Development of novel M1 antagonist scaffolds through the continued optimization of the MLPCN probe ML012

Article abstract of DOI:10.1016/j.bmcl.2012.06.018

This Paper describes the continued optimization of an MLPCN probe molecule M₁ antagonist (ML012) through an iterative parallel synthesis approach. After several rounds of modifications of the parent compound, we arrived at a new azetidine scaffold that displayed improved potency while maintaining a desirable level of selectivity over other muscarinic receptor subtypes. Data for representative molecules 7w (VU0452865) and 12a (VU0455691) are presented.

Full text of DOI:10.1016/j.bmcl.2012.06.018

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5035–5040
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of novel M1 antagonist scaffolds through the continued
optimization of the MLPCN probe ML012
Bruce J. Melancon a,c,d, , Thomas J. Utley a,c,d, Christian Sevel a,c,d, Margrith E. Mattmann a,c,d
,
⇑
Yiu-Yin Cheung a,c,d, Thomas M. Bridges a,c,d, Ryan D. Morrison a,c,d, Douglas J. Sheffler a,c,d
,
,
Colleen M. Niswender a,c,d, J. Scott Daniels a,c,d, P. Jeffrey Conn a,c,d, Craig W. Lindsley a,b,c,d
Michael R. Wood a,b,c,d
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center for Accelerated Probe Development (MLPCN), 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 Paper describes the continued optimization of an MLPCN probe molecule M1 antagonist (ML012)
through an iterative parallel synthesis approach. After several rounds of modifications of the parent com-
pound, we arrived at a new azetidine scaffold that displayed improved potency while maintaining a
desirable level of selectivity over other muscarinic receptor subtypes. Data for representative molecules
7w (VU0452865) and 12a (VU0455691) are presented.
Received 17 May 2012
Revised 31 May 2012
Accepted 4 June 2012
Available online 15 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Muscarinic acetylcholine receptor 1
M1 antagonist
ML012
VU0455691
VU0452865
Acetylcholine (ACh) is a critical neurotransmitter with diverse
functions both within the central nervous system (CNS) and in
peripheral signaling pathways.1–4 ACh operates by interacting with
two very distinct groups of receptors; a set of ligand-gated ion
channels—the nicotinic acetylcholine receptors (nAChRs)—and a
set of family A, G protein-coupled receptors (GPCRs)—the musca-
rinic acetylcholine receptors (mAChRs). The muscarinic family of
acetylcholine receptors is divided into five subtypes (M1–5).5 These
subtypes can be further classified into two subsets based on their G
protein-coupling partners, with the M1,3,5 receptors preferentially
coupling to Gq (stimulating PLC and intracellular calcium mobiliza-
tion) and the M2,4 receptors preferentially coupling to Gi/o (inhibit-
ing adenylate cyclase (AC), thereby decreasing cAMP production).4
The specific subtypes of mAChRs are expressed throughout the
body with varying degrees of expression levels based on the partic-
ular site or organ.6 As a result, mAChRs play significant roles in a
wide range of physiological functions such as memory and atten-
tion, motor control, nociception, regulation of sleep-wake cycles,
cardiovascular function, secretory functions, and mediators of
inflammation, renal and gastrointestinal (GI) function, among
many others.6,7
It has been postulated that M1,4,5 receptors are the relevant sub-
types for CNS therapies; however, specific functions for each recep-
tor subtype are still being investigated.7 This is a direct result of the
highly conserved orthosteric binding site for the endogenous ligand
(ACh) that is shared across all five subtypes of mAChRs. This similar-
ity has stymied the discovery and development of muscarinic
ligands with high selectivity for a particular subtype.8 Yet, this lack
of selectivity has not precluded the development of pharmaceuti-
cals with activity at mAChRs for a range of indications. Many of
these non-selective compounds have undesirable side-effects that
are attributed to activity at the other mAChRs (often M2 and M3),
limiting their clinical impact. For example, xanomeline, a reported
M1- and M4-selective agonist, showed robust clinical efficacy in
Phase II trials for Alzheimer’s disease and schizophrenia,9,10 but also
has nearly equivalent agonist activity at M3.8 Even in the absence of
off-target mAChR activity, the debate still remains whether a single
mAChR subtype (M1 or M4) is responsible for the positive outcomes
in these trials; although, recent studies using mAChR genetic knock-
out (KO) mice have shed additional light on this topic.11 More highly
selective mAChR ligands would allow for more direct pharmacolog-
ical insight and a better understanding of the individual roles for
⇑
Corresponding author.
