Continued optimization of the M5NAM ML375: Discovery of VU6008667, an M5NAM with high CNS penetration and a desired short half-life in rat for addiction studies

Article abstract of DOI:10.1016/j.bmcl.2017.02.020

This letter describes the continued optimization of M₅NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375?has an excessively long elimination half-life in rat (t_1/2?=?80?h), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M₅NAM of comparable potency to ML375, but with a rat t_1/2of less than 4?h. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11 (VU6008667), an equipotent M₅NAM, with high CNS penetration, excellent selectivity versus M_1–4and the desired short half-life (t_1/2?=?2.3?h) in rat.

Full text of DOI:10.1016/j.bmcl.2017.02.020

Accepted Manuscript
Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an
M5 NAM with high CNS penetration and a desired short half-life in rat for ad-
diction studies
Kevin M. McGowan, Kellie D. Nance, Hykeyung P. Cho, Thomas M. Bridges,
Carrie K. Jones, P. Jeffrey Conn, Carrie K. Jones, Craig W. Lindsley
PII:
S0960-894X(17)30136-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.020
BMCL 24692
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 January 2017
6 February 2017
7 February 2017
Please cite this article as: McGowan, K.M., Nance, K.D., Cho, H.P., Bridges, T.M., Jones, C.K., Jeffrey Conn, P.,
Jones, C.K., Lindsley, C.W., Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an M5
NAM with high CNS penetration and a desired short half-life in rat for addiction studies, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.020
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Continued optimization of the M5 NAM ML375: Discovery
of VU6008667, an M5 NAM with high CNS penetration
and a desired short half-life in rat for addiction studies
Kevin M. McGowan, Kellie D. Nance, Hyekyung P. Cho, Thomas M. Bridges, P. Jeffrey Conn, Carrie K. Jones,
and Craig W. Lindsley

Bioorganic & Medicinal Chemistry Letters
Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an M5
NAM with high CNS penetration and a desired short half-life in rat for addiction
studies
Kevin M. McGowan,a,bǂ Kellie D. Nance,a,cǂ Hykeyung P. Cho,a,b Thomas M. Bridges,a,b Carrie K. Jonesa,b
P. Jeffrey Conn,a,b Carrie K. Jonesa,b and Craig W. Lindsleya,b,c,d*
aVanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University School of Medicine, 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
dVanderbilt Institute of Chemical Biology and Vanderbilt Center for Addiction Research, Vanderbilt University, Nashville, TN 37232, USA
ǂThese authors contributed equally
*To whom correspondence should be addressed: craig.lindsley@vanderbilt.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
This letter describes the continued optimization of M5 NAM ML375 (VU0483253). While a
valuable in vivo tool compound, ML375 has an excessively long elimination half-life in rat (t1/2
=
Revised
Accepted
Available online
80 hours), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement).
Thus, we required an M5 NAM of comparable potency to ML375, but with a rat t1/2 of less than
4 hours. Steep SAR plagued this chemotype, and here we detail aniline replacements that
offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a
single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11
(VU6008667), an equipotent M5 NAM, with high CNS penetration, excellent selectivity versus
M1-4 and the desired short half-life (t1/2 = 2.3 hours) in rat.
Keywords:
M5
Muscarinic acetylcholine receptor
pharmacokinetics
CNS penetration
2017 Elsevier Ltd. All rights reserved.
Structure-Activity Relationship (SAR)
Recently, we reported on the discovery of highly selective
muscarinic acetylcholine receptor subtype 5 (M5) inhibitors (both
negative allosteric modulators (NAMs), represented by ML375
(VU0483253), 1, and VU6000181, 2)1,2 and an orthosteric
antagonist, ML381 (VU0488130), 33 (Figure 1). Based on these
chemotypes, we also performed a ligand-based virtual screening
campaign, but no highly subtype selective M5 inhibitors were
identified.4 Pharmacological recapitulation of the strong genetic
data (from M5 knock-out mice) linking this receptor to a role in
addiction5-7 with a selective small molecule inhibitor is of great
interest. Excitingly, we have now achieved that validation in
models of cocaine addiction with ML375.8 However, the
excessively long elimination half-life of ML375 in rat (t1/2 = 80
hours)1 is problematic in the context of addiction studies
involving reinstatement paradigms and washout periods. Both 2
and 3 possessed PK profiles not suitable as in vivo tool
compounds.2,3 Ideally, we desired an M5 NAM with high CNS
penetration and reasonable potency but with a short to moderate
half-life in rat (t1/2 < 4 hours) to further assess this novel
mechanism for the treatment of addiction. Yet, these series
Figure 1. Structures of M5 NAMs ML375 (1, VU0483253), VU6000181 (2)
and the highly selective orthosteric M5 antagonist ML381 (3, VU0488130).
typify the worst caveats of allosteric modulator SAR
–
tremendously steep SAR.1-3,9-11 In this Letter, we detail the
continued optimization of ML375 that ultimately afforded an M5
NAM with high CNS penetration and the desired short half-life
in rat useful for evaluation in rat models of drug addiction.
As SAR to almost all portions of the ML375 scaffold resulted
in largely inactive analogs (Figure 2), we directed our focus to
further modifications to the 9b-phenyl moiety and explored
installing aniline moieties to replace the chlorine atom. This was

