Challenges in the development of an M4PAM in vivo tool compound: The discovery of VU0467154 and unexpected DMPK profiles of close analogs

Article abstract of DOI:10.1016/j.bmcl.2016.11.086

This letter describes the chemical optimization of a novel series of M₄positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration).

Full text of DOI:10.1016/j.bmcl.2016.11.086

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Challenges in the development of an M4 PAM in vivo tool compound: The
discovery of VU0467154 and unexpected DMPK profiles of close analogs
Michael R. Wood a,c,1, Meredith J. Noetzel a,b,1, Michael S. Poslusney a,1, Bruce J. Melancon a,b
,
James C. Tarr a, Atin Lamsal a, Sichen Chang a, Vincent B. Luscombe a,b, Rebecca L. Weiner a,b
,
Hyekyung P. Cho a,b, Michael Bubser a, Carrie K. Jones a,b,e, Colleen M. Niswender a,b,e
,
Michael W. Wood d, Darren W. Engers a,b, Nicholas J. Brandon d, Mark E. Duggan d,
a,b,c,
P. Jeffrey Conn a,b,e, Thomas M. Bridges a,b, , Craig W. Lindsley
⇑
⇑
a Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
c Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
d Neuroscience Innovative Medicines, Astra Zeneca, 141 Portland Street, Cambridge, MA 02139, USA
e Vanderbilt Kennedy Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This letter describes the chemical optimization of a novel series of M4 positive allosteric modulators
(PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis,
and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first
report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of
VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species
differences in PAM pharmacology and subtle structural changes affecting CNS penetration).
Ó 2016 Elsevier Ltd. All rights reserved.
Received 26 October 2016
Revised 28 November 2016
Accepted 29 November 2016
Available online xxxx
Keywords:
M4
Muscarinic acetylcholine receptor
Positive allosteric modulator (PAM)
Schizophrenia
Structure-activity relationship (SAR)
M4 (muscarinic acetylcholine receptor subtype 4) positive allos-
teric modulators (PAMs) represent an exciting therapeutic strategy
to treat multiple domains of schizophrenia,1–18 as well as other
CNS disorders,19,20 via a new molecular mechanism.21 However,
the great potential has been hampered by limited chemical diver-
sity centered on a 3-amino-thieno[2,3-b]pyridine core, as in 1–4,
(Fig. 1), which engenders steep SAR, species differences (rat versus
human M4 PAM potency, affinity/cooperativity and subtype selec-
tivity), poor solubility, and/or low CNS penetration.1–18 Early
in vivo tool compounds, such as 2,1,2 energized the field but were
not optimal. Recently, we disclosed VU0467154 (5) based on a
novel 5-amino-thieno[2,3-c]pyridazine core that has proven to be
a valuable rodent in vivo M4 PAM tool compound with robust
efficacy in a broad range of preclinical mouse and rat models of
psychosis and cognition, as well as Huntington’s disease.11,17,18
Here, we describe for the first time the optimization campaign
(SAR and intriguing DMPK profiles) that advanced PAMs 2–4, into
5 with exceptional rodent M4 PAM potency, selectivity, DMPK pro-
file and CNS penetration; however, a significant species disconnect
(35Â less potent on human M4) precluded advancement as a clin-
ical candidate.11,17,18
Within the 3-amino-thieno[2,3-b]pyridine series, balancing M4
PAM potency and solubility was a distinct challenge;1–18 therefore,
initial efforts focused on replacements for the pyridine ring, as very
few substituents were tolerated. Regioisomeric pyridines were
evaluated, along with isomeric pyrimidines, but all lost consider-
able M4 PAM potency at both human and rat M4. Based on the
attractive physiochemical properties and high dielectric constant
of the pyridazine ring, we prepared a 5-amino-thieno[2,3-c]pyri-
dazine congener 6 of 2 (Fig. 2). This modification proved favorable
(M4 PAM potency enhanced 4-fold, free fraction increased up to 9-
fold, CLhep improved as well as Kp,uu and cLogP with no change in
molecular weight), but the PMB amide proved to be a metabolic
‘hot spot’ (CYP-mediated oxidative demethylation) and would
⇑
Corresponding authors at: Vanderbilt Center for Neuroscience Drug Discovery,
Vanderbilt University Medical Center, Nashville, TN 37232, USA.