E-mail address: bruce.j.melancon@vanderbilt.edu (B.J. Melancon).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.018

5036
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5035–5040
O
S
O
O
S NH
O
We have previously reported on the selective M1 antagonist,
NH
ML012 (VU0255035, Fig. 1), and progress on optimization of the
ML012 scaffold. ML012 showed 45- to 159-fold selectivity for M1
over the other subtypes.12 ML012 also reduced pilocarpine-in-
duced seizures in rodents at doses that had no negative impact
on contextual fear conditioning, a behavioral model of hippocam-
pal-dependent cognitive function. These findings demonstrated
that selective M1 antagonists have therapeutic potential over
non-selective muscarinic antagonists. Given the potential for M1
antagonists in such indications as Parkinson’s disease, movement
disorders, and Fragile X syndrome,18,19 we engaged in an optimiza-
N
N
N
N
N
N
N
N
N
S
O
O
1
ML012
VU0255035
hM1 IC50 = 690 nM
VU0415248
hM1 IC50 = 400 nM
Figure 1. MLPCN Probe ML012 and VU0415248, a selective M1 antagonist.
Table 1
Structures and potencies of M1 antagonist analogs 2a–d with cyclic constraints
tion campaign of ML012. Our efforts yielded compound
1
(VU0415248, Fig. 1), a more potent antagonist with better selectiv-
ity for M1.20 These efforts also expanded the structure–activity
relationship (SAR) of ML012 and other compounds in this series.
Herein, we report further modifications which provided a panel
of compounds with improved potency and good selectivity for
the M1 muscarinic receptor, and more importantly, divergent SAR
from the ML012 series.
O
S
O
N
N
N
N
N
N
S
O
2a-d
Compd Aza ring
pIC50
SEMb
hM1 IC50
%EC min
SEMb
In our previous work on ML012 optimization, the central linker
was modified through the introduction of methyl substitution and
fluorination at the alpha position of the beta-alanine moiety. Of
these modifications, none provided a desirable increase in potency
or selectivity and many abolished activity altogether.20 Concur-
rently, we prepared a limited series of cyclic constrained analogs
and screened these compounds for antagonism at M1 (Table 1).
For compounds 2a–c, potency was significantly decreased while
some slight activity at M1 remained (for 2a, 33% activity and for
2c, 47% activity). We were encouraged that an azetidine analog,
2d, was tolerated, albeit threefold less active than ML012. This pro-
vided an opportunity to enter into new chemical space and inves-
tigate compounds for improved potency and selectivity over
ML012. Previous modifications to the Western thiadiazole of
ML012 led us to determine that an oxadiazole was a suitable
replacement and generally maintained potency.20 We procured 3
(Fig. 2), which contained the desired oxadiazole and a central azeti-
dine linker, and found that it was equipotent to 1 (vida supra), our
improved M1 antagonist.
With 3 in hand, we explored the SAR at both termini of the mol-
ecule through an iterative parallel synthesis approach. These
routes, illustrated in Scheme 1, made use of the commercially
available azetidine central linkers 4 and 8. For the Eastern SAR,
azetidine 4 and aryl sulfonyl chlorides were reacted to provide sul-
fonamide 5, followed by saponification which yielded acid 6.
Amide coupling with substituted piperazines provided the target
compound (7). As with our previous findings from the optimization
of ML012, Eastern SAR around the pyridine ring was unforgivingly
steep.20 Indeed, only compound 3 (hM1 IC50 = 430 nM) or substitu-
constraintsa
(l
M)b
N
2a
2b
>10
33.4 3.9
Inactive
N
2c
>10
2.2
47.1 4.0
10.1 3.3
N
N
2d
5.68 0.07
a
Examples 2a and 2c were prepared and screened as racemic mixtures.