Table 1. Structures and activities for hM5 NAM 1 and analogs 4.
hM5
IC50 (M)a
[% ACh Min ±SEM]
1.11
hM5 pIC50
(±SEM)a
Cpd
NR1R2
Figure 2. Summary of unproductive SAR with the M5 NAM ML375 (1,
VU0483253). Only fluoro substituents retained activity, but led to higher
clogPs and very poor physiochemical properties.
“Cl”
1
ML375
4a
[2.3+0.3]
5.98+0.04
attractive for three reasons: 1) the ability to form a salt (and
hopefully improve physiochemical and DMPK properties), 2) the
ease of installation onto chiral 1 (which we had in bulk) and 3)
the diversity of amines for the requisite Buchwald coupling. In
the event (Scheme 1), analogs 4 were prepared in a single step,
employing a microwave-assisted Buchwald coupling in moderate
to good yields (45-92%). In short order, over 100 analogs 4
6.04
[10.5+2.8]
5.22+0.04
5.37+0.04
5.20+0.03
5.84+0.04
4.28
[13.6+1.0]
4b
6.36
[6.9+1.7]
4c
1.46
[3.1+0.2]
4d
Scheme 1. Synthesis of M5 NAM analogs 4.a
5.70
4e
[2.6+0.1]
5.84+0.04
5.74+0.04
1.82
[3.1+0.2]
4f
3.42
[3.2+0.2]
4g
5.47+0.03
5.40+0.4
5.14+0.05
4.01
[3.6+0.2]
4h
4i
aReagents and conditions: (a) HNR1R2 (3.0 equiv.), 5 mol% Pd(OAc)2, 15
mol% SPhos, toluene (0.25 M), microwave, 20 min, 45-92%.
7.29
[26.3+6.2]
8.21
[20.1+7.7]
4k
4l
5.11+0.10
5.35+0.05
were synthesized, purified, and screened in our hM5 functional
assay (intracellular calcium mobilization); however, and true to
the steep SAR in observed with this chemotype,1,2 only about
10% of the analogs displayed M5 NAM activity (Table 1).
While none of the analogs 4 proved to more potent than 1,
several (4d, 4f and 4m) were comparable in terms of hM5 NAM
activity (hM5 IC50s in the 1.3 to 1.8 M range and rat M5 potency
with 2-fold), but superior in terms of aqueous solubility as the
respective HCl salts (cf 10 mg/mL versus <0.01 mg/mL for 1).
However, only lipophilic amine moieties provided M5 NAMs in
this subseries 4, which resulted in high clogPs (>4), high
predicted hepatic clearance (rCLhep >65 mL/min/kg, hCLhep >18
mL/min/kg) based on microsomal CLint data, and low fraction
unbound (rat and human fu,plasma <0.01). The series also displayed
a robust in vitro/in vivo correlation (IVIVC) for clearance in rat,
displaying CLps of >70 mL/min/kg and very short elimination
half-lives (t1/2 <30 min). Within analogs 4, CNS distribution was
variable in rat, affording brain:plasma partition coefficients (Kps)
either greater than 1, or below the limit of quantitation (no
detectable brain levels). Thus, while we were excited to identify
tractable SAR for this M5 NAM chemotype and improvements in
aqueous solubility, the uniformly poor disposition in rat
precluded analogs 4 from further consideration as in vivo tools
for addiction studies. Although, the improved solubility of
4.55
[27.0+7.7]
1.32
[2.6+0.9]
4m
4n
4o
5.89+0.08
5.17+0.08
6.92
[25.5+9.3]
3.09
[3.3+0.3]
5.52+0.05
>10
[26.2+3.5]
3.50
4p
4q
>5
[6.0+0.3]
5.46+0.03
5.06+0.14
9.49
[7.5+2.4]
4r
aMean of three independent determinations performed in triplicate via an
intracellular calcium mobilization assay with recombinant hM5 cells in the
presence of an ACh EC80.
At this point, and with over 600 analogs synthesized and
assayed in the ML375 series, it was not immediately clear where
to go in the optimization effort to provide a short half-life analog
congeners
4
should provide better tools for in vitro
electrophysiology studies.