E-mail addresses: thomas.m.bridges@vanderbilt.edu (T.M. Bridges), craig.
lindsley@vanderbilt.edu (C.W. Lindsley).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2016.11.086
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wood M.R., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.086

2
M.R. Wood et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Me
N
NH2
NH2
NH2
Me
N
NH2
CN
Cl
O
O
b
O
a
Cl
O
N
N
N
O-Na+
NHR
S
S
N
N
N
S
HN
Me
S
HN
MeO
7
8
9
Scheme 1. Synthesis of M4 PAM analogs 9a. aReagents and conditions: (a) Methyl
thioglycolate, MeOH, 1 M aq. NaOH, 150 °C, microwave, 30 min, 78%; (b) NH2CH2Ar,
HATU, DMF, DIEPA, 2 h, 45–92%.
1, LY2033298
2, VU0152100 (ML108)
OMe
Me
Me
NH2
NH2
O
Cl
Me
O
covered, and several displayed excellent CNS penetration (Kp and
S
HN
F
S
O
N
HN
N
Kp,uu). The direct thioether analog 9a of 6 displayed an ꢀ10-fold
N
F
increase in rat M4 PAM potency (EC50 = 11.2 nM), but diminished
CNS exposure (Kp = 0.13). Oxidation to the corresponding sulfoxide
9b lost potency (EC50 = 139 nM), which was restored by the methyl
sulfone 9c (EC50 = 51.3 nM; however, CNS penetration was poor,
Kp = 0.04). Moving the sulfone from the 4-position to the 3-posi-
tion, as in 9d, was poorly tolerated (EC50 = 417 nM), while steric
bulk, as in 9e–g, retained good rat M4 PAM potency (EC50s 22–
48 nM), but with low, variable Kps (0.05–0.11). Thus, steric bulk
alone was not sufficient to balance PAM potency and Kp, therefore
increased lipophilicity, in the form of fluorine atoms, was then
evaluated. Addition of a single fluorine atom alpha to the methyl
sulfone (9h) maintained PAM potency (EC50 = 58 nM), and CNS
penetration improved (Kp = 0.51, Kp,uu = 2.78). Consecutive addi-
tion of fluorines to the methyl moiety of 9c afforded various fluo-
romethyl sulfones 9i–j with potent rat M4 PAM activity (EC50s 17–
23 nM), but unexpected and highly variable CNS exposure. The
mono-fluoromethyl analog 9i was not detected in the CNS, while
brain distribution of the difluoromethyl congener 9j was modest
(Kp = 0.11, Kp,uu = 0.10) and that of the trifluoromethyl derivative
5 was exceptional (Kp = 0.49, Kp,uu = 1.1). Finally, an unusual
pentaflurosulfur (SF5) analog, 9l, was a potent rat M4 PAM
(EC50 = 30 nM) with excellent CNS distribution (Kp = 1.4, Kp,
uu = 1.0). Of these, 5 and 9l stood out as potential candidates as
rodent in vivo tool compounds, but we wanted to assess their activ-
ity at human M4 (as species differences are common amongst M4
PAM ligands) to determine if these could translate into clinical can-
didates.8–11,13,14 Unfortunately, as shown in Table 2, there was a 6-
to 35-fold rightward shift in human PAM potency, precluding these
analogs from consideration as clinical candidates.
Next, we assessed physiochemical properties and DMPK profiles
of 9j, 5 and 9l in battery of in vitro and in vivo assays (Table 3).