Values represent the mean standard error mean of at least three independent
determinations performed in triplicate.
b
O
N
O
N
N
S
O
N
N
N
O
3
VU0414910
hM IC = 430 nM
1
50
Figure 2. VU0414910, an M1 antagonist.
each of the five mAChRs. We envision two ways to obtain mAChR
subtype selectivity with synthetic ligands: 1) simultaneous binding
to the orthosteric site and into adjacent areas which may be less
structurally conserved among the other mAChRs12 or 2) binding to
a completely distinct region of the mAChR at an allosteric site,
imparting a level of selectivity to the ligand not found relative to
the other four mAChRs. This allosteric approach has been highly
successful for a number of the individual mAChRs: M1,13–15 M4,16
and M5. 17
tion with
a
(5-bromopyridin-2-yl)piperazine moiety (hM1
IC50 = 280 nM, structure not shown) maintained activity.21 Focus-
ing on the Western SAR and starting with azetidine 8, amide cou-
pling to give 9 was followed by deprotection of the N-Boc with TFA
in DCM to provide bis-TFA salt 10. Sulfonation with aryl sulfonyl
O
O
O
O
O
a
b
HCl•HN
BocN
ArSO2N
ArSO2N
c
O
OH
6
4
5
N
ArSO2N
N
O
Ar'
O
7
O
a
c
d
N
N
HN
2•TFA
N
OH
BocN
N
Ar'
Ar'
8
9
10
Scheme 1. Reagents: (a) ArSO2Cl, NEt3, DCM; (b) NaOH (aq), MeOH; (c) amine, EDCI, HOBt, DIEA, DMF; (d) TFA/DCM (1:1).

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5035–5040
5037
Table 2
Structures and activities of M1 antagonist analogs 7a–7aa
O
N
O
N
N
S
Ar
O
N
7a-7aa
Compd Ar
pIC50
SEMa
hM1 IC50 %EC min
Compd Ar
pIC50
hM1 IC50 %EC min Compd Ar
pIC50
SEMa
hM1 IC50 %EC min
a
a
a
(l
M)
SEMa
SEMa
(
lM)
SEMa
(l
M)
SEMa
OCF3
N
7a
7b
7c
5.60 0.07 2.5
5.77 0.04 1.7
5.55 0.04 2.8
8.4 1.0
7j
5.91 0.09 1.2
5060 0.11 2.5
5.64 0.02 2.3
12.3 1.0 7s
5.88 0.06 1.3
8.2 1.0
N
N
Cl
F
S
N
9.5 0.8
7k
7l
12.7 1.6 7t
15.3 1.5 7u
>10
>10
20.6 1.6
40.7 0.8
MeO
F3C
N
CF3
F
Cl
Cl
O
11.4 1.0
N
N
7d
7e
7f
5.39 0.05 4.1
20.7 2.3
41.3 1.6
38.5 0.7
7m
7n
7o
>10
6.08 0.08 0.84
6.08 0.11 0.83
33.0 0.9 7v
3.5 0.2 7w
5.3 0.8 7x
6.90 0.07 0.13
6.94 0.14 0.11
6.13 0.01 0.74
3.6 0.6
2.7 0.3
3.5 0.4
O
N
NH
N
Cl
Cl
N
>10
>10
O
N
N
O
Cl
N
Cl
Cl
7g
7h
5.75 0.07 1.8
5.77 0.02 1.7
8.8 1.1
8.0 0.8
7p
7q
6.43 0.07 0.37
6.30 0.04 0.50
4.9 0.2 7y
5.7 0.8 7z
5.81 0.02 1.6
5.67 0.11 2.2
6.0 0.9
9.8 1.6
N
N
N
Cl
N
N
N
N
Cl
Cl
O
7i
5.64 0.01 2.3
10.5 1.2
7r
6.51 0.05 0.31
4.9 0.8 7aa
5.26 0.01 5.5
12.7 0.7
Cl
a
Values represent the mean standard error mean of at least three independent determinations performed in triplicate.
chlorides generated sulfonamides (7), shown in Table 2 with the
requisite (pyridin-4-yl)-piperazine and a variety of Ar groups.