of 1.
The M5 NAM 2, harboring a 9b-(4-methoxy-3-
mL/min/kg). Moreover, 11 was highly brain penetrant (Kp = 4.1,
Kp,uu = 0.88). In a rat IV (1 mg/kg)/PO (3 mg/kg, solution dose)
PK study, 11 displayed the desired diminished elimination half-
life (t1/2 = 2.3 hr) driven by a smaller (yet still large) volume (Vss
= 7.4 L/kg) and higher clearance (CLp = 82 mL/min/kg), with
moderate oral bioavailability (17% F). These results reveal a
methylphenyl) moiety, was of comparable potency to 1, but
demonstrated poor rat PK (t1/2 <30 min, CLp >70 mL/min/kg), as
well as limited CNS penetration.1,2 Thus, we postulated that a
hybrid of NAMs 1 and 2, with incorporation of a 3-methyl group
into the 9b-phenyl ring of 1, might engender a metabolic shunt,
and thus provide
a balance between M5 NAM activity,
physiochemical properties and disposition (i.e., reduced rat t1/2).
Scheme 2. Synthesis of M5 NAM analogs 9-11.a
Figure 3. Molecular pharmacology profile of M5 NAMs 9-11. A) hM5
concentration response curves (CRCs) for 9, 10 and 11; B) mAChR CRCs for
hM1-5, highlighting the exquisite selectivity for (S)-11 as an M5 NAM, as well
as rat M5, showing no significant species differences. Data represent means
from at least three independent determinations performed in triplicate via
intracellular calcium mobilization assays in recombinant Chinese Hamster
Ovary (CHO) cells stably transfected with the individual mAChRs.
aReagents and conditions: (a) nBuLi, THF, -78 °C, 2h, 50%; (b)
ethylenediamine, pTsOH, 4Å sieves, toluene, 150°C µW 25 min, 70%; (c)
3,4-difluorobenzoyl chloride, DIPEA, DCM, 80%; (d) SFC, (CHIRALPAK
IE, 20 mm × 250 mm column at 40 °C, back-pressure regulated at 100 bar,
IPA cosolvent, 30% isocratic prep over 7 min at 80 mL/min).
dramatic impact of the addition of a single methyl group to 1 as a
metabolic shunt, decreasing rat t1/2 by ~35-fold while maintaining
M5 NAM potency and favorable CNS penetration. Again, an M5
NAM with this PK profile was key for addiction studies in rat
examining reinstatement paradigms and washout, where 1 could
be problematic.
The synthesis of the 9b-(4-chloro-3-methylphenyl)-containing
M5 NAM was straightforward (Scheme 2).1,12 Lithium-halogen
exchange on 5, and addition of anhydride 6 affords benzoic acid
7 in 37% yield. Condensation with ethylenediamine, under
microwave irradiation, provides the 1,2,3,9b-tetrahydro-5H-
imidazo[2,1-a]isoindol-5-one core 8 in 70%. Finally, acylation
with 3,4-difluorobenzoyl chloride delivers racemic 9. Chiral
resolution by super criterial fluid chromatography gave (R)-10
and (S)-11.1,12 Racemic 9 was indeed an M5 NAM (IC50 = 1.8
M, pIC50 = 5.75+0.03, 2.9+0.29 % ACh min). As has held true
for this series, the (R)-enantiomer 10 was devoid of M5 NAM
activity (IC50 >10 M), while the (S)-enantiomer 11 (human M5
IC50 = 1.2 M, pIC50 = 5.93+0.02, 2.3+0.03 % ACh min and rat
M5 IC50 = 1.6 M, pIC50 = 5.78+0.02, 2.6+0.03 % ACh min) was
the active enantiomer (Figure 3A), and equipotent to 1.
Moreover, 11 (VU6008667) was selective for M5 over M1-4
(Figure 3B). Next, we sought to assess if incorporation of a
putative metabolic shunt into the framework of 1, with 11, would
lower the 80 hour half-life in rat.
In summary, the continued optimization of the long half-life
M5 NAM ML375 resulted in the discovery of (S)-11
(VU6008667), an equipotent human and rat M5 NAM with a
desired short half-life in rat (t1/2 = 2.3 hr). For many of the
addiction studies underway on our labs, a short half-life M5 NAM
was required. New studies are underway to directly compare
ML375 to (S)-11 in a variety of paradigms and with multiple
drugs of abuse, and results will be reported in due course.
Acknowledgments
We thank the NIH for funding via the National Institute of Drug
Abuse (1R01DA037207). 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.).
The in vitro DMPK profile of 11 was similar to 1 in terms of
plasma protein binding (fu,plasma rat = 0.014,human = 0.006) and
rat brain homogenate binding (fu,brain = 0.003), but the predicted
hepatic clearance, as we had hoped, was an order of magnitude
higher (rat CLhep = 67 mL/min/kg and human CLhep = 15
References