Molecular weights were all similar and both the difluoromethyl
sulfone 9j and the trifluoromethyl sulfone 5 had similar TPSAs,
but the SF5 analog, 9l, had greatly reduced TPSA. In vitro clearance
(predicted CLhep for both human and rat) was similar across the
three PAMs, but plasma protein binding did differentiate the PAMs.
9j, with a cLogP of 1.98, had the most favorable fraction unbound
(fu (rat, human) of 0.066 and 0.053) as well as fraction unbound in
brain (fu rat brain, 0.062). In contrast, the highly lipophilic SF5
(cLogP = 5.13) congener 9l, displayed poor fraction unbound (fu
(rat, human) of 0.004 and 0.010). 5 represented the middle ground
with moderate fraction unbound in plasma (fu (rat, human) of
0.031 and 0.019) and in brain (fu rat brain, 0.067) with a n optimal
cLogP (2.49) predictive of good CNS exposure. The CYP450 profiles
were very clean, except for weak inhibition of 2D6 by 9j and 9l.
In vivo rat PK (1 mg/kg i.v.) again distinguished 5, with low CLp
N
3
4
, VU0448088 (ML253)
, ML173
Me
N
NH2
Me
O
N
S
HN
SO2CF3
5,
VU0467154
Fig. 1. Structures of representative M4 PAMs 1–4, highlighting the conserved 3-
amino-thieno[2,3-b]pyridine chemotype, and the optimized rodent in vivo tool M4
PAM, VU0467154 (5) with a novel pyridazine core.
Me
N
Me
N
NH2
NH2
Me
O
O
N
S
HN
S
HN
Me
OMe
OMe
2, VU0152100
6, VU0464090
hM4 EC50 = 520 nM, 80% ACh Max
Kp = 0.38, Kpu,u = 1.1
rCLhep = 58 mL/min/kg
hCLhep =16 mL/min/kg
cLogP = 2.94
hM4 EC50 = 150 nM, 83% ACh Max
Kp = 0.28 Kpu,u = 2.98
rCLhep = 37 mL/min/kg
hCLhep =13 mL/min/kg
cLogP = 1.88
fu (r, h) = 0.015, 0.004
fu (r, h) = 0.022, 0.035
Fig. 2. Structures of the first in vivo M4 PAM tool compound 2, and the new 5-
amino-thieno[2,3-c]pyridazine congener 6, which displayed improved physiochem-
ical, pharmacological and DMPK properties.
need to be replaced with an alternate amide.22 At this point, mul-
tiple avenues of inquiry were pursued, and here we will focus on
amide moieties containing sulfur (thioethers, sulfoxides, sulfones
and SF5) moieties.
The synthesis of analogs 9 proved to be straightforward. Con-
densation of commercially available 3-chloro-5,6-dimethylpyri-
dazine-4-carbonitrile 7 with methyl thioglycolate under basic
conditions smoothly affords the sodium carboxylate 8 in 78%
yield.11 A HATU-mediated amide coupling with various benzyl
amines then delivered analogs 9 in yields ranging from 45 to
92%. In all cases, either the desired benzylamine or the correspond-
ing nitrile, which was easily reduced to the requisite benzyl amine,
was commercially available (Scheme 1).
(7.8 mL/min/kg),
a long half-life (t1/2 = 5.7 h), excellent oral
bioavailability (61%F, from 3 mg/kg suspension dose) and with
excellent brain distribution (Kp = 0.49 and Kp,uu = 1.1. Interestingly,
and despite varying in vivo DMPK profiles, all three afforded robust
reversal of amphetamine-induced hyperlocomotion (AHL) when
evaluated in single-point oral dosing at either 10 mg/kg (9j,
49.3% and 5, 55.3%) or at 30 mg/kg (9l, 66.3%) – our standard phar-
SAR was driven on rat M4, as the objective was an in vivo POC
tool compound, but key compounds were assessed on human M4
as well. As shown in Table 1, many potent rat M4 PAMs were dis-
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M.R. Wood et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
Table 1
Structures and activities for rat M4 PAM 5 and analogs 9.
