.; Niswender, C.M.; Daniels, J.S.;
Melancon, B.J.; Tarr, J.C.; Wess, J.; Duggan, M.E.; Brandon, N.J.;
Dunlop, J.; Wood, M.W.; Wood, M.R.; Lindsley, C.W.; Conn,
P.J.; Jones C.K. ACS Chem. Neurosci. 2014, 5, 920-942.
12. Smith, E.; Chase, P.; Niswender, C.M.; Conn, P.J.; Lindsley, C.W.;
Madoux, F.; Acosta, M.; Scampavia, L.; Spicer, T.; Hodder, P.
J. Biomol. Screening 2015, 20, 858-868.
13. Wood, M.R.; Noetzel, M.J.; Tarr, J.C.; Rodriguez, A.L.; Lamsal,
A.; Chang, S.; Foster, J.J.; Smith, E.; Hodder, P.S.; Engers, D.W.;
Niswender, C.M.; Brandon, N.J.; Wood, M.W.; Duggan, M.E.;
Conn, P.J.; Bridges, T.M.; Lindsley, C.W. Bioorg. Med. Chem.
Lett. 2016, 26, 4282-4286.
14. Wood, M.R.; Noetzel, M.J.; Engers, J.L.; Bollinger, K.A.;
Melancon, B.J.; Tarr, J.C.; Han, C.; West, M.; Gregro, A.R.;
Lamsal, A.; Chang, S.; Ajmera, S.; Smith, E.; Chase, P.; Hodder,
P.S.; Bubser, M.; Jones, C.K.; Hopkins, C.R.; Emmitte, K.A.;
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17. Experimental for the synthesis of 8p (VU0476406), 5-amino-N-(4-
(2-fluoropyridine-3-yl)benzyl)-3,4-dimethylthieno[2,3-c]
pyridazine-6-carboxamide. To a 5 mL microwave vial equipped
with a stir bar was added 5-amino-N-(4-bromobenzyl)-3-
dimethylthieno[2,3-c]pyridazine6-carboxamide (50 mg, 0.13
mmol), (2-fluoropyridin-3-yl)boronic acid (36 mg, 0.26 mmol),
.
10 mol% Pd(dppf)Cl2 CH2Cl2 (10.3 mg, 0.013 mmol). The
microwave vial was sealed, evacuated and back-filled three times
with argon. Then, an aqueous solution of potassium phosphate
(380 L, 1 M K3PO4 in H2O, 0.38 mmol) was added, followed by
THF (1.3 mL). The biphasic mixture was heated at 160 oC for 20
min, and after cooling, the suspension was diluted with DCM and
filtered through celite. Silica gel chromatography afforded 8p as
an orange solid (40 mg, 80%). LCMS: RT = 0.623 min,
>99%@254 nm, >99%@215 nm; m/z (M+1) = 408. 1H NMR
(400 MHz, CDCl3, d (ppm)): 8.7 (t, J=4.0 Hz, 1H), 8.2 (d, J=4.0