Table 2 illustrates the Western SAR of azetidine-linked aryl
piperazines. While not performed extensively, we did investigate
other ways to functionalize the N-H of the azetidine, including sul-
fonamide and amide bond formation. Amides were not a fruitful
ML012 (hM1 IC50 = 0.37
such as 4-chloropyridin-3-yl sulfonamide 7q and 4-chloro-
5-methylpyridin-3-yl sulfonamide 7r were potent (hM1 IC50
0.50 M and hM1 IC50 = 0.31 M, respectively), as was 2-chloro-
pyridin-3-yl sulfonamide 7s, albeit to a lesser extent. 4-methoxy-
pyridin-3-yl and 4-trifluoromethylpyridin-3-yl sulfonamides 7t
lM). Substituted pyridine replacements
=
l
l
endeavor, resulting in all compounds with >10
l
M potency at M1
and 7u were weak antagonists (hM1 IC50 >10
ACh activity remaining at the highest concentration tested
(30 M)). This loss of potency could be attributed to the increased
steric bulk at the 4-position of the pyridine. The largest improve-
ments in potency were seen with compounds 7v and 7w (hM1
lM, 20 and 40%
(data not shown). Aryl sulfonamides were tolerated in a much
broader sense than seen previously with the ML012 scaffold,19
with many compounds displaying IC50 values in the low to mid-
micromolar range. Simple benzene sulfonamides 7a–d were 2- to
5-fold less potent than ML012 (used as our standard in this con-
l
IC50 = 0.13 lM and hM1 IC50 = 0.11 lM, respectively, Table 2) with
text, hM1 IC50 = 0.81
weak antagonists with both compounds displaying hM1 IC50
>10 M, 38 and 41% ACh activity remaining at the highest concen-
tration tested (30 M). Other heterocyclic aromatics were modest
l
M). 2-Pyridyl sulfonamides 7e and 7f were
an eightfold increase. It seemed that many of these azetidine
analogs of ML012 began to show a divergent SAR profile from
the parent molecule. Finally, compounds 7x–aa exhibited a steric
l
l
intolerance at the a-position of the isoquinoline and were less
antagonists with low to mid-micromolar potencies (see 7g–m, Ta-
ble 2). 2,3-Dichlorination of the benzene ring (7n) gave a com-
pound that was nearly equipotent to ML012 as did piperonyl
sulfonamide, 7o. In the beta-alanine-linked ML012 series, 3-pyri-
dyl sulfonamides were inactive,19 yet a small subset of 3-pyridyl
sulfonamides, including compound 7p, were more potent than
potent antagonists of M1.
Isoquinoline sulfonamide 7w represented a new and attractive
scaffold on which to work. The strategic introduction of a critical
nitrogen atom improved potency over benzothiadiazole and ben-
zoxadiazole sulfonamides 2d and 3 and likely mimics an interac-
tion at the N-1 nitrogen of both structures. Previously, this trend

5038
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5035–5040
Table 3
to the original scaffold. To insure that we had found the optimal
Eastern heterocycle, we held the Western isoquinoline sulfon-
amide and the azetidine linker constant. Following our iterative
parallel synthesis approach described in Scheme 1 (compounds
4–7, vida supra), we generated a small set of Eastern replacements
to the pyridin-4-yl piperazine (Table 3). We anticipated that only
basic moieties would be tolerated in Ar substitutions on compound
11 from the SAR profiles previously described. Indeed, 11a proved
to be a modest antagonist of M1 (hM1 IC50 = 1.5 lM). Improved
potencies were observed for a more basic set of substituents (
11b–e).
Next, replacements for the piperazine were explored. We uti-
lized common isosteres for this aza-heterocycle, shown in Fig. 3.
Many of these variants did not provide antagonists with any appre-
ciable activity. The one notable exception was the 3,7-diazabicy-
clo[3.3.0]octane 12a. This isostere was remarkably potent (hM1
Structures and activities of M1 antagonist analogs 11a–e with eastern ring
replacements
O
N
N
O
S
N
N
Ar
O
11a-e
hM1 IC50
a
Compd
Ar
pIC50 SEMa
5.83 0.14
(
lM)
%EC min SEMa
7.3 1.4
N
11a
1.5
N
N
11b
6.74 0.18
0.18
3.6 0.7
Cl
IC50 = 0.23 lM, Table 4).
N
11c
11d
11e
6.16 0.09
6.29 0.01
6.97 0.08
0.69
0.51
0.11
4.7 0.5
7.3 1.4
3.1 0.1
Probing the ML012 scaffold for SAR revealed that rather stark
modifications to the central linker and piperazine would afford a
new SAR paradigm to be explored. Holding the central azetidine
and the newly discovered 3,7-diazabicyclo[3.3.0]octane substitu-
tions constant, we performed another round of explorations on
the Western side of compound 12a (VU0455691, Table 4). Revisit-
ing many of the same aryl sulfonamides as described in Table 3 and
5-quinoline sulfonamide 12b was a weak antagonist (hM1 IC50
N
N
a
Values represent the mean standard error mean of at least three independent
determinations performed in triplicate.