NH2
O
N
S
HN
N
Ar
9
b
Cpd
Ar
rM4 EC50 (nM)a [% ACh Max SEM]
11.2 [75.6 1.7]
rM4 pEC50 ( SEM)
7.95 0.09
Rat Kp (Kp,uu
0.13 (0.48)
)
9a
SMe
9b
9c
139 [74.2 1.9]
51.3 [72.6 2.2]
6.86 0.04
7.29 0.08
ND
O
S
Me
0.04 (0.14)
O
S
S
Me
O
O
9d
9e
417 [74.6 2.1]
47.9 [76.8 2.7]
6.38 0.17
7.32 0.06
ND
O
Me
0.07 (0.67)
O
S
O
9f
22.4 [67.1 3.1]
44.7 [72.1 2.2]
57.7 [69.2 3.5]
7.65 0.05
7.35 0.06
7.24 0.10
0.05 (0.29)
0.11 (0.34)
0.51 (2.8)
O
O
S
S
O
9g
9h
O
O
S
Me
O
F
9i
9j
5
21.9 [62.5 5.5]
17.8 [65.2 2.4]
17.8 [68.1 1.6]
23.4 [76.7 2.6]
30.2 [75.7 1.6]
7.66 0.04
7.75 0.14
7.75 0.06
7.63 0.05
7.52 0.03
BLQ
O
F
S
O
0.11 (0.10)
O
F
S
F
O
O
c
0.49 (1.1)
O
F
F
F
S
9k
9l
BLQ
O
F
S
O
F
F
1.4 (1.0)
F
S
F
F
F
F
a
Calcium mobilization assays with rM4/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.
b
Total and calculated unbound brain:plasma partition coefficients determined at 0.25 h post-administration of an IV cassette dose (0.20–0.25 mg/kg) to male, SD rat
(n = 1); in conjunction with in vitro rat plasma protein and brain homogenate binding assay data. ND = not determined. BLQ = below limit of quantitation.
c
Mean values obtained from discrete studies using a 1.5 h sample time point.11,13,14
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4
M.R. Wood et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Table 2
macodynamic assay for M4 PAM optimization. While we have
recently reported results from dose-response AHL (as well as
MK-801-induced hyperlocomotion) studies with 5 in rat and
mouse (Kp for mouse of 0.64), here we show full dose response
AHL for 9j and 9l (Fig. 3), and, while not ideal tools, both 9j and
9l are efficacious in vivo. Data for 5 was previously reported, with
46% reversal at 10 mg/kg p.o., 53% reversal at 30 mg/kg p.o. and a
minimum effective dose (MED) at 3 mg/kg p.o. (33% reversal).11
To vet these analogs as potential rodent in vivo tool compounds,
we next evaluated selectivity across the other muscarinic receptors
(M1–3,5) and broader ancillary pharmacology in Eurofin’s Lead Pro-
filing panel (68 GPCRs, ion channels and transporter radioligand
binding assay panel).23 All three M4 PAMs, 9j, 5 and 9l, had no
Structures and human activities for M4 PAM analogs 9.
NH2
O
N
S
HN
N
Ar
9
Cpd Ar
hM4 EC50 (nM)a [% ACh
Max SEM]
hM4 pEC50
( SEM)
9a
60.2 [71.9 1.7]
7.22 0.09
SMe
9c
9i
363 [73.8 2.2]
6.44 + 0.08
O
S
Me
O
209 [62.5 5.5]
251 [72.8 2.4]
631 [56.4 2.4]
550 [66.1 1.6]
6.68 0.04
6.60 0.14
6.20 0.06
6.26 0.03
O
F
S
O
9j
5
O
F
S
F
O
O
O
F
F
F
S
9l
F
S
F
F
F
F
a
Calcium mobilization assays with hM4/Gqi5-CHO cells performed in the pres-
ence of an EC20 fixed concentration of acetylcholine; values represent means from
three (n = 3) independent experiments performed in triplicate.
Table 3
In vitro and in vivo DMPK properties of 9j, 5 and 9l.