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7.25-7.18 (m, 2H), 7.06-7.02 (m, 2H), 4.34 (ddd, J = 12.2, 7.8, 1.8
Hz, 1H), 4.01-3.93 (m, 1H), 3.79 (ddd, J = 9.6, 7.5, 1.8 Hz, 1H),
3.31 (ddd, J = 17.9, 9.9, 7.6 Hz, 1H), 2.35 (s, 3H); 13C NMR (100
MHz, CDCl3) δ (ppm): 172 2, 167 0, 145 9, 136 9, 136 8, 135.3,
133.6, 132.0, 130.7, 129.6, 129.1, 128.7, 124.9, 124.1, 123.8,
123.3, 118.2, 118.0, 117.0, 116.8, 87.5, 52.4, 39.8, 20.7. Specific
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12. Preparation of 12 (VU6008667): To a stirring solution of 4-
bromo-1-chloro-2-methyl-benzene 5 (4.92 mL, 37.1 mmol) in
THF (75 mL) cooled to -78 oC was added n-butyllithium (14.9
o
mL, 37.1 mmol) dropwise. The solution was stirred at -78 C for
30 min and then was added to a stirring solution of phthalic
o
anhydride 6 (5.00 g, 33.8 mmol) in THF (90 mL) at -78 C. The
reaction was stirred at -78 oC for 2 hrs before being quenched with
1N HCl (100 mL). The solution was allowed to warm to rt, the
layers were separated and the aqueous layer was extracted with
EtOAc (3 x 100 mL). The combined organic layers were passed
through a phase separator and concentrated. The crude material
was purified using Teledyne ISCO Combi-Flash system (solid
loading, 40G column, 40-80% EtOAc, 20 min run) to give 2-(4-
chloro-3-methyl-benzoyl)benzoic acid 7 (4.67 g, 50.4% yield) as a
white solid. 1H NMR (400 MHz, CDCl3) δ (ppm): 8 10 (d, J = 7.8
Hz, 1H), 7.68 (dt, J = 7.6, 1.2 Hz, 1H), 7.64-7.62 (m, 1H), 7.59
(dt, J = 7.9, 1.2 Hz, 1H), 7.44 (dd, J = 8.2, 2.2 Hz, 1H), 7.39-7.35
(m, 2H), 2.39 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm):
170.1, 136.8, 135.7, 133.6, 131.8, 131.7, 131.2, 131.1, 129.9,
129.6, 128.5, 127.9, 127.8, 20.3. To a solution of 2-(4-chloro-3-
methyl-benzoyl)benzoic acid 7 (175.0 mg, 0.640 mmol) in toluene
(1.59 mL) in a microwave vial was added ethylenediamine (0.09
mL, 1.27 mmol) followed by p-toluenesulfonic acid (8.48 mg,
0.040 mmol). The reaction subjected to microwave irradiation at
o
150 C for 30 min. The reaction was diluted with EtOAc (10 mL)
and quenched with saturated aqueous NaHCO3 (5 mL). The layers
were separated and the organic layer was washed with water (2 x 5
mL). The organic layer was dried (MgSO4) before concentration
to dryness. The crude material was purified using Teledyne ISCO
Combi-Flash system (solid loading, 24G column, 80-100%
EtOAc, 18 min run) to give 9b-(4-chloro-3-methyl-phenyl)-2,3-
dihydro-1H-imidazo[2,1-a]isoindol-5-one 8 (135 mg , 70.9%
1
yield) as a clear oil. H NMR (400 MHz, CDCl3) δ (ppm): 7 82-
7.77 (m, 1H), 7.55-7.53 (m, 1H), 7.50-7.45 (m, 3H), 7.34-7.28 (m,
2H), 3.86-3.78 (m, 1H), 3.68-3.64 (m, 1H), 3.29-3.15 (m, 2H),
2.37 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 172 8, 147 8,
137.8, 136.7, 134.7, 133.0, 131.9, 130.1, 129.6, 129.0, 125.3,
124.7, 123.5, 89.3, 50.9, 41.9, 20.4. To a stirring solution of 9b-(4-
chloro-3-methyl-phenyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-
5-one 8 (1.40 g, 4.69 mmol) in DCM (46.8 mL) was added 3,4-
difluorobenzoyl chloride (0.884 mL, 7.03 mmol) followed by
diisopropylethylamine (2.05 mL, 11.72 mmol). The reaction was
stirred for 1.5 hrs. The reaction was quenched with saturated
aqueous NH4Cl (50 mL) and the layers were separated. The
aqueous layer was extracted with DCM (2 x 50 mL) and the
combined organic layers were passed through a phase separator
and concentrated to dryness. The crude material was dissolved in
DMSO and purified using the Gilson LC system (30 x 100 mm
column, 55-95%, 0.1% TFA water and ACN, 10 min run, 6 runs).
The desired fractions were neutralized with saturated aqueous