>10
lM, 26% activity remaining) yet 6-methylisoquinoline-5-sul-
fonamide 12c was a micromolar antagonist (hM1 IC50 = 2.7
l
M).
Unfortunately, many of these aryl sulfonamides that were potent
in the piperazine series were less so in the 3,7-diazabicyclo-
[3.3.0]octane series. This was exemplified by compounds 12d–i
which had modest antagonist activity at M1 (Table 4).
OH
N
N
N
N
N
N
The development of selective allosteric compounds for mAChRs
is well documented in our laboratories.13–17 Selective orthosteric
compounds are more difficult to achieve due to a high conservation
of the binding site between the five mAChR subtypes. In the case of
ML012 and VU0415248, both compounds were selective orthoster-
Figure 3. Piperazine replacements for M1 antagonists.
was also observed in the optimization of ML012 to arrive at
VU0451248.20 With this structure in hand, we moved to probe
the Eastern SAR once more, given the breadth of changes made
Table 4
Structures and activities of M1 antagonist analogs 12a–i with Western ring replacements
O
N
O
N
S
N
Ar
O
12a-i
N
a
a
Compd
Ar
pIC50 SEMa
6.64 0.15
hM1 IC50
0.23
(lM)
%EC min SEMa
2.8 0.4
Compd
Ar
pIC50 SEMa
5.72 0.04
hM1 IC50
1.9
(lM)
%EC min SEMa
13.5 1.8
O
N
O
12a
12f
N
N
12b
12c
>10
2.7
25.8 0.9
14.8 1.5
12g
12h
12i
5.81 0.06
5.50 0.07
5.83 0.02
1.5
3.2
1.5
6.6 1.1
18.9 1.1
9.9 0.8
Cl
Cl
5.57 0.05
F
S
N
N
N
12d
12e
5.55 0.02
5.71 0.01
2.8
2.0
16.2 0.6
12.6 1.0
N
Cl
O
N
a
Values represent the mean standard error mean of at least three independent determinations performed in triplicate.

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5035–5040
5039
Figure 4. A, Compound 7o (VU0456704), 12a (VU0455691), and atropine compete with [3H]NMS binding at M1. B, 12a (VU0455691) competitively antagonizes M1 response
to ACh in a concentration-dependent manner in a calcium mobilization assay. C, Schild regression of the concentration ratios derived from 12a (VU0455691) antagonism of
ACh (slope of this regression is 0.94 0.01. Kd = 12 2.5 nM. R2 = 0.993). D, 7o (VU0456704) competitively antagonizes the M1 response to ACh in a concentration-dependent
manner in a calcium mobilization assay. E, Schild regression of the concentration ratios derived from 7o (VU0456704) antagonism of ACh (slope of this regression is
0.96 0.02. Kd = 54 8.5 nM. R2 = 0.999). Values represent the mean S.E.M. of three experiments conducted in triplicate.
ic antagonists of M1 mAChR.20 These new series of antagonists
merited a reexamination of the nature of their interaction with
the M1 mAChR due to the changes made to the original ML012
scaffold. With this in mind, equilibrium radioligand binding stud-
ies using 1-[N-methyl-3H]scopolamine ([3H]NMS) were performed
using two representative compounds, one from the azetidine
linked piperazine series (VU0456704, 7o) and one from the
[3.3.0] series (VU0455691, 12a). Both compounds showed com-
plete displacement of the orthosteric ligand [3H]NMS, Figure 4A,
consistent with a competitive interaction. Additionally, functional
Schild analyses of compounds 7o and 12a were also performed.12
Both 7o and 12a displayed a parallel rightward shift in the ACh
concentration response curves as the concentration of 7o or 12a
was increased, as shown in Figure 4B and D (analysis of Schild data
showed a linear regression of essentially unity, Fig. 4C and E).
These data further support an orthosteric binding mode as well
as a competitive orthosteric ligand in functional assays.
Our most potent compounds from the piperazine and the 3,7-
diazabicyclo[3.3.0]octane series (7w and 12a, respectively) were
chosen for profiling against both human and rat M1–5 receptors

5040
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5035–5040
forms 2C9, 2D6, 3A4; IC50 >30 lM for 1A2). This compound was
also measured in a rat brain homogenate binding experiment
and exhibited desirable levels of% unbound (fu = 0.08).
In conclusion, we have further expanded the SAR surrounding
ML012 which has culminated in the development of selective
orthosteric M1 antagonists 7w (VU0452865) and 12a (VU0455691).
These antagonists utilized a novel scaffold relative to ML012 and
clearly displayed a unique and separate SAR from the previous ser-
ies. These compounds represent valuable in vitro tools with im-
proved selectivity over ML012. Continuing work on the SAR
described here may yet improve the DMPK properties of these clas-
ses of antagonists. This work will be reported in due course. ML012
22
is an MLPCN probe and is freely available upon request.
Acknowledgments
The authors thank Seaside Therapeutics, NIMH (RO1MH082867),
NIH (U54MH084659) and NINDS (P50NS071669) for support of
our Center in the development of subtype selective mAChR
antagonists.
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Figure 5. Compound 7w (VU0452865) and compound 12a (VU0455691) selectively
antagonize M1 when compared to M2–5 receptors. CRCs were obtained in the
presence of an EC80 concentration of ACh for each receptor in calcium mobilization
assays. Data were normalized to the maximum response of 30
presented as a percentage of the EC80 ACh response.
lM ACh and are
in a calcium mobilization assay. Both compounds compounds were
highly selective for human M1 over the other human muscarinic
subtypes (hM2-5 IC50s >10 lM, data not shown), despite their
orthosteric behavior observed in binding and functional assays.
Figure 5 shows these data for the rat (r) M1–5 receptors. One would
anticipate that ML012 and 1 would have more structural flexibility
in the linker region relative to 7w and 12a. Indeed, when the same
quinoline sulfonamide of 1 is made in the piperazine and the 3,7-
diazabicyclo[3.3.0]octane series (7h and 12b, Tables 2 and 4,
respectively) resulting compounds are inactive or weak antago-
nists at best. The structural rigidity provided by the azetidine lin-
ker seems to require
a regioisomeric quinoline sulfonamide
relative to 1 to maintain potency at M1. Additionally, we suspect
this key nitrogen interaction is vital for selectivity at M1 over M
2–5. We proceeded to evaluate our most divergent compound from
ML012 and 1, compound 12a, for its pharmacokinetic properties.
This compound was extremely hydrophilic which is not surprising,
given its remarkably low cLogP (cLogP = 0.78) and, consequently,
exhibited a high% unbound in plasma protein binding assays (hu-
man PPB fu = 0.97, rat PPB fu = 0.60). Unfortunately, 12a also dis-
played an IV plasma clearance value in the rat of 68.6 mL/min/kg,
which correlated well with its moderate to high in vitro hepatic
microsome intrinsic clearance (CLINT: 107 mL/min/kg) and pre-
dicted hepatic clearance (CLHEP: 42.3 mL/min/kg). 12a was also
measured for its ability to inhibit the more common cytochrome
P450 enzymes. Three of the four P450 enzymes tested were inhib-
18. Veeraragavan, S.; Bui, N.; Perkins, J. R.; Yuva-Paylor, L. A.; Carpenter, R. L.;
Paylor, R. Psychopharmacology 2011, 217, 143.
19. Healy, A.; Rush, R.; Ocain, T. ACS Chem. Neurol. 2011, 2, 402.
20. Melancon, B. J.; Bridges, T. M.; Lamers, A. P.; Sulikowski, G. A.; Sheffler, D. J.;
Niswender, C. M.; Noetzel, M. J.; Utley, T. J.; Daniels, J. S.; Morrison, R. D.; Jones,
C. K.; Conn, P. J.; Lindsley, C. W.; Wood, M. R. Bioorg. Med. Chem. Lett. 2012, 22,
1044.
21. (1-(Benzo[c][1,2,5]oxadiazol-4-ylsulfonyl)azetidin-3-yl)(4-(pyridin-2-
yl)piperazin-1-yl)methanone had an IC50 = 5 lM.
22. For information on the MLPCN and information on how to request probe
compounds, such as ML012, see: http://mli.nih.gov/mli/mlpcn/.
ited at low micromolar concentrations of 12a (IC50 <2.5 lM, iso-