Property
9j
5
9l
MW
cLogP
TPSA
426.4
1.98
114
444.4
2.49
114
438.4
5.13
79.8
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
48.1
28.1
15.6
9.34
0.066
0.053
0.062
47.7
28.4
19.8
10.2
0.031
0.019
0.067
95.9
40.5
52.8
15.0
0.004
0.010
0.004
Human fuplasma
Rat fubrain
Cytochrome P450 (IC50, lM)
1A2
2C9
2D6
3A4
>30
>30
20.8
>30
>30
>30
>30
>30
>30
>30
23.9
>30
In vivo PK (Sprague-Dawley rats)
IV (1 mg/kg)
CLp (mL/min/kg)
t ½ (h)
Vss (L/kg)
16
2.4
2.9
7.8
5.7
3.1
12
3.6
3.1
Tissue distribution (1 mg/kg, IP) sample time (0.25 h)
Fig. 3. Reversal of amphetamine-induced hyperlocomotion with A) 9j
(VU0468182), B) 9l (VU0469785). The M4 PAMs were administered 30 min after
habituation in the chamber, and either vehicle or 0.75 mg/kg amphetamine
administered s.c. at t = 60 min. For each dose group, n = 7–8 rats
Kp,
0.11
0.10
49.3
0.49
1.1
55.3
1.0
0.75
66.3
brain
Kpuu,
brain
% reversal in AHL (10 mg/kg, p.o.)
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M.R. Wood et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
5
activity (EC50s > 30
played no ancillary pharmacology at any of the 68 targets in the
Eurofin panel (no % inhibition >50%@10 M). Both 9j and 9l
showed a less clean profile, showing significant off-target activities
at multiple targets (>50% inhibition@10 M which were then con-
firmed in full CRC). 9j proved to be a ligand for hCav1.2 (5.7 M),
DAT (175 nM), SERT (600 nM) and 5-HT3 (1.6 M). Similarly, 9l
proved to be a ligand for DAT (150 nM), SERT (520 nM), Ghrelin
receptor (6.5 M), BDZ (1.5 M), GABA (2.1 M) and 5-HT3
(1.5 M) Therefore, 5 emerged as the ideal rodent in vivo M4
l
M) at both rat and human M1-3,5 and 5 dis-
Network (U54MH084659 to C.W.L.) and U01MH087965 (Vander-
bilt 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.).
l
l
l
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22. Manuscript in preparation.
PAM tool compound with clean ancillary pharmacology and excel-
lent rodent PK. A subsequent, broader ancillary pharmacology
panel identified single off-target activity for 5, the human adeno-
sine transporter, with an IC50 of 240 nM. The full in vitro and
in vivo pharmacological profile of
5 (VU0467154) has been
described in detail.11
In summary, we have detailed for the first time the progression
from the prototypical 3-amino-thieno[2,3-b]pyridine core M4 PAM
core to the 5-amino-thieno[2,3-c]pyridazine core, which imparts
compounds with improved potency, physiochemical properties
and DMPK profiles. However, the sulfone series detailed here still
suffers from significant species differences in M4 PAM potency, in
favor of rat, and unpredictable variations in CNS penetration based
on fluorine content and/or lipophilicity. This effort led to the study
of an unusual SF5 congener with potent PAM activity and in vivo effi-
cacy, but the high cLogP proved problematic. Importantly, this gave
rise to the highly valuable rodent M4 PAM in vivo tool compound 5
(VU0467154), with a balanced profile and demonstrated utility in
target validation studies. Further optimization efforts en route to
M4 PAM clinical candidates, with equivalent human and rat M4
PAM potencies within this series, will be reported in due course.
23. For information on the Eurofin LeadProfiling Screen see: www.eurofin.com.
Acknowledgments
We thank the NIH for funding via the NIH Roadmap Initiative
1X01 MH077607 (C.M.N.), the Molecular Libraries Probe Center
Please cite this article in press as: Wood M.R., